It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles ...It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles in the endosymbiotic cyanobacterium TDX16 (prokaryote) that was released from its senescent/necrotic host cell of green alga Haematococcus pluvialis (eukaryote). Microscopic observations showed that organelle biogenesis in TDX16 initiated with cytoplasm compartmentalization, followed by de-compartmentalization, DNA allocation, and re-compartmentalization, as such two composite organelles-the primitive chloroplast and primitive nucleus sequestering minor and major fractions of cellular DNA respectively were formed. Thereafter, the eukaryotic cytoplasmic matrix was built up from the matrix extruded from the primitive nucleus;mitochondria were assembled in and segregated from the primitive chloroplast, whereby the primitive nucleus and primitive chloroplast matured into the nucleus and chloroplast respectively. While mitochondria subsequently turned into double-membraned vacuoles after matrix degradation. Results of pigment analyses, 16S rRNA and genome sequencing revealed that TDX16 is a phycocyanin-containing cyanobacterium resembling Chroococcidiopsis thermalis, which had acquired 9,017,401 bp DNAs with 10,301 genes from its host. Accordingly, we conclude that organelle biogenesis in TDX16 is achieved by hybridizing the acquired eukaryotic DNAs with its own one and expressing the hybrid genome. The formation of organelles in cyanobacterium TDX16 is the first case of organelle biogenesis in prokaryotes observed so far, which sheds an unprecedented light on eukaryotes and their connections with prokaryotes, and thus has broad implications on biology.展开更多
Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture ...Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture media of LPS-treated Raw264.7 cells was able to stimulate growth in MEF1F2 and NIH3T3 mouse fibroblast cell lines. We identified five secreted and LPS-induced chemokines, CCL2, CCL5, CCL12, CxCL2, and CxCL10, by microarray analysis and tested their stimulatory activity. We used commercially available bacterially expressed proteins, and found only CCL12, CxCL2 and CxCL10 stimulated growth in MEF1F2 and NIH3T3 cells. The saturation density of the cells was also increased. They were not able to stimulate growth in v-Src transformed MEF1F2 or SWAP-70 transformed NIH3T3 cells. We examined signaling pathways activated by these three factors. We found that ERK and p38 MAP kinase were activated and were required for the activity to stimulate the cell growth. Other pathways including phosophatidylinositol-3 kinase (PI3K), NFκB pathways were not activated. These results suggest that Raw264.7 cells secretes growth stimulation factors for fibroblasts when differentiated to macrophages implicating that fast growth of them is related to inflamation although the reason is still unclear.展开更多
Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, despite its importance, the specific role of insulin resistance in the et...Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, despite its importance, the specific role of insulin resistance in the etiology of these diseases is poorly understood. At the same time, ethanol (ETOH) is a potent vasoconstrictor that primarily induces down regulation of mitogen activated protein kinases (MAPKs) which could exacerbate insulin resistance and possibly lead to cardiovascular diseases. This article describes how chronic ETOH exposure interferes with insulin signaling in hypertensive vascular smooth muscle cells (HVSMCs) which leads to the alteration of MAPKs, the major signaling molecules. Elevated (50 - 800 mM) chronic exposure (24 hr) of HVSMCS to ETOH prior to insulin stimulation decreased insulin-induced ERK 1/2 (MAPKs) and AKT expression. Similar experiments were conducted using normotensive cells from rat. These cells showed reductions in insulin-induced ERK 1/2 phosphorylation as well, but only at higher concentrations of ETOH (400 - 800 mM). These alterations in insulin signaling could provide an alternative molecular mechanism that may increase the risk of insulin resistance, thus increasing the possibility of cardiovascular diseases.展开更多
The differences in satellite DNA methylation pattern of corn seedlings with various spontaneous chromosome aberration yields and changes in methylation pattern of these DNA sequences under different exposure modes of ...The differences in satellite DNA methylation pattern of corn seedlings with various spontaneous chromosome aberration yields and changes in methylation pattern of these DNA sequences under different exposure modes of acute UV-C and chronic gamma-irradiations have been investigated. The obtained experimental data and the conducted correlation analysis demonstrated the significant correlation between the satellite DNA methylation pattern varieties and chromosome aberration yields under various stress exposure modes. The role of satellite DNA methylation pattern variability and its changing in key responses to stress such as mobile elements’ activation, cell’s passage of checkpoints, and homological repair was discussed.展开更多
Cytoskeleton exists in all eukaryotes and is involved in many significant cytobiological processes, especially the movements and developmental changes of plant cells. The cytoskeleton consists of microtubule (MT), mic...Cytoskeleton exists in all eukaryotes and is involved in many significant cytobiological processes, especially the movements and developmental changes of plant cells. The cytoskeleton consists of microtubule (MT), microfilament (MF), and intermediate filament (IF). MT and MF are vital components of plant cytoskeleton. Crosslinking factor acts as a bridge between MF and MT. They play an important role in cellular life process and have always been a hot topic and key point in plant cytobiology, and the IF is a difficult point in this field. In this paper, the latest research on the cytoskeleton of plants is introduced, which focuses on the structure and dynamics of MT, MF, and IF, and summarizes the crosslinking factors between MT and MF. Also, the paper prospects the future research direction of plant cytoskeleton and the possible research hotspot, which provides a certain reference for people to continue to explore the function of plant cytoskeleton in the future.展开更多
