Cell interaction with the extracellular matrices produced by endothelial cells and fibroblasts

The extracellular matrices (ECM) produced by cultured bovine corneal endothelial cells and chick embryo fibroblasts were compared for their induction of cell attachment, proliferation and differentiation. The corneal endothelial ECM (cECM) induced a comparable and rapid attachment and flattening of both human Ewing's sarcoma and colon carcinoma cells which utilize fibronectin and laminin as adhesive glycoproteins, respectively. In contrast, the ECM produced by fibroblasts (fECM) readily supported the attachment and flattening of Ewing's sarcoma cells but had only a small effect on the carcinoma cells. Vascular endothelial cells were stimulated to proliferate by both types of matrices, but to a lesser extent by the fECM. In contrast, the formation of a closely apposed, non-overlapping and contact-inhibited endothelial cell monolayer was only dictated by the cECM. Vascular endothelial cells cultured on fECM grew on top of each other and incorporated [3H]thymidine even late at confluency. Neurite outgrowth (ciliary ganglion cells) and network formation (adult rat oligodendrocytes) were promoted by both types of matrices but in a more consistent manner with the cECM. It is likely that the small amounts of laminin deposited by chick embryo fibroblasts into their ECM are responsible for its efficient induction of neurite outgrowth and for the limited degree of carcinoma cell attachment and flattening. It is thus demonstrated that differences in chemical composition and supramolecular arrangement between cECM and fECM result not only in differences in the attachment, spreading and proliferative responses of cells but also in the expression of their characteristic morphological appearance and differentiated functions.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.     

Molecular mass analysis of murine immunosuppressive immunoglobulin G-binding factors (IgG-BFs) produced by T-cell hybrids

Induced and constitutive murine IgG-binding factors (IgG-BFs) have been purified by affinity chromatography from supernatants of T-cells preincubated with or without murine monoclonal IgG1 and IgG2b, respectively. IgG-BF Mr values have been studied by SDS-polyacrylamide gel electrophoresis (PAGE) after treatment with SDS under conditions which do not noticeably alter their immunosuppressive activities on the secondary in vitro IgG antibody response. Suppression was recovered at Mr values of 80000, 40000 and 20000. When induced IgG-BF was tested, the isotype-specific suppressive activity was found only at 40 kDa. The 20-kDa moiety appeared to derive from the 40-kDa component and the material found at 80 kDa exerted non-specific immunosuppressive effects. We conclude therefore that isotype-specific IgG-BF has an apparent Mr of 40000.     

Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.     

Some arylamidases from vetch seeds]

Hydrolysis of L-phenylalanyl-p-nitroanilide (PPA) and glycyl-p-nitroanilide by extracts from resting vetch seeds was shown to be the effect of two different arylamidases. One of them, PPAase, was 2000-fold purified on hydroxylapatide, DEAE-cellulose and by gel filtration through Sephadex G-100. The preparation obtained was nearly homogenous chromatographycally. Molecular weight of PPAase, as shown by means of gel filtration, was 66000, K(m) was calculated to be 1.64-10-4 M. PPAase was inhibited by SH-reagents and partially by o-oxyquinoline. Some increase in the enzyme activity was observed in the presence of Ca2+, Mg2+ and Mn2+ in low concentrations. The enzyme hydrolysed amino acid arylamides with hydrophobic side groups and some dipeptides, which had at least one hydrophobic amino acid and did not contain amino acids with polar group.     

Skeletal Cell Differentiation Is Enhanced by Atmospheric Dielectric Barrier Discharge Plasma Treatment

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.     

Variability of Bacterial Community Composition on Leaves Between and Within Plant Species

The phyllosphere is one of the largest habitats for terrestrial microorganisms. To gain a better insight into the factors underlying the composition of bacterial communities inhabiting leaf surfaces we performed culture-dependent and independent (Denaturing Gradient Gel Electrophoresis) analyses on the bacteria associated with the leaves of three plant species: Amygdalus communis, Citrus paradisi, and Nicotiana glauca. We found that the culturable classes Bacilli and Actinobacteria were the predominant classes on the phyllosphere of all three plant species. In contrast to this consistency on the bacterial class level, we found a significant variation on the bacterial species-level based on the culturable methods. Although some variation was detected among individual plants within one plant species, the inter-specific variability exceeded the intra-specific variability. C. paradisi leaf surface had the highest predicted total species richness (Chao 2 and ICE) and the highest species diversity (βw) among the three plant species. Our findings demonstrate that environmental conditions, mainly the plant species within a site, govern the bacterial community composition on leaf surfaces.