Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture

It has classically been accepted that the healing of narrow wounds in epithelia occurs by the formation of a contractile actin cable, while wide wounds are resurfaced by lamellipodia-dependent migration of border cells into the denuded area. To further investigate the general validity of this idea, we performed systematic experiments of the roles of wound geometry, wound size, and extracellular matrix (ECM) in wound healing in monolayers of bovine corneal endothelial cells, a system shown here to predominantly display any of the two healing mechanisms according to the experimental conditions. We found that, in this system, it is the absence or presence of the ECM on the wound surface that determines the specific healing mode. Our observations demonstrate that, independent of their size and geometry, wounds created maintaining the ECM heal by migration of cells into the wound area, while ECM removal from the wound surface determines the predominant formation of an actin cable. While the latter mechanism is slower, the actin cable permits the maintainance of the epithelial phenotype to a larger extent during the healing process, as also confirmed by our finding of a more conserved localization of cadherin and vinculin. We also introduce a model that simulates experimental findings about the dynamics of healing mechanisms, both for the maintenance or removal of the ECM on the wound surface. The findings of this study may contribute to the understanding of physiological and pathological aspects of epithelial wound healing and to the design of therapeutic strategies.     

[Ni(L)(MeCN)]complex cation as a template for the assembly of extended I3 · I5 and I5 · I7 polyiodide networks {L=2,5,8-trithia[9](2,9)-1,10-phenanthrolinophane}. Synthesis and structures of [Ni(L)(MeCN)]I8 and [Ni(L)(MeCN)]I12

The compounds [Ni(L)(MeCN)]I₈ (1) and [Ni(L)(MeCN)]I₁₂ (2) have been obtained from the reactions of the complexes [Ni(L)(L^′)][BF₄]_(2 + n) {L=2,5,8-trithia[9](2,9)-1,10-phenanthrolinophane; L^′=MeCN, Cl⁻, Br⁻, I⁻; n=charge of L^′} with an excess of I₂ (molar ratios of 6, 10 and 20 have been used), in the presence of the stoichiometric amount of I⁻ (as Bu₄ⁿNI) necessary to balance the charge of the complex cation [Ni(L)(L^′)]^(2 + n)+. An X-ray diffraction analysis shows that, independently of the nature of L^′, both 1 and 2 contain the complex cation [Ni(L)(MeCN)]²⁺, which is therefore capable of templating two different polyiodide networks based on interacting I₃⁻/I₅⁻ and I₅⁻/I₇ units, respectively. The solid state FT-Raman spectra of 1 and 2 are discussed based on their structural features.     

Characterization of a thy1.2 GFP transgenic mouse reveals a tissue-specific organization of the spinal dorsal horn

In this study, transgenic mice in which membrane-linked enhanced green fluorescent protein (mGFP) is expressed from the Thy1.2 promoter were used. In these mice, a subpopulation of small to medium sized DRG neurons double stained for IB4 but not for CGRP. Most of the peripheral terminals traversed the dermis and ramify within the epidermis and form superficial terminals. Within the spinal cord, these afferents terminated exclusively within the substantia gelatinosa (SG). A second fibre type in the skin also expressed mGFP, and formed club-shaped endings towards the bases of hairs. Injury to the sciatic nerve resulted in mGFP loss from the SG ipsilateral to the nerve injury, but also in the corresponding region contralaterally. Together, these findings reveal the specificity of connectivity of a defined subpopulation of DRG sensory neurons innervating the epidermis and this will facilitate analysis of their physiological functions.     

Multilocus genotyping of human Giardia isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children

Background

Giardia intestinalis is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. G. intestinalis consists of eight genetically distinct genotypes or assemblages, designated A–H, and assemblages A and B can infect humans. Giardiasis has been classified as a possible zoonotic disease but the role of animals in human disease transmission still needs to be proven. We tried to link different assemblages and sub-assemblages of G. intestinalis isolates from Swedish human patients to clinical symptoms and zoonotic transmission.

Methodology/Principal Findings

Multilocus sequence-based genotyping of 207 human Giardia isolates using three gene loci: ß-giardin, glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was combined with assemblage-specific tpi PCRs. This analysis identified 73 patients infected with assemblage A, 128 with assemblage B, and six with mixed assemblages A+B. Multilocus genotypes (MLGs) were easily determined for the assemblage A isolates, and most patients with this genotype had apparently been infected through anthroponotic transmission. However, we also found evidence of limited zoonotic transmission of Giardia in Sweden, since a few domestic human infections involved the same assemblage A MLGs previously reported in Swedish cats and ruminants. Assemblage B was detected more frequently than assemblage A and it was also more common in patients with suspected treatment failure. However, a large genetic variability made determination of assemblage B MLGs problematic. Correlation between symptoms and assemblages was found only for flatulence, which was significantly more common in children less than six years of age infected with assemblage B.

Conclusions/Significance

This study shows that certain assemblage A subtypes are potentially zoonotic and that flatulence is connected to assemblage B infections in young children. Determination of MLGs from assemblages A and B can be a valuable tool in outbreak situations and to help identify possible zoonotic transmission.     

Quantitative SUMO-1 modification of a vaccinia virus protein is required for its specific localization and prevents its self-association

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.     

Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources

Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, d-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and d-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.     

Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.