Gastric parietal cells are important in acid secretion, but it is unclear which cells throughout the gastric gland have the highest secretion potency. Here, we used immunohistochemical methods with anti-H+, K+-ATPase,...Gastric parietal cells are important in acid secretion, but it is unclear which cells throughout the gastric gland have the highest secretion potency. Here, we used immunohistochemical methods with anti-H+, K+-ATPase, phosphoryl ezrin and CD44 antibodies to study the distribution of gastric acid secretion activity. Stomach tissues from freely fed and starved rats were cryofixed for light microscopy or fixed by high-pressure freezing for electron microscopy. Parietal cells from freely fed animals corresponded to the active secretion phase and to the inactive resting phase from starved rats. Anti-H+, K+-ATPase and anti-phosphoryl ezrin labeling were observed on the membrane of the intracellular canaliculi and the tubulovesicle from freely fed rats, while cells from starved animals showed weak labeling with anti-phosphoryl ezrin antibody staining. Morphometrical analysis at the electron microscopic level was performed on active and inactive acid secretory phases between the upper and base regions of the gland. H+, K+-ATPase and CD44 were distributed on both sites of the microvillous and tubulovesicle membrane in the same cells, but phosphoryl ezrin localized predominantly on the microvillous membrane in active cells of the glandular neck and upper base. Therefore, the highest secreting potency appeared to be in cells of the glandular neck and upper base.展开更多
Radiation induced reactive oxygen/nitrogen species (ROS/RNS) are reported to cause lung injuries such as pneumonitis and fibrosis which may be fatal at times. Current study is designed to analyse the radioprotective e...Radiation induced reactive oxygen/nitrogen species (ROS/RNS) are reported to cause lung injuries such as pneumonitis and fibrosis which may be fatal at times. Current study is designed to analyse the radioprotective efficacy of P. hexandrum active principles (G-002M) on lungs of mice exposed to high dose of gamma irradiation (7 Gy). Cellular profiles and inflammatory cell infiltrates of irradiated bronchoalveolar lavage fluid (BALF) have shown correlations with lung pathology. Cell counts were determined in BALF of control, 7 Gy radiation exposed and radiation with G-002M pretreated mice. ROS/Nitric Oxide (NO) production was measured by 2,7?dichlorodihydrofluorescein diacetate (DCF-DA) and diaminofluorescein diacetate (DAF-2DA) through microscopy and flow cytometry respectively. Immunostaining of inducible nitric oxide synthase (iNOS) in BALF cells and lung sections was also observed microscopically. iNOS ex- pression was observed in lungs by western blotting. BALF was also processed to estimate total protein, LDH, and phospholipids content. Catalase, reduced Glutathione (GSH), Glutathione reductase (GR) and lipid peroxidation were estimated in lung tissues. Pre-administration of G-002M significantly decreased radiation mediated neutrophils count in BALF of irradiated mice. ROS generation, iNOS expression, total protein, LDH and phospholipids were found less affected in G-002M pretreated group in comparison to radiation alone group. Radiation exposure to mice was found apparently leading to parenchymal fibrosis, an architectural distortion of the lung tissue with edema, infiltration of inflammatory blood cells with increased immunolabeling of iNOS. G-002M pretreatment significantly countered radiation mediated increased lipid peroxidation and decreased GR, catalase and GSH in mice. Current study demonstrates possible role of P. hexandrum (G-002M) in minimizing lung damage induced by radiation mediated ROS/RNS generation.展开更多
The cystatins are a super family of cysteine protease inhibitors which are ubiquitous in their biologic occurrence. Cystatin C, a type II cystatin, is primarily a secreted protein found in most biological fluids. Besi...The cystatins are a super family of cysteine protease inhibitors which are ubiquitous in their biologic occurrence. Cystatin C, a type II cystatin, is primarily a secreted protein found in most biological fluids. Besides acting as inhibitors of cathepsin, the cystatins have been found to have some non-inhibitor related functions and multiple physiological roles. Much interest has been generated for the cystatins as metastasis “suppressor-like” proteins, as they have been shown to inhibit metastasis for multiple cancer types. The sites and actions of the cystatins related to tumor suppressor actions are still unclear, however. In this work, we have examined the uptake of cystatin by metastatic melanoma cells in culture. Our results indicate cystatin uptake is mediated by a non-canonical endocytotic pathway in B16 murine melanoma cells.展开更多
