In vitro reproductive toxicity of polychlorinated biphenyls: effects on oocyte maturation and developmental competence in cattle

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread group of endocrine disrupting compounds in the ecosystem. High concentrations of these substances are known to be present in sewage sludge from industrial, agricultural, and domestic origin that is spread in increasing amounts on arable land and pasture as fertilizer and is found in water, representing an increasing risk for the reproductive health of farm animals. Objective of this study was to determine the impact of PCBs on maturation and developmental competence of cattle oocytes. Since PCBs are a family of 209 molecules present in the environment as a mixture, Aroclor-1254, a pool of more than 60 congeners, was used in these experiments as its composition is considered to be environmentally relevant. Cumulus-oocytes complexes were exposed during IVM to serial concentrations of Aroclor-1254 (between 1 microg/ml and 0.0001 microg/ml) and compared with control groups. Aroclor decreased the percentage of oocytes that reached metaphase II stage after 24 hr, at doses as low as 0.01 microg/ml. Groups treated with 0.001 microg/ml or above, showed an impaired fertilization rate and a dramatic increase of polyspermy. Moreover, exposure during maturation resulted in a reduced proportion of oocytes that cleaved and developed until blastocyst stage although no differences in embryo cell numbers were observed. The present study indicates that very low PCBs concentrations are sufficient to disrupt bovine oocyte maturation, its fertilization, and developmental competence. These results also provide a set of reference data for the assessment of the risk posed by these substances to animal reproductive health, though further work will be necessary to equate in vitro doses to in vivo exposures.     

Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.     

Topographical analysis of Schizolobium parahyba chymotrypsin inhibitor (SPCI) by atomic force microscopy

Atomic Force Microscopy (AFM) has been a useful tool for molecular surface analysis and to estimate topographical properties of proteins. Here we report a topographical study of a chymotrypsin inhibitor from Schizolobium parahyba seeds (SPCI) by AFM. The underlying structure of SPCI oligomers has been resolved in nanometer order resolution. SPCI oligomerize in hexagonal, ellipsoid, comet, pyramidal, and "Z" shaped. The hexagonal was the most observed oligomer shape.     

Hypothalamic insulin signaling is required for inhibition of glucose production

Circulating insulin inhibits endogenous glucose production. Here we report that bidirectional changes in hypothalamic insulin signaling affect glucose production. The infusion of either insulin or a small-molecule insulin mimetic in the third cerebral ventricle suppressed glucose production independent of circulating levels of insulin and of other glucoregulatory hormones. Conversely, central antagonism of insulin signaling impaired the ability of circulating insulin to inhibit glucose production. Finally, third-cerebral-ventricle administration of inhibitors of ATP-sensitive potassium channels, but not of antagonists of the central melanocortin receptors, also blunted the effect of hyperinsulinemia on glucose production. These results reveal a new site of action of insulin on glucose production and suggest that hypothalamic insulin resistance can contribute to hyperglycemia in type 2 diabetes mellitus.     

New strategies for treatment of Candida vaginal infections

New strategies for treatment of vaginal candidiasis have been recently exploited, due to widespread occurrence of this disease, in particular as recurrent infections, limitations of safe and efficacious antifungals as well as the lack of reliable preventative approaches. In this review new chemotherapeutic and immunotherapeutic strategies, based on the improved understanding of the immunopathogenesis of this prevalent human infection, will be discussed. The role of killer antibodies (or their molecular derivatives), i.e. antibodies that show antibiotic activity bearing the internal image of a yeast killer toxin (KT), characterized by a wide spectrum of microbicidal activity, and of the specific cell wall KT receptor as putative new therapeutic agents and preventative or therapeutic vaccines, respectively, will be particularly outlined.     

Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.     

In vitro leishmanicidal activity of a monoclonal antibody mimicking a yeast killer toxin

The microbicidal effect of a monoclonal antiidiotypic antibody, mimicking the activity of a yeast killer toxin, characterized by a wide antimicrobial spectrum, has been evaluated in vitro against two relevant species of protozoan parasites, Leishmania major and Leishmania infantum. The antiidiotypic antibody exerted a significant and dose-dependent antileishmanial activity against parasite promastigotes in comparison to an irrelevant isotype-matched monoclonal antibody. This is the first demonstration that an antibody, which had been already shown to be fungicidal and bactericidal, may also exert a direct microbicidal activity against protozoa.     

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.