Control of human coagulation by recombinant serine proteases. Blood clotting is activated by recombinant factor XII deleted of five regulatory domains.

The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge. This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.     

Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.     

Retinoblastoma protein dephosphorylation is an early event of cellular response to prooxidant conditions

The modification of intracellular redox conditions with diethylmaleate (DEM), a glutathione-depleting agent, induces a p53-independent growth arrest mediated by the accumulation of p21(waf1) mRNA and protein. The same treatment also induces the retinoblastoma protein (pRb) dephosphorylation. This dephosphorylation (i) is very fast, being observed already 5 min after the exposure of the cells to DEM, (ii) is dependent on the prooxidant effects of DEM, being prevented by the treatment with N-acetylcysteine and (iii) is completely reversible, since the rephosphorylation of pRb is promptly obtained upon the removal of the glutathione-depleting agent from the culture medium. The dephosphorylation of pRb is independent of the accumulation of p21(waf1) induced by DEM; in fact, p21(waf1) levels start to increase much later after DEM treatment and accordingly cyclin-dependent kinase activities are not yet induced when pRb is already dephosphorylated following DEM treatment. Finally, pRb dephosphorylation is catalyzed by phosphatases activated by DEM treatment.     

Techniques and statistical data analysis in molecular population genetics

Following the development of PCR methods, molecular techniques have become widely used for detecting genetic variation in natural populations. Most nucleotide changes can be detected by these techniques. Many of these changes probably reflect silent substitutions that are likely to be selectively neutral, making them particularly suitable to population genetic studies. In this paper, we review the published literature on molecular population genetics, with respect to the genome assayed (nuclear, mitochondrial or chloroplast), the organisms studied, the molecular techniques used, and the biological problems addressed. Several molecular techniques are then compared using experimental results obtained from a population genetic study of the Mytilus complex in the North Atlantic and Mediterranean. Finally, the most appropriate theoretical tools to analyse molecular population genetic data are discussed.     

The fa leptin receptor mutation and the heritability of respiratory frequency in a Brown Norway and Zucker intercross.

We sought to determine whether the fa (leptin receptor) mutation was a major determinant of the putative obesity effects on respiratory frequency in an intercross between the Brown Norway (low breathing frequency, nonobese strain) and the Zucker (moderately high breathing frequency, with the fa mutation) strains. The hypothesis was that rats bearing one (heterozygote) or two (homozygote) alleles of the Glu296Pro point mutation (fa) would have a uniformly high respiratory frequency in the second filial (F2) generation, compared with wild-type animals. In addition to breathing frequency, tidal volume and minute ventilation were assessed during baseline, acute hypoxic (10% O2-0% CO2-balance nitrogen), hypercapnic (93% O2-7% CO2), hyperoxic (100% O2-0% CO2), and combined (10% O2-3% CO2-balance nitrogen) challenges in fa homozygote (fa/fa; n = 24), fa heterozygote (fa/wt; n = 33), and wild-type (wt/wt; n = 19) animals. Phenotypes were adjusted with stepwise regression analyses for the effects of age, sex, length, and litter size. Broad-sense heritability was estimated by examining the variance of the traits in first filial and F2 generations. ANOVAs were used to determine the mode of inheritance of the fa allele in the F2 generation. As anticipated, weight demonstrated the greatest overall broad-sense heritability (77%) and was the result of the recessive mutation. Breathing parameters during the hypoxic, hypercapnic, and combined challenges demonstrated a wide range of heritability from 5 to 96%, with a very nonuniform proportion of heritability explained by the leptin receptor. At best, for frequency 4.5 min into the hypercapnic hypoxic challenge, approximately 20% of the total heritability (approximately 67%) could be attributed to an effect of the leptin receptor mutation. We conclude that, unlike its major effect on weight, the effect of the fa allele is not a major gene involved in the regulation of breathing frequency.     

Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1‐associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1‐bound miR‐222‐3p, miR‐497‐5p and miR‐21‐5p were significantly higher, and let‐7a‐5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1‐HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF‐related miRNAs compared with the whole serum.     

Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases

Stem cell-based treatments have been extensively explored in the last few decades to develop therapeutic strategies aimed at providing effective alternatives for those human pathologies in which surgical or pharmacological therapies produce limited effects. Among stem cells of different sources, mesenchymal stem cells (MSCs) offer several advantages, such as the absence of ethical concerns, easy harvesting, low immunogenicity and reduced tumorigenesis risks. Other than a multipotent differentiation ability, MSCs can release extracellular vesicles conveying proteins, mRNA and microRNA. Thanks to these properties, new therapeutic approaches have been designed for the treatment of various pathologies, including ocular diseases. In this review, the use of different MSCs and different administration strategies are described for the treatment of diabetic retinopathy, glaucoma, and retinitis pigmentosa. In a large number of investigations, positive results have been obtained by in vitro experiments and by MSC administration in animal models. Most authors agree that beneficial effects are likely related to MSC paracrine activity. Based on these considerations, many clinical trials have already been carried out. Overall, although some adverse effects have been described, promising outcomes are reported. It can be assumed that in the near future, safer and more effective protocols will be developed for more numerous clinical applications to improve the quality of life of patients affected by eye diseases.     

A novel metal-free low molecular weight superoxide dismutase mimic

2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes.     

Cell proliferation and cytoarchitectural remodeling during spinal cord reconnection in the fresh-water turtle <Emphasis Type="Italic">Trachemys dorbignyi</Emphasis>

In fresh-water turtles, the bridge connecting the proximal and caudal stumps of transected spinal cords consists of regenerating axons running through a glial cellular matrix. To understand the process leading to the generation of the scaffold bridging the lesion, we analyzed the mitotic activity triggered by spinal injury in animals maintained alive for 20–30 days after spinal cord transection. Flow cytometry and bromodeoxyuridine (BrdU)-labeling experiments revealed a significant increment of cycling cells around the lesion epicenter. BrdU-tagged cells maintained a close association with regenerating axons. Most dividing cells expressed the brain lipid-binding protein (BLBP). Cells with BrdU-positive nuclei expressed glial fibrillary acidic protein. As spinal cord regeneration involves dynamic cell rearrangements, we explored the ultra-structure of the bridge and found cells with the aspect of immature oligodendrocytes forming an embryonic-like microenvironment. These cells supported and ensheathed regenerating axons that were recognized by immunocytological and electron-microscopical procedures. Since functional recovery depends on proper impulse transmission, we examined the anatomical axon-glia relationships near the lesion epicenter. Computer-assisted three-dimensional models revealed helical axon-glial junctions in which the intercellular space appeared to be reduced (5–7 nm). Serial-sectioning analysis revealed that fibril-containing processes provided myelinating axon sheaths. Thus, disruption of the ependymal layer elicits mitotic activity predominantly in radial glia expressing BLBP on the lateral aspects of the ependyma. These cycling cells seem to migrate and contribute to the bridge providing the main support and sheaths for regenerating axons.