Oxidative damage and redox metal homeostasis loss are two contributing factors in brain aging and widely distributed neurodegenerative diseases. Oxidative species in company with excessive amounts of intracellular fre...Oxidative damage and redox metal homeostasis loss are two contributing factors in brain aging and widely distributed neurodegenerative diseases. Oxidative species in company with excessive amounts of intracellular free iron result in Fenton-type reaction with subsequent production of highly reactive hydroxyl radicals which initiate peroxidation of biomolecules and further formation of non-degradable toxic pigments called lipofuscin that amasses in long-lived postmitotic cells such as neurons. Dietary flavonoid baicalein can counteract the detrimental consequences through exertion of a multiplicity of protective actions within the brain including direct ROS scavenging activity and iron chelation. In this study, we evaluated the neuroprotective effects of baicalein in menadione (superoxide radical generator)-treated SK-N-MC neuroblastoma cell line. Our results showed that treatment of cells with menadione led to lipofuscin formation due to elevated intracellular iron contents and accumulation of oxidative products such as MDA and PCO. Also, menadione caused apoptotic cell death in SK-N-MC cells. However, pretreatment with baicalein (40 μM) reversed the harmful effects by chelating free iron and preventing biomolecules peroxidations. Moreover, baicalein prevented cell death through modulation of key molecules in apoptotic pathways including suppression of Bax and caspase-9 activities and induction of bcl2 expression. Key structural features such as presence of hydroxyl groups and iron-binding motifs in baicalein make it the appropriate candidate in antioxidant-based therapy in age-related neurodegenerative diseases.展开更多
The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin αIIb domain to induce receptor activation and integrin αIIbβ3 mediated cell adhes...The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin αIIb domain to induce receptor activation and integrin αIIbβ3 mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin αVβ3 or chimeric αVβ3 carrying αIIb cytoplasmic tail, we report that the interaction of CIB with β3 integrins is not αIIbβ3 specific but binds αIIb as well as αV cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the αIIb cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric αV/αIIbβ3-CIB interaction. This demonstrates that αIIb cytoplasmic domain loop-like conformation is required for interaction with CIB. Moreover, mutations of β3 cytoplasmic domain residues Tyr-747 and/or Tyr-759 to phenylalanine residues (Y747F, Y759F, and Y747,759F) as well as residues Ser-752 to proline or alanine (S752P and S752A), do not affect the αIIbβ3 or αVβ3 interaction with CIB. Since tyrosine residues Tyr-747 and/or Tyr-759 are the sites of tyrosine phosphorylation of β3 subunit, these results suggest that the β3 integrin-CIB interaction occurs through aβ3-phosphorylation independent mechanism. Likewise, ablation of conformation-dependent affinity change in β3 Ser752Pro mutation had no affect on CIB-β3 interaction. In summary, our results demonstrate that the αIIb-subunit integrin and CIB interaction is non-exclusive and requires the loop-like αIIb-cytoplasmic domain conformation. An interaction of CIB with αV-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.展开更多
Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sort...Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.展开更多
The purpose of the work was to assess the combined effect of drought and salinity (50, 100, 200 mМ NaCl) on the meso- and ultrastructure of mesophyll cells of wheat seedlings. Stress development was estimated by a de...The purpose of the work was to assess the combined effect of drought and salinity (50, 100, 200 mМ NaCl) on the meso- and ultrastructure of mesophyll cells of wheat seedlings. Stress development was estimated by a decrease in the relative water content (RWC) and CO2-dependent O2 evolution (An) in leaves. The decrease in the RWC and in An occurred rapidly in the absence of salt in the substrate and slowly in the presence of salt, especially at a treatment of 100 mM NaCl. The resumption of watering led to the recovery of the both parameters in all variants except one with 200 mM NaCl. Structural studies showed that a weak drought stress (RWC 60%) without salinity led to the destruction of cell membranes and hyaloplasm, which did not occur in all salt treatments. By contrast, the ultrastructure of nuclei in weak drought without salinity remained unchanged, whereas in all salt treatments chromatin changed substantially. Heterochromatin underwent a strong condensation followed by the fusion into a united mass with the simultaneous loss of electron density. A strong water stress (RWC 40%) in all variants led to cell destruction and the hydrolysis of cell compounds. Under the drought without salinity, vacuoles disappeared, whereas in salt-treated samples they were retained and filled with organelles being at different degrees of degradation. Cell nuclei under strong drought stress lost their rounded shape, nuclear envelopes were destroyed, and at the end only a finely dispersed substance remained. Thus, under the combined action of drought and salt, there is some critical level of salt concentration in substrate above which the effect of NaCl changes to the adverse, which enhances the action of drought. Among structural components of mesophyll cells, the most sensitive parts to NaCl are nuclei and their chromatin.展开更多
Cleft palate or harelip is one of the most common congenital defects in humans and harelip with cleft lip in whites is estimated 1 out of every 7000 to 1000 births. In recent years, surgeons and doctors have tried to ...Cleft palate or harelip is one of the most common congenital defects in humans and harelip with cleft lip in whites is estimated 1 out of every 7000 to 1000 births. In recent years, surgeons and doctors have tried to resolve this problem immediately after the birth by operation, but sometimes we encounter things that cannot be easily solved by a surgical procedure. Our goal is to use stem cells to eliminate the disease and prevent operation, because children are often afraid of surgery and pain later on. Also, the need for general anesthesia for bone remodeling involves complications, such as long-term pain and nerve disorders. By the advent of the use of alternative stem extraction techniques, a better alternative to the invasive method of cleft lip and palate therapy has been developed.展开更多
