Extracellular myocilin affects activity of human trabecular meshwork cells

The trabecular meshwork (TM), a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. The protein product has been localized to both intra and extracellular sites, but its function still remains unclear. This study was to determine whether extracellular myocilin presented in the matrix affects adhesion, morphology, and migratory and phagocytic activities of human TM cells in culture. Cell adhesion assays indicated that TM cells, while adhering readily on fibronectin, failed to attach on recombinant myocilin purified from bacterial cultures. Adhesion on fibronectin was also compromised by myocilin in a dose dependent manner. Myocilin in addition triggered TM cells to assume a stellate appearance with broad cell bodies and microspikes. Loss of actin stress fibers and focal adhesions was observed. TM cell migration on fibronectin/myocilin to scratched wounds was reduced compared to fibronectin controls. Myocilin, however, had little impact on phagocytic activities of TM cells. Cell attachment on fibronectin and migration of corneal fibroblasts, a control cell type, were not altered by myocilin. These results demonstrate that extracellular myocilin elicits anti-adhesive and counter-migratory effects on TM cells. Myocilin in the matrix of tissues could be exerting a similar influence on TM cells in vivo, impacting the flexibility and resilience required for maintenance of the normal aqueous outflow.     

Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression

Tumor necrosis factor (TNF)-alpha plays a key role in the pathogenesis of septic shock syndrome, and myocardial TNF-alpha expression may contribute to this pathophysiology. We examined the myocardial expression of TNF-alpha-related cytokines and chemokines in mice exposed to lipopolysaccharide (LPS) and tested the effects of anti-TNF therapy on myocardial cytokine expression. Cytokine mRNA levels were measured by RNase protection assay, and protein levels in the plasma and myocardium were assessed by enzyme-linked immunosorbent assays. LPS (4 microg/g body wt ip) induced marked cytokine expression, including TNF-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein (MCP)-1, in both the plasma and myocardium. Pretreatment with adenovirus-mediated TNF receptor fusion protein (AdTNFR1; 10(9) plaque-forming units iv) decreased plasma cytokine levels. In contrast, whereas myocardial IL-1beta expression was also suppressed, expression of IL-6 and MCP-1 was not inhibited by AdTNFR1. In summary, anti-TNF treatment differentially altered the cytokine expression in the plasma and myocardium during endotoxemia. Inability to block myocardial expression of IL-6 and MCP-1 suggests a possible mechanism for the failure of anti-TNF therapies in the treatment of endotoxin shock.     

Seasonal changes in the freezing behavior of xylem ray parenchyma cells in four boreal hardwood species

The freezing behavior of xylem ray parenchyma cells in several boreal hardwood species, namely, Betula platyphylla, Populus canadensis, P. sieboldii, and Salix sachalinensis, was examined by differential thermal analysis (DTA), cryo-scanning electron microscopy (Cryo-SEM), and freeze-fracture replica electron microscopy. Although DTA profiles of samples harvested in summer and in winter suggested that the xylem ray parenchyma cells in all four species responded to freezing stress by extracellular freezing, Cryo-SEM showed clearly that the xylem ray parenchyma cells in all these species responded to freezing stress by shallow supercooling in summer and by extracellular freezing in winter. It is suggested that DTA failed to reveal the true freezing behavior of xylem ray parenchyma cells because of an overlap of temperature ranges between the high-temperature exotherm and the low-temperature exotherm and/or because of the limited extent of the LTE. The seasonal changes in freezing behavior of xylem ray parenchyma cells in all these boreal species, which are results of seasonal cold acclimation, support the hypothesis that a gradual shift of freezing behavior in xylem ray parenchyma cells from shallow supercooling in hardwood species that grow in tropical zones to extracellular freezing in hardwood species that grow in cold areas might be a result of the evolutionary adaptation of hardwood species to cold climates. Copyright 1999 Academic Press.     

