Purification and initial characterization of guinea pig testicular acrosin

Guinea pig (GP) acrosin was purified following acid extraction of testicular acetone powder, pH precipitation of the soluble extract, gel filtration on Sephadex G-100, ion-exchange chromatography on SP-Sephadex, and affinity chromatography on Concanavalin A-Sepharose. Final purification was achieved by re-chromatography on Sephadex G-100. Enzymatic activity was detected by following the hydrolysis of N-benzyloxycarbonylarginyl amide of 7-amino-4-trifluoromethylcoumarin at 37 degrees C, pH 8.0, before and after activation. GP testicular acrosin exhibited a molecular weight of 48,000 by gel filtration and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE in gels containing 0.1% gelatin, protease activity was observed to comigrate with the major protein detected by silver staining. The purified GP acrosin showed cross-reactivity with a monospecific polyclonal rabbit antiserum directed against boar sperm acrosin and exhibited reversible pH-dependent activation. The physiochemical characteristics of the purified protein, including the amino acid composition, resemble those reported for acrosins from other species.     

Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Hydroxyproline-2-epimerase of pseudomonas: Active-site peptides

Hydroxyproline-2-epimerase was treated with 14C-iodoacetate under conditions that produced almost complete inactivation of the enzyme and concomitant incorporation of almost one molar equivalent of iodoacetate. Both processes were prevented by saturating concentrations of substrate. From reaction mixtures in which both incorporation and inactivation were 85 to 90% complete, two radioactive tryptic peptides were isolated by paper chromatography-electrophoresis. The incorporated radioactivity was divided between the peptides in an approximately 2:1 ratio. Analysis of the isolated peptides suggested that they both contained 9 amino acids and had similar composition; one appeared to be a lysine, the second an arginine peptide. Attempts to sequence each peptide failed, apparently because of the conversion of the S-carboxymethylcysteine to S-carboxymethylcysteine sulfone, indicating that the cysteine residue was N-terminal in each peptide.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.     

The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.