Transporter associated with antigen processing (TAP)-like (TAPL;ABCB9) is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that function in the ER membrane. To determine the cellular localization,...Transporter associated with antigen processing (TAP)-like (TAPL;ABCB9) is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that function in the ER membrane. To determine the cellular localization, TAPL and truncated forms of it were tagged with GFP at their car-boxyl termini. Intracellular localization of these fusion proteins was compared between transient and stable expression in CHO-K1 cells. When they were expressed transiently, the fluorescence of the fusion proteins was detected on the intracellular membrane, mainly in the ER, and all the fusion proteins, i.e., TAPL(M1 -A766)-GFP, TAPL(M1-S275)-GFP, TAPL(M1-K182 )-GFP, TAPL(M 1-R141)-GFP and TAPL(M1-G75), were co-localized with an ER marker, PDI. However, the fluorescence of all of them except for TAPL(M1-G 75)-GFP and TAPL(M1-S275)-GFP overlapped with a lysosome marker, cathepsin D, upon stable expression. Lysosomal localization was similarly observed with TAPL(M1- A766)-DsRed, which was stably expressed. These results suggest that TAPL is sorted to the lysosomal membrane when expressed stably in CHO-K1 cells. Furthermore, the lysosomal targeting signal may comprise the N-terminal four transmembrane helices since the N-terminal two transmembrane helices may not be enough to function as such a signal.展开更多
Intracellular Notch (ICN) initiates DNA transcription in cooperation with CSL that acts as repressor in the absence of ICN. The ICN mediates recruitment of MAML protein, leading to the formation of minimal transcripti...Intracellular Notch (ICN) initiates DNA transcription in cooperation with CSL that acts as repressor in the absence of ICN. The ICN mediates recruitment of MAML protein, leading to the formation of minimal transcriptional complex, MAML/ICN/CSL/DNA. Crystal structure reveals that different conformations exist between the free (CSL/DNA) and bound (ICN/MAML/CSL/DNA) forms. The significance of this modulation of the CSL/DNA molecular complex can be better understood by experimental approaches that aim to elucidate the cause and timing of these events. There are four orthologues of human ICN (ICN1-4). We studied interactions between human full-length ICN1 and CSL/DNA without involvement of MAML, in vitro, and found that 1) the EMSA profile of CSL/DNA is altered in the presence of ICN1 as a consequence of an intrinsic change(s) in CSL/DNA, and not due to the formation of an ICN/CSL/DNA molecular complex;2) ICN1 destabilizes CSL/DNA. These findings indicate that human ICN1 functions to modulate the CSL/DNA molecular complex for subsequent recruitment of MAML, and that modulated CSL/DNA cannot accommodate ICN1 in the absence of MAML. The latter in turn, implies that the formation of the MAML/ICN1/CSL/DNA is likely to be a collective event, wherein preassembly of MAML and ICN1 as a binary complex co-localizes at the CSL/DNA promoter site, or the MAML/ICN1/CSL complex is pre-assembled prior to binding to the promoter, rather than ICN1 arriving at CSL/DNA ahead of MAML and/or other associated transcription factors. The novel finding that ICN1 destabilizes the CSL/DNA complex opens new possibilities of transcriptional regulation by Notch.展开更多
Mechanical stimulations have been shown to regulate cellular mechanical properties. However, the stimulation patterns for effective regulation are as yet unclear. We investigated the effects of application of differin...Mechanical stimulations have been shown to regulate cellular mechanical properties. However, the stimulation patterns for effective regulation are as yet unclear. We investigated the effects of application of differing numbers of mechanical stimulation sets, each set consisting of 8% extension and compression to cells via deformation of cell culture elastic chamber, on cellular elasticity. Elasticity increased with only a single step-like stretch and with a single step-like stretch after 1 set of mechanical stimulation, whereas elasticity did not change with a single step-like stretch after 10 sets of mechanical stimulation. These results indicate that the increase in cellular elasticity with the single step-like stretch depends on the number of applied mechanical stimulations. Immunofluorescence staining showed that phosphorylation and dephosphorylation of myosin regulatory light chain (MRLC), which regulates intracellular contractile force and cellular elasticity, accompanied cellular elasticity changes. These findings suggest that cellular elasticity changes under cyclic and step-like stretches are mediated by MRLC.展开更多
To date, White Spot Syndrome (WSS) produced by the White Spot Syndrome Virus (WSSV) causes one of the most severe diseases infecting penaeid shrimps worldwide. Although a vast amount of studies has elucidated pathogen...To date, White Spot Syndrome (WSS) produced by the White Spot Syndrome Virus (WSSV) causes one of the most severe diseases infecting penaeid shrimps worldwide. Although a vast amount of studies has elucidated pathogenesis in live infection models, there is still little information about the interaction of WSSV infections using in vitro models in the whiteleg shrimp Litopenaeus vannamei (L. vannamei) hemocytes. In this study, a WSSV infection kinetics was performed using total hemocytes isolated from healthy L. vannamei organisms and maintained in in vitro conditions using isotonic solution for shrimp (ISS). The infected experimental cells received ≈ 30,000 viral copies of WSSV. The viability of the hemocytes (control and infected group) was measured during the kinetics with trypan blue exclusion method and cells were maintained up to 6 hpi (post-infection) with non-significant differences of viability between both groups. WSSV replication was assessed using RT- PCR at the RNA expression level of the early viral gene Ie1 and transcripts were detected as early as 30 min pi. Hemocytes from WSSV group showed disrupted integrity, degranulation and irregular shape. This study provides evidence of the capability of WSSV to infect and replicates in L. vannamei hemocytes using in vitro assays in short times as 30 min.展开更多
Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the con...Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift;a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.展开更多
Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction;2) The numb...Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction;2) The number of cells indicated for therapy that is relatively high, with the need to optimize the expansion, number and time wise;3) The utilization of third party donors;4) Culture passage number(P);and 5) Source of the cells. This study was designed to determine the superiority of the Platelet Lysates(PL)over FBS on the expansion of MSC, the optimal cell’ plating density and days between each pass, and to investigate if donor total nucleated cells(TNC)obtained from the washouts of discharged bags and filters of hematopoietic stem cell transplantation(HSCT)can be expanded to be used at clinical grade. TNC were removed, plated and after the first passage were cultivated in different concentrations with FBS or PL, and the number of days to reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or with FBS and after seven days counted to analyze how much they had grown in that period. The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC and to allow the proliferation of enough number of MSC at P2 for clinical use.展开更多
Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many ...Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.展开更多
文摘It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles in the endosymbiotic cyanobacterium TDX16 (prokaryote) that was released from its senescent/necrotic host cell of green alga Haematococcus pluvialis (eukaryote). Microscopic observations showed that organelle biogenesis in TDX16 initiated with cytoplasm compartmentalization, followed by de-compartmentalization, DNA allocation, and re-compartmentalization, as such two composite organelles-the primitive chloroplast and primitive nucleus sequestering minor and major fractions of cellular DNA respectively were formed. Thereafter, the eukaryotic cytoplasmic matrix was built up from the matrix extruded from the primitive nucleus;mitochondria were assembled in and segregated from the primitive chloroplast, whereby the primitive nucleus and primitive chloroplast matured into the nucleus and chloroplast respectively. While mitochondria subsequently turned into double-membraned vacuoles after matrix degradation. Results of pigment analyses, 16S rRNA and genome sequencing revealed that TDX16 is a phycocyanin-containing cyanobacterium resembling Chroococcidiopsis thermalis, which had acquired 9,017,401 bp DNAs with 10,301 genes from its host. Accordingly, we conclude that organelle biogenesis in TDX16 is achieved by hybridizing the acquired eukaryotic DNAs with its own one and expressing the hybrid genome. The formation of organelles in cyanobacterium TDX16 is the first case of organelle biogenesis in prokaryotes observed so far, which sheds an unprecedented light on eukaryotes and their connections with prokaryotes, and thus has broad implications on biology.
文摘Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture media of LPS-treated Raw264.7 cells was able to stimulate growth in MEF1F2 and NIH3T3 mouse fibroblast cell lines. We identified five secreted and LPS-induced chemokines, CCL2, CCL5, CCL12, CxCL2, and CxCL10, by microarray analysis and tested their stimulatory activity. We used commercially available bacterially expressed proteins, and found only CCL12, CxCL2 and CxCL10 stimulated growth in MEF1F2 and NIH3T3 cells. The saturation density of the cells was also increased. They were not able to stimulate growth in v-Src transformed MEF1F2 or SWAP-70 transformed NIH3T3 cells. We examined signaling pathways activated by these three factors. We found that ERK and p38 MAP kinase were activated and were required for the activity to stimulate the cell growth. Other pathways including phosophatidylinositol-3 kinase (PI3K), NFκB pathways were not activated. These results suggest that Raw264.7 cells secretes growth stimulation factors for fibroblasts when differentiated to macrophages implicating that fast growth of them is related to inflamation although the reason is still unclear.
文摘Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, despite its importance, the specific role of insulin resistance in the etiology of these diseases is poorly understood. At the same time, ethanol (ETOH) is a potent vasoconstrictor that primarily induces down regulation of mitogen activated protein kinases (MAPKs) which could exacerbate insulin resistance and possibly lead to cardiovascular diseases. This article describes how chronic ETOH exposure interferes with insulin signaling in hypertensive vascular smooth muscle cells (HVSMCs) which leads to the alteration of MAPKs, the major signaling molecules. Elevated (50 - 800 mM) chronic exposure (24 hr) of HVSMCS to ETOH prior to insulin stimulation decreased insulin-induced ERK 1/2 (MAPKs) and AKT expression. Similar experiments were conducted using normotensive cells from rat. These cells showed reductions in insulin-induced ERK 1/2 phosphorylation as well, but only at higher concentrations of ETOH (400 - 800 mM). These alterations in insulin signaling could provide an alternative molecular mechanism that may increase the risk of insulin resistance, thus increasing the possibility of cardiovascular diseases.
文摘The differences in satellite DNA methylation pattern of corn seedlings with various spontaneous chromosome aberration yields and changes in methylation pattern of these DNA sequences under different exposure modes of acute UV-C and chronic gamma-irradiations have been investigated. The obtained experimental data and the conducted correlation analysis demonstrated the significant correlation between the satellite DNA methylation pattern varieties and chromosome aberration yields under various stress exposure modes. The role of satellite DNA methylation pattern variability and its changing in key responses to stress such as mobile elements’ activation, cell’s passage of checkpoints, and homological repair was discussed.