Spread of X-chromosome inactivation into chromosome 15 is associated with Prader–Willi syndrome phenotype in a boy with a t(X;15)(p21.1;q11.2) translocation

X-chromosome inactivation (XCI) is an essential mechanism in females that compensates for the genome imbalance between females and males. It is known that XCI can spread into an autosome of patients with X;autosome translocations. The subject was a 5-year-old boy with Prader?CWilli syndrome (PWS)-like features including hypotonia, hypo-genitalism, hypo-pigmentation, and developmental delay. G-banding, fluorescent in situ hybridization, BrdU-incorporated replication, human androgen receptor gene locus assay, SNP microarrays, ChIP-on-chip assay, bisulfite sequencing, and real-time RT-PCR were performed. Cytogenetic analyses revealed that the karyotype was 46,XY,der(X)t(X;15)(p21.1;q11.2),?15. In the derivative chromosome, the X and half of the chromosome 15 segments showed late replication. The X segment was maternal, and the chromosome 15 region was paternal, indicating its post-zygotic origin. The two chromosome 15s had a biparental origin. The DNA methylation level was relatively high in the region proximal from the breakpoint, and the level decreased toward the middle of the chromosome 15 region; however, scattered areas of hypermethylation were found in the distal region. The promoter regions of the imprinted SNRPN and the non-imprinted OCA2 genes were completely and half methylated, respectively. However, no methylation was found in the adjacent imprinted gene UBE3A, which contained a lower density of LINE1 repeats. Our findings suggest that XCI spread into the paternal chromosome 15 led to the aberrant hypermethylation of SNRPN and OCA2 and their decreased expression, which contributes to the PWS-like features and hypo-pigmentation of the patient. To our knowledge, this is the first chromosome-wide methylation study in which the DNA methylation level is demonstrated in an autosome subject to XCI.     

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

AimsInsulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methodsTo localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2^+/+ mice. IA-2^?/? mice served as a negative control.Key findingsWestern blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.SignificanceThe IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.     

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.     

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G₂ arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G₂ arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G₂ arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic low-level DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12, 27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7, 21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3, 22, 33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16, 38, 41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16, 30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD6 pathway. Srs2 is known to act as an antirecombinase by eliminating recombination intermediates. This can occur independently of PCNA sumoylation, and when srs2Δ cells are UV irradiated or other antirecombinases, such as Sgs1, are concomitantly deleted, toxic recombination structures accumulate (1, 10). Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20, 40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18, 34, 35).The UV spectrum present in sunlight is a primary environmental cause of exogenous DNA damage. Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in humans (17). In the natural environment, organisms are exposed to chronic low-dose UV light (CLUV), as opposed to the acute high doses commonly used in laboratory experiments. Hence, understanding the cellular response to CLUV exposure is an important approach complementary to the more traditional laboratory approaches for clarifying the biological significance of specific DNA damage response pathways. A recently developed experimental assay for the analysis of CLUV-induced DNA damage responses was used to show that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance of CLUV exposure by preventing the generation of excessive ssDNA when replication forks arrest, thus suppressing counterproductive checkpoint activation (13).Mutants of SRS2 were first isolated by their ability to suppress the radiation sensitivity of rad6 and rad18 mutants (defective in PRR) by a mechanism that requires a functional HR pathway (23, 36). In this study, we analyzed the function of Srs2 in CLUV-exposed PRR-deficient cells. We established that Srs2 acts in conjunction with SUMO-modified PCNA to lower the threshold for checkpoint activation and maintenance by suppressing the function of HR in rad18Δ cells exposed to CLUV. We also showed that Srs2 is separately involved in an HR-dependent recovery process following cessation of CLUV exposure and that this second role for Srs2, unlike its primary role in checkpoint activation and maintenance, is regulated by CDK1-dependent phosphorylation. Thus, Srs2 is involved in both CLUV-induced checkpoint-mediated arrest and recovery from CLUV exposure in PRR-deficient cells, and these two functions, while both involving HR, are separable and thus independent.     

Studies on the microsomal electron-transport system of anaerobically grown yeast. V. Purification and characterization of NADPH-cytochrome c reductase.

A flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70,000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-1,4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 micron M, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1.6.2.4].     

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.