Effects of tetraiodothyronine and triiodothyronine on hamster cheek pouch microcirculation

The aim of the present study was to assess the effects of topically applied triiodothyronine (T(3)) and thyroxine (T(4)) on the arterioles of hamster cheek pouch microcirculation in vivo. Microvessels were visualized using a fluorescent microscopy technique. Topical application of T(3) (3.08, 30.8, 61.5, 307, 615, and 6,150 nM/l) consistently induced dose-dependent dilation of arterioles within 2.0 +/- 0.5 min of administration. The application of T(4) (150, 257, 514, and 5,140 nM/l) caused different dose-dependent effects: dilation at the three lower doses within 16 +/- 2 min and rhythmic diameter changes at the highest dose. Aging of hamsters did not alter the arteriolar responses to T(3) and T(4). T(3)-induced dilation was countered by the inhibition of nitric oxide synthase with N(G)-nitro-L-arginine-methyl ester or N(G)-nitro-L-arginine. Iopanoic acid (IPA), which inhibits types I and II 5'-deiodinase, abolished the dilation elicited by 514 nM T(4) but did not affect T(3)-dependent dilation. 6-Propyl-2-thiouracil (PTU), which inhibits type I 5'-deiodinase only, did not affect the dilation induced by T(4). IPA and PTU did not impair arteriolar dilation induced by acetylcholine or sodium nitroprusside. These results indicate that T(3) induces arteriolar dilation, likely through nitric oxide release. The local conversion of T(4) to T(3) appears to be crucial for the dilation induced by T(4).     

Identification of a feline coronavirus type I strain from a cat with feline infectious peritonitis by RT-pCR and phylogenetic analysis

A case of feline infectious peritonitis (FIP) in an 11-month-old European shorthair cat is reported. The infected cat displayed loss of weight, respiratory distress, ascitis, anemia and died within 15 days after the first appearance of clinical signs. Lesions typical of a mixed form (effusive and non-effusive) of FIP were observed and by RT-PCR a feline coronavirus (FCoV) type I strain was detected in several tissues. The RT-PCR results were confirmed by sequence analysis of the amplified products. Phylogeny carried out on fragments of the M and S genes showed that the FCoV strain segregates with typical type I FCoVs.     

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.     

Reconstituted basement-membrane matrix modulates fibroblast activities in vitro

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth-Holm-Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio of type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement-membrane matrix seems to influence the activities of fibroblasts significantly.     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.     

Developmental variations of plasma gamma-glutamyltransferase fractions in humans and in laboratory mammalians

Plasma samples from human cord blood, and fetuses, newborns, and adults of different mammalians species were analyzed by gel-filtration chromatography, to ascertain whether gamma-glutamyltransferase (GGT) fractions reflect liver maturation. Human cord blood plasma showed higher b-, m-, and s-GGT fraction as compared to adult women. In rat and mouse fetuses and in newborns, b-GGT was the most abundant fraction. As in adult humans, in adult rats, mice, rabbits, sheep, and mini pigs, f-GGT was the most abundant fraction. GGT fractions are a common feature of all mammalian species tested. Their pattern changes seem to reflect liver postnatal maturation, function.     

Effectiveness of the postponed isolation (post-frozen isolation) method for PCR-quality <Emphasis Type="Italic">Sarcoptes</Emphasis> mite gDNA

The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin (‘postponed isolation’ method) are suitable sources of PCR-quality genomic DNA, and to test the effectiveness of this method in comparison with the ‘direct isolation’ method, often used through force of habit. Hundreds of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a ~450 bp product (ITS-2) and multi-locus 10× genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era.     

Procollagen type III N-terminal endopeptidase in fibroblast culture.

A peptidase activity capable of excising in a single fragment the N-terminal extension of the precursor of collagen type III (p-N-collagen type III) was observed in calf tendon fibroblast culture medium. A new procedure was developed for detecting this peptidase (p-N-collagen type III peptidase). It is based on the use of 14C-labelled p-N-collagen type III obtained by carboxymethylation of the half-cystine residues with iodo-[14C]acetamide. The released labelled N-terminal extension is soluble in 27% (v/v) ethanol, whereas the uncleaved substrate and the collagen are precipitated under these conditions. The endopeptidase nature of p-N-collagen type III peptidase is supported by the similarity in molecular weight of the product of cleavage of p-N-collagen III by the enzyme to those obtained by cleavage with bacterial collagenase. An apparent Km of 0.3 X 10(-6)M was established. The pH optimum of p-N-collagen type III peptidase is similar to that of p-N-collagen type I peptidase, i.e. about 7.5. Both peptidases are inhibited by dithiothreitol and by Cu2+ and Zn2+, but not by other bivalent ions. p-N-collagen type III peptidase does not cleave p-N-collagen I or p-N-gelatin I. Partial purification of p-N-collagen type III peptidase from fibroblast culture medium was performed by sieve chromatography on Ultrogel AcA-34 to yield two peaks of activity, of mol.wts. 170000 and 100000. Part of the activity was retained on affinity chromatography on concanavalin A--Sepharose. Studied as a function of the age of the culture, p-N-collagen type III peptidase activity produced by tendon fibroblasts parallels that of p-N-collagen type I peptidase and collagen synthesis.