文摘Cytoskeleton exists in all eukaryotes and is involved in many significant cytobiological processes, especially the movements and developmental changes of plant cells. The cytoskeleton consists of microtubule (MT), microfilament (MF), and intermediate filament (IF). MT and MF are vital components of plant cytoskeleton. Crosslinking factor acts as a bridge between MF and MT. They play an important role in cellular life process and have always been a hot topic and key point in plant cytobiology, and the IF is a difficult point in this field. In this paper, the latest research on the cytoskeleton of plants is introduced, which focuses on the structure and dynamics of MT, MF, and IF, and summarizes the crosslinking factors between MT and MF. Also, the paper prospects the future research direction of plant cytoskeleton and the possible research hotspot, which provides a certain reference for people to continue to explore the function of plant cytoskeleton in the future.
文摘Gastric parietal cells are important in acid secretion, but it is unclear which cells throughout the gastric gland have the highest secretion potency. Here, we used immunohistochemical methods with anti-H+, K+-ATPase, phosphoryl ezrin and CD44 antibodies to study the distribution of gastric acid secretion activity. Stomach tissues from freely fed and starved rats were cryofixed for light microscopy or fixed by high-pressure freezing for electron microscopy. Parietal cells from freely fed animals corresponded to the active secretion phase and to the inactive resting phase from starved rats. Anti-H+, K+-ATPase and anti-phosphoryl ezrin labeling were observed on the membrane of the intracellular canaliculi and the tubulovesicle from freely fed rats, while cells from starved animals showed weak labeling with anti-phosphoryl ezrin antibody staining. Morphometrical analysis at the electron microscopic level was performed on active and inactive acid secretory phases between the upper and base regions of the gland. H+, K+-ATPase and CD44 were distributed on both sites of the microvillous and tubulovesicle membrane in the same cells, but phosphoryl ezrin localized predominantly on the microvillous membrane in active cells of the glandular neck and upper base. Therefore, the highest secreting potency appeared to be in cells of the glandular neck and upper base.
文摘Radiation induced reactive oxygen/nitrogen species (ROS/RNS) are reported to cause lung injuries such as pneumonitis and fibrosis which may be fatal at times. Current study is designed to analyse the radioprotective efficacy of P. hexandrum active principles (G-002M) on lungs of mice exposed to high dose of gamma irradiation (7 Gy). Cellular profiles and inflammatory cell infiltrates of irradiated bronchoalveolar lavage fluid (BALF) have shown correlations with lung pathology. Cell counts were determined in BALF of control, 7 Gy radiation exposed and radiation with G-002M pretreated mice. ROS/Nitric Oxide (NO) production was measured by 2,7?dichlorodihydrofluorescein diacetate (DCF-DA) and diaminofluorescein diacetate (DAF-2DA) through microscopy and flow cytometry respectively. Immunostaining of inducible nitric oxide synthase (iNOS) in BALF cells and lung sections was also observed microscopically. iNOS ex- pression was observed in lungs by western blotting. BALF was also processed to estimate total protein, LDH, and phospholipids content. Catalase, reduced Glutathione (GSH), Glutathione reductase (GR) and lipid peroxidation were estimated in lung tissues. Pre-administration of G-002M significantly decreased radiation mediated neutrophils count in BALF of irradiated mice. ROS generation, iNOS expression, total protein, LDH and phospholipids were found less affected in G-002M pretreated group in comparison to radiation alone group. Radiation exposure to mice was found apparently leading to parenchymal fibrosis, an architectural distortion of the lung tissue with edema, infiltration of inflammatory blood cells with increased immunolabeling of iNOS. G-002M pretreatment significantly countered radiation mediated increased lipid peroxidation and decreased GR, catalase and GSH in mice. Current study demonstrates possible role of P. hexandrum (G-002M) in minimizing lung damage induced by radiation mediated ROS/RNS generation.
文摘The cystatins are a super family of cysteine protease inhibitors which are ubiquitous in their biologic occurrence. Cystatin C, a type II cystatin, is primarily a secreted protein found in most biological fluids. Besides acting as inhibitors of cathepsin, the cystatins have been found to have some non-inhibitor related functions and multiple physiological roles. Much interest has been generated for the cystatins as metastasis “suppressor-like” proteins, as they have been shown to inhibit metastasis for multiple cancer types. The sites and actions of the cystatins related to tumor suppressor actions are still unclear, however. In this work, we have examined the uptake of cystatin by metastatic melanoma cells in culture. Our results indicate cystatin uptake is mediated by a non-canonical endocytotic pathway in B16 murine melanoma cells.
文摘Oxidative damage and redox metal homeostasis loss are two contributing factors in brain aging and widely distributed neurodegenerative diseases. Oxidative species in company with excessive amounts of intracellular free iron result in Fenton-type reaction with subsequent production of highly reactive hydroxyl radicals which initiate peroxidation of biomolecules and further formation of non-degradable toxic pigments called lipofuscin that amasses in long-lived postmitotic cells such as neurons. Dietary flavonoid baicalein can counteract the detrimental consequences through exertion of a multiplicity of protective actions within the brain including direct ROS scavenging activity and iron chelation. In this study, we evaluated the neuroprotective effects of baicalein in menadione (superoxide radical generator)-treated SK-N-MC neuroblastoma cell line. Our results showed that treatment of cells with menadione led to lipofuscin formation due to elevated intracellular iron contents and accumulation of oxidative products such as MDA and PCO. Also, menadione caused apoptotic cell death in SK-N-MC cells. However, pretreatment with baicalein (40 μM) reversed the harmful effects by chelating free iron and preventing biomolecules peroxidations. Moreover, baicalein prevented cell death through modulation of key molecules in apoptotic pathways including suppression of Bax and caspase-9 activities and induction of bcl2 expression. Key structural features such as presence of hydroxyl groups and iron-binding motifs in baicalein make it the appropriate candidate in antioxidant-based therapy in age-related neurodegenerative diseases.
