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European Review For Medical and... Feb 2020The aim of this study was to explore the influence of hydrogen sulfide (H2S) on cardiomyocyte apoptosis in rats with myocardial ischemia-reperfusion injury via the c-Jun...
OBJECTIVE
The aim of this study was to explore the influence of hydrogen sulfide (H2S) on cardiomyocyte apoptosis in rats with myocardial ischemia-reperfusion injury via the c-Jun N-terminal kinase (JNK) pathway.
MATERIALS AND METHODS
A total of 60 normal female Sprague-Dawley (SD) rats aged 38 weeks were divided into 3 groups, including the sham operation group (n=20), ischemia group (n=20) and ischemia + sodium hydrosulfide (NaHS) group (n=20). Subsequently, differences in cardiac function, the morphology of myocardial tissues, protein expression of JNK2, the content of plasma H2S and malondialdehyde (MDA), the activity of superoxide dismutase (SOD), cystathionine-γ-lyase (CSE) and glutathione peroxidase (GSH-Px) were analyzed among rats in all groups.
RESULTS
Left ventricular diastolic pressure (LVDP) and maximum rate of pressure rise/fall (± dP/dtmax) were the highest in of rats of the sham operation group and the lowest in the ischemia group. Meanwhile, they were significantly elevated in the ischemia + NaHS group compared with those in the ischemia group (p<0.01). Left ventricular end-diastolic pressure (LVEDP) was the lowest in rats of the sham operation group and the highest in the ischemia group. Similarly, it decreased markedly in the ischemia + NaHS group compared with the ischemia group (p<0.01). Compared with the sham operation group, the perinuclear space in the myocardium was gradually larger, the arrangement of fibers became significantly more disordered, and the damage of mitochondrial cristae and membrane was remarkably more severe in rats in the ischemia group. Compared with the ischemia group, the above-mentioned conditions of rat cardiomyocytes were markedly improved (p<0.01). Meanwhile, the content of H2S and activity of CSE in the cardiomyocytes were altered in rats of the ischemia + NaHS group. Western blotting results indicated that, compared with the sham operation group, both the ischemia group and ischemia + NaHS group showed significantly up-regulated protein expression level of phosphorylated JNK2, with the highest level in the ischemia group. The content of MDA in rat myocardial tissues was markedly higher in the ischemia group than that of the ischemia + NaHS group, with the lowest level in the sham operation group (p<0.01). Additionally, the activity of SOD and GSH-Px in rat myocardial tissues was remarkably worse in the ischemia group than that of the ischemia + NaHS group, and it was the strongest in the sham operation group (p<0.01).
CONCLUSIONS
H2S inhibits the activity of the JNK pathway, decreases its phosphorylation level and down-regulates the protein expression level of JNK2, thereby protecting against myocardial ischemia-reperfusion injury.
Topics: Animals; Female; Hydrogen Sulfide; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley
PubMed: 32141574
DOI: 10.26355/eurrev_202002_20383 -
Journal of Virology Mar 2020Herpesvirus nucleocapsids leave the nucleus by a vesicle-mediated translocation mediated by the viral nuclear egress complex (NEC). The NEC is composed of two conserved...
Herpesvirus nucleocapsids leave the nucleus by a vesicle-mediated translocation mediated by the viral nuclear egress complex (NEC). The NEC is composed of two conserved viral proteins, designated pUL34 and pUL31 in the alphaherpesvirus pseudorabies virus (PrV). It is required for efficient nuclear egress and is sufficient for vesicle formation and scission from the inner nuclear membrane (INM). Structure-based mutagenesis identified a lysine at position 242 (K242) in pUL31, located in the most membrane distal part of the NEC, to be crucial for efficient nucleocapsid incorporation into budding vesicles. Replacing the lysine by alanine (K242A) resulted in accumulations of empty vesicles in the perinuclear space, despite the presence of excess nucleocapsids in the nucleus. However, it remained unclear whether the defect in capsid incorporation was due to interference with a direct, electrostatic interaction between the capsid and the NEC or structural restrictions. To test this, we replaced K242 with several amino acids, thereby modifying the charge, size, and side chain orientation. In addition, virus recombinants expressing pUL31-K242A were passaged and screened for second-site mutations. Compensatory mutations at different locations in pUL31 or pUL34 were identified, pointing to an inherent flexibility of the NEC. In summary, our data suggest that the amino acid at position 242 does not directly interact with the nucleocapsid but that rearrangements in the NEC coat are required for efficient nucleocapsid envelopment at the INM. Herpesviruses encode an exceptional vesicle formation and scission machinery, which operates at the inner nuclear membrane, translocating the viral nucleocapsid from the nucleus into the perinuclear space. The conserved herpesviral nuclear egress complex (NEC) orchestrates this process. High-resolution imaging approaches as well as the recently solved crystal structures of the NEC provided deep insight into the molecular details of vesicle formation and scission. Nevertheless, the molecular mechanism of nucleocapsid incorporation remained unclear. In accordance with structure-based predictions, a basic amino acid could be pinpointed in the most membrane-distal domain of the NEC (pUL31-K242), indicating that capsid incorporation might depend on a direct electrostatic interaction. Our follow-up study, described here, however, shows that the positive charge is not relevant but that the overall structure matters.
Topics: Active Transport, Cell Nucleus; Animals; Capsid Proteins; Cell Line; Cell Nucleus; Chlorocebus aethiops; DNA Mutational Analysis; Follow-Up Studies; Herpesvirus 1, Suid; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Nuclear Envelope; Nucleocapsid; Protein Conformation; Vero Cells; Viral Proteins; Virion
PubMed: 32051272
DOI: 10.1128/JVI.01910-19 -
Journal of Virology Mar 2020Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from...
Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from betaherpesviruses and gammaherpesviruses. pUS3 homologs are multifunctional and are involved in many processes, including modification of the cytoskeleton, inhibition of apoptosis, and immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM), pointing to a modulatory function for pUS3 during deenvelopment. The HSV and pseudorabies virus (PrV) US3 genes are transcribed into two mRNAs encoding two pUS3 isoforms, which have different aminoterminal sequences and abundances. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform, or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture, while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested but disclosed a more severe defect in Madin-Darby bovine kidney cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV. A plethora of substrates and functions have been assigned to the alphaherpesviral pUS3 kinase, including a role in nuclear egress. In PrV, two different pUS3 isoforms are expressed, which differ in size, abundance, and intracellular localization. Their respective role in replication is unknown, however. Here, we show that efficient nuclear egress of PrV requires the smaller isoform and intact kinase activity, whereas absence of the larger isoform has no significant effect on viral replication. Thus, there is a clear distinction in function between the two US3 gene products of PrV.
Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Cattle; Cell Nucleus; Chlorocebus aethiops; Cytoskeleton; Genome, Viral; Herpesvirus 1, Suid; Kidney; Mutation; Nuclear Envelope; Phenotype; Protein Isoforms; Protein Serine-Threonine Kinases; Rabbits; Vero Cells; Viral Proteins; Virus Assembly
PubMed: 31941788
DOI: 10.1128/JVI.02029-19 -
Cells Nov 2019Excretory and secretory products are crucial for parasite infectivity and host immunomodulation, but the functioning and ultrastructure of the excretory gland cell (EC)...
Excretory and secretory products are crucial for parasite infectivity and host immunomodulation, but the functioning and ultrastructure of the excretory gland cell (EC) that produces these products are still scarcely understood and described. In light of growing reports on anisakiasis cases in Europe, we aimed to characterise the EC of larval and adult . In the latter, EC starts 0.85 mm from the head tip, measuring 1.936 × 0.564 mm. Larval EC shows a long nucleus with thorn-like extravaginations toward the cytoplasm, numerous electron-dense and -lucent secretory granules spanning from the perinuclear to subplasmalemmal space, an elevated number of free ribosomes, small, spherical mitochondria with few cristae and a laminated matrix, small and few Golgi apparatuses, and few endoplasmic reticula, with wide cisternae complexes. Ultrastructure suggests that anaerobic glycolysis is the main metabolic pathway, obtained through nutrient endocytosis across the pseudocoelomic surface of the EC plasmalemma and its endocytic canaliculi. Thorn-like extravaginations of EC karyotheca likely mediate specific processes (Ca signaling, gene expression, transport, nuclear lipid metabolism) into the extremely wide EC cytosol, enabling focal delivery of a signal to specific sites in a short time. These functional annotations of parasitic EC should help to clarify anisakiasis pathogenesis.
Topics: Anaerobiosis; Animals; Ascaridoidea; Exocrine Glands; Glycolysis; Larva; Microscopy, Confocal; X-Ray Microtomography
PubMed: 31744245
DOI: 10.3390/cells8111451 -
Small (Weinheim An Der Bergstrasse,... Dec 2019The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties...
The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.
Topics: Animals; Biomechanical Phenomena; Capillaries; Cell Movement; Collagen; Cytoskeleton; Hydrogels; Mice; Myosin Type II; NIH 3T3 Cells; Vimentin
PubMed: 31721440
DOI: 10.1002/smll.201903180 -
Animals : An Open Access Journal From... Nov 2019The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory...
The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory proteins are transferred via extracellular vesicles (EVs) to the spermatozoa for their maturation and long-term viability. The aim of the present study was to investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their EVs in the epithelia of the efferentes duct in a turtle species, the soft-shelled freshwater turtle by using light and transmission electron microscopy. The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells (CC) and moderate to weak in the non-ciliated cells (NCC) of DE. The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC, whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC. Many EVs, as sources of MVBs, were located within the blebs that were attached to the cilia of CC, within the apical blebs from NCC, and the lateral spaces of CC and NCC. There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE. Collectively, the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. These EVs were associated with spermatozoa in the DE lumen of this turtle. Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa.
PubMed: 31683774
DOI: 10.3390/ani9110888 -
The Journal of Cell Biology Dec 2019Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear...
Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.