文摘The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin αIIb domain to induce receptor activation and integrin αIIbβ3 mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin αVβ3 or chimeric αVβ3 carrying αIIb cytoplasmic tail, we report that the interaction of CIB with β3 integrins is not αIIbβ3 specific but binds αIIb as well as αV cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the αIIb cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric αV/αIIbβ3-CIB interaction. This demonstrates that αIIb cytoplasmic domain loop-like conformation is required for interaction with CIB. Moreover, mutations of β3 cytoplasmic domain residues Tyr-747 and/or Tyr-759 to phenylalanine residues (Y747F, Y759F, and Y747,759F) as well as residues Ser-752 to proline or alanine (S752P and S752A), do not affect the αIIbβ3 or αVβ3 interaction with CIB. Since tyrosine residues Tyr-747 and/or Tyr-759 are the sites of tyrosine phosphorylation of β3 subunit, these results suggest that the β3 integrin-CIB interaction occurs through aβ3-phosphorylation independent mechanism. Likewise, ablation of conformation-dependent affinity change in β3 Ser752Pro mutation had no affect on CIB-β3 interaction. In summary, our results demonstrate that the αIIb-subunit integrin and CIB interaction is non-exclusive and requires the loop-like αIIb-cytoplasmic domain conformation. An interaction of CIB with αV-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.
文摘Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.
文摘The purpose of the work was to assess the combined effect of drought and salinity (50, 100, 200 mМ NaCl) on the meso- and ultrastructure of mesophyll cells of wheat seedlings. Stress development was estimated by a decrease in the relative water content (RWC) and CO2-dependent O2 evolution (An) in leaves. The decrease in the RWC and in An occurred rapidly in the absence of salt in the substrate and slowly in the presence of salt, especially at a treatment of 100 mM NaCl. The resumption of watering led to the recovery of the both parameters in all variants except one with 200 mM NaCl. Structural studies showed that a weak drought stress (RWC 60%) without salinity led to the destruction of cell membranes and hyaloplasm, which did not occur in all salt treatments. By contrast, the ultrastructure of nuclei in weak drought without salinity remained unchanged, whereas in all salt treatments chromatin changed substantially. Heterochromatin underwent a strong condensation followed by the fusion into a united mass with the simultaneous loss of electron density. A strong water stress (RWC 40%) in all variants led to cell destruction and the hydrolysis of cell compounds. Under the drought without salinity, vacuoles disappeared, whereas in salt-treated samples they were retained and filled with organelles being at different degrees of degradation. Cell nuclei under strong drought stress lost their rounded shape, nuclear envelopes were destroyed, and at the end only a finely dispersed substance remained. Thus, under the combined action of drought and salt, there is some critical level of salt concentration in substrate above which the effect of NaCl changes to the adverse, which enhances the action of drought. Among structural components of mesophyll cells, the most sensitive parts to NaCl are nuclei and their chromatin.
文摘Cleft palate or harelip is one of the most common congenital defects in humans and harelip with cleft lip in whites is estimated 1 out of every 7000 to 1000 births. In recent years, surgeons and doctors have tried to resolve this problem immediately after the birth by operation, but sometimes we encounter things that cannot be easily solved by a surgical procedure. Our goal is to use stem cells to eliminate the disease and prevent operation, because children are often afraid of surgery and pain later on. Also, the need for general anesthesia for bone remodeling involves complications, such as long-term pain and nerve disorders. By the advent of the use of alternative stem extraction techniques, a better alternative to the invasive method of cleft lip and palate therapy has been developed.
文摘Transporter associated with antigen processing (TAP)-like (TAPL;ABCB9) is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that function in the ER membrane. To determine the cellular localization, TAPL and truncated forms of it were tagged with GFP at their car-boxyl termini. Intracellular localization of these fusion proteins was compared between transient and stable expression in CHO-K1 cells. When they were expressed transiently, the fluorescence of the fusion proteins was detected on the intracellular membrane, mainly in the ER, and all the fusion proteins, i.e., TAPL(M1 -A766)-GFP, TAPL(M1-S275)-GFP, TAPL(M1-K182 )-GFP, TAPL(M 1-R141)-GFP and TAPL(M1-G75), were co-localized with an ER marker, PDI. However, the fluorescence of all of them except for TAPL(M1-G 75)-GFP and TAPL(M1-S275)-GFP overlapped with a lysosome marker, cathepsin D, upon stable expression. Lysosomal localization was similarly observed with TAPL(M1- A766)-DsRed, which was stably expressed. These results suggest that TAPL is sorted to the lysosomal membrane when expressed stably in CHO-K1 cells. Furthermore, the lysosomal targeting signal may comprise the N-terminal four transmembrane helices since the N-terminal two transmembrane helices may not be enough to function as such a signal.