Topics: Animals; Cell Movement; Cell Nucleus; Collagen; Cytoskeleton; DNA Damage; Fibroblasts; Intermediate Filament Proteins; Intermediate Filaments; Mice; Microscopy, Atomic Force; Microscopy, Confocal; Necrosis; Vimentin
PubMed: 31676718
DOI: 10.1083/jcb.201902046 -
Journal of Virology Dec 2019The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the...
The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the cytoplasm by facilitating the budding of capsids into the perinuclear space (PNS). The NEC of alphaherpesvirus can induce the formation of virion-like vesicles from the nuclear membrane in the absence of other viral proteins. However, whether the NEC of gammaherpesvirus harbors the ability to do so in mammalian cells remains to be determined. In this study, we first constructed open reading frame 67 (ORF67)-null and ORF69-null mutants of murine gammaherpesvirus 68 (MHV-68) and demonstrated that both ORF67 and ORF69 play critical roles in nuclear egress and hence viral lytic replication. Biochemical and bioimaging analyses showed that ORF67 and ORF69 interacted with each other and were sufficient to induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. Thus, we designated ORF67 and ORF69 components of MHV-68 NEC. Furthermore, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 through homology modeling and verified their function in nuclear egress, providing insights into the molecular basis of NEC formation in gammaherpesviruses. Increasing amounts of knowledge indicate that the nuclear egress complex (NEC) is critical for the nuclear egress of herpesvirus capsids, which can be viewed as a vesicle-mediated transport pathway through the nuclear membrane. In this study, we identified open reading frame 67 (ORF67) and ORF69 as components of the NEC in murine gammaherpesvirus 68 (MHV-68) and demonstrated that they efficiently induce virion-like vesicles from the nuclear membrane in mammalian cells. This is the first time that the NEC of a gammaherpesvirus has been found to demonstrate such an essential characteristic. In addition, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 as well as nuclear egress. Notably, these amino acids are conserved in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), providing a structural basis to design antigammaherpesvirus drugs.
Topics: Active Transport, Cell Nucleus; Animals; Capsid; Cell Nucleus; Cytoplasm; DNA, Viral; Gammaherpesvirinae; HEK293 Cells; HeLa Cells; Herpesviridae; Herpesviridae Infections; Herpesvirus 4, Human; Herpesvirus 8, Human; Humans; Loss of Function Mutation; Mice; Nuclear Envelope; Open Reading Frames; Viral Envelope Proteins; Viral Proteins; Virion; Virus Replication
PubMed: 31554685
DOI: 10.1128/JVI.01422-19 -
International Journal of Nanomedicine 2019We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP-(NV)) in rats....
BACKGROUND
We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP-(NV)) in rats. FNDP-(NV) primary deposition was found in the liver, yet liver function tests remained normal.
PURPOSE
The present study aimed to gain preliminary insights on discrete localization of FNDP-(NV) in liver cells of the hepatic lobule unit and venous micro-vasculature. Kinetics of FDNP-(NV) uptake into liver cells surrogates in culture was conducted along with cell cytokinesis as markers of cells' viability.
METHODS
Preserved liver specimens from a pilot consisting of two animals which were stained for cytoskeletal elements (fluorescein-isothiocyanate-phalloidin) were examined for distribution of FNDP-(NV) by fluorescent microscopy (FM) and Confocal-FM (CFM) using near infra-red fluorescence (NIR). Hepatocellular carcinoma cells (HepG-2) and human umbilical vein endothelial cells (HUVEC) were cultured with FNDP-(NV) and assayed for particle uptake and location using spectrophotometric technology and microscopy.
RESULTS
HepG-2 and HUVEC displayed rapid (<30 mins) onset and concentration-dependent FNDP-(NV) internalization and formation of peri-nuclear corona. FM/CFM of liver sections revealed FNDP-(NV) presence throughout the hepatic lobules structures marked by spatial distribution, venous microvascular spaces and parenchyma and non-parenchyma cells.
CONCLUSION
The robust presence of FNDP-(NV) throughout the hepatic lobules including those internalized within parenchyma cells and agglomerates in the liver venous micro-circulation were not associated with macro or micro histopathological signs nor vascular lesions. Cells cultures indicated normal cytokinesis in cells containing FNDP-(NV) agglomerates. Liver parenchyma cells and the liver microcirculation remain agnostic to presence of FNDP-(NV) in the sinusoids or internalized in the hepatic cells.
Topics: Animals; Biocompatible Materials; Hep G2 Cells; Hepatocytes; Human Umbilical Vein Endothelial Cells; Humans; Imaging, Three-Dimensional; Kinetics; Liver; Microscopy, Fluorescence; Nanodiamonds; Particle Size; Rats, Sprague-Dawley; Tissue Distribution
PubMed: 31496697
DOI: 10.2147/IJN.S209663 -
The Journal of Clinical Investigation Aug 2019Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and...
Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.
Topics: Animals; Carrier Proteins; Hepatocytes; Lipid Metabolism; Lipoproteins, VLDL; Membrane Proteins; Mice; Mice, Knockout; Molecular Chaperones; Non-alcoholic Fatty Liver Disease; Nuclear Envelope
PubMed: 31408437
DOI: 10.1172/JCI129769