文摘Intracellular Notch (ICN) initiates DNA transcription in cooperation with CSL that acts as repressor in the absence of ICN. The ICN mediates recruitment of MAML protein, leading to the formation of minimal transcriptional complex, MAML/ICN/CSL/DNA. Crystal structure reveals that different conformations exist between the free (CSL/DNA) and bound (ICN/MAML/CSL/DNA) forms. The significance of this modulation of the CSL/DNA molecular complex can be better understood by experimental approaches that aim to elucidate the cause and timing of these events. There are four orthologues of human ICN (ICN1-4). We studied interactions between human full-length ICN1 and CSL/DNA without involvement of MAML, in vitro, and found that 1) the EMSA profile of CSL/DNA is altered in the presence of ICN1 as a consequence of an intrinsic change(s) in CSL/DNA, and not due to the formation of an ICN/CSL/DNA molecular complex;2) ICN1 destabilizes CSL/DNA. These findings indicate that human ICN1 functions to modulate the CSL/DNA molecular complex for subsequent recruitment of MAML, and that modulated CSL/DNA cannot accommodate ICN1 in the absence of MAML. The latter in turn, implies that the formation of the MAML/ICN1/CSL/DNA is likely to be a collective event, wherein preassembly of MAML and ICN1 as a binary complex co-localizes at the CSL/DNA promoter site, or the MAML/ICN1/CSL complex is pre-assembled prior to binding to the promoter, rather than ICN1 arriving at CSL/DNA ahead of MAML and/or other associated transcription factors. The novel finding that ICN1 destabilizes the CSL/DNA complex opens new possibilities of transcriptional regulation by Notch.
文摘Mechanical stimulations have been shown to regulate cellular mechanical properties. However, the stimulation patterns for effective regulation are as yet unclear. We investigated the effects of application of differing numbers of mechanical stimulation sets, each set consisting of 8% extension and compression to cells via deformation of cell culture elastic chamber, on cellular elasticity. Elasticity increased with only a single step-like stretch and with a single step-like stretch after 1 set of mechanical stimulation, whereas elasticity did not change with a single step-like stretch after 10 sets of mechanical stimulation. These results indicate that the increase in cellular elasticity with the single step-like stretch depends on the number of applied mechanical stimulations. Immunofluorescence staining showed that phosphorylation and dephosphorylation of myosin regulatory light chain (MRLC), which regulates intracellular contractile force and cellular elasticity, accompanied cellular elasticity changes. These findings suggest that cellular elasticity changes under cyclic and step-like stretches are mediated by MRLC.
基金funded by the“Laboratorio de Referencia,Analisis y Diagnostico de Sanidad Acuícola del Centro de Investigaciones Biologicas del Noroeste”(#15789)by the Project Conacyt-Ciencia Basica 2013“Actividad antiinflamatoria y cicatrizante del Pepino de Mar(Isostichopus badionotus)en un modelo murino:caracterizacion de la actividad farmacologica y los mecanismos moleculares involucrados”(#221734).
文摘To date, White Spot Syndrome (WSS) produced by the White Spot Syndrome Virus (WSSV) causes one of the most severe diseases infecting penaeid shrimps worldwide. Although a vast amount of studies has elucidated pathogenesis in live infection models, there is still little information about the interaction of WSSV infections using in vitro models in the whiteleg shrimp Litopenaeus vannamei (L. vannamei) hemocytes. In this study, a WSSV infection kinetics was performed using total hemocytes isolated from healthy L. vannamei organisms and maintained in in vitro conditions using isotonic solution for shrimp (ISS). The infected experimental cells received ≈ 30,000 viral copies of WSSV. The viability of the hemocytes (control and infected group) was measured during the kinetics with trypan blue exclusion method and cells were maintained up to 6 hpi (post-infection) with non-significant differences of viability between both groups. WSSV replication was assessed using RT- PCR at the RNA expression level of the early viral gene Ie1 and transcripts were detected as early as 30 min pi. Hemocytes from WSSV group showed disrupted integrity, degranulation and irregular shape. This study provides evidence of the capability of WSSV to infect and replicates in L. vannamei hemocytes using in vitro assays in short times as 30 min.
文摘Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift;a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.
文摘Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction;2) The number of cells indicated for therapy that is relatively high, with the need to optimize the expansion, number and time wise;3) The utilization of third party donors;4) Culture passage number(P);and 5) Source of the cells. This study was designed to determine the superiority of the Platelet Lysates(PL)over FBS on the expansion of MSC, the optimal cell’ plating density and days between each pass, and to investigate if donor total nucleated cells(TNC)obtained from the washouts of discharged bags and filters of hematopoietic stem cell transplantation(HSCT)can be expanded to be used at clinical grade. TNC were removed, plated and after the first passage were cultivated in different concentrations with FBS or PL, and the number of days to reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or with FBS and after seven days counted to analyze how much they had grown in that period. The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC and to allow the proliferation of enough number of MSC at P2 for clinical use.
文摘Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.
