Poisoning in Ferrets by Tissues of Alkyl Mercury-Fed Chickens

Chickens were fed alkyl mercury-dressed wheat (mercury content about 8 mg/kg) for 35–44 days and were then immediately sacrificed. No signs of untoward effects were observed. Muscle of the chickens, and a minor proportion of liver, were fed to two groups of two ferrets (Mustela furo L. × M. putorius L.), the mercury content of the diet being 7 and 5 mg/kg, respectively. The ferrets of the first group died after 35 and 36 days and those of the second after 58 days. The experimental ferrets showed a marked weight loss, attributable to muscular atrophy in addition to a reduced food intake. Clinical signs appeared in two to three weeks and were primarily neurological such as ataxia, trembling and paralysis. The signs could be correlated with pronounced degenerative changes of the central and peripheral nervous systems involving mainly the cerebellum and peripheral nerves and, to a lesser extent, the cerebrum and the spinal cord. Hypoplasia of the lymphatic tissue of the spleen and degeneration of the graafian follicles were seen as well. High mercury levels were found in the kidneys, liver and brain and also in skeletal muscle and the gonads of the ferrets (Table 2). Methyl mercury constituted the major part of the tissue mercury in the ferrets (as well as in the chickens). The results provide direct evidence of the transfer and accumulation of alkyl mercury in a toxic form through a food chain. The ecological implications are discussed.     

An EG-VEGF-Dependent Decrease in Homeobox Gene NKX3.1 Contributes to Cytotrophoblast Dysfunction: A Possible Mechanism in Human Fetal Growth Restriction

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that endocrine gland–derived vascular endothelial growth factor (EG-VEGF), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesize that EG-VEGF-dependent changes in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a homeobox gene cDNA array on placental explants of 8–12 wks gestation after stimulation with EG-VEGF in vitro for 24 h. The homeobox gene array identified a greater-than-five-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX 3.1 showed a greater-than-two-fold decrease in mRNA expression compared with untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional roles are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared with control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR-8/SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 downstream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.     

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.     

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

BACKGROUND INFORMATION: Pancreatic beta-cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL-1beta (interleukin 1beta) induces distinct changes of beta-cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL-1beta and known pathways for IL-1beta actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL-1beta were monitored by measurements of nitrite and PGE(2) (prostaglandin E(2)). RESULTS: IL-1beta caused an enhancement of islet intracellular insulin degradation and an increase in the lysosomal incorporation of beta-cell secretory granules. The effects of IL-1beta were abolished by aminoguanidine, a selective inhibitor of inducible NOS (nitric oxide synthase), or by rofecoxib, a specific inhibitor of COX-2 (cyclo-oxygenase 2). In the absence of IL-1beta, nitroarginine, which is a selective inhibitor of constitutive NOS, caused a decrease in intracellular degradation of insulin in parallel with a decreased production of NO and PGE(2) by the islets. CONCLUSIONS: The correlation between the enhanced intracellular insulin degradation and lysosomal changes caused by IL-1beta suggests that insulin degradation may be effected by crinophagy. Under physiological conditions, significant beta-cell degradation of insulin may depend on the activity of COX-2, possibly stimulated by endogenous NO.     

Potentials of mean force for protein structure prediction vindicated, formalized and generalized

Understanding protein structure is of crucial importance in science, medicine and biotechnology. For about two decades, knowledge-based potentials based on pairwise distances--so-called "potentials of mean force" (PMFs)--have been center stage in the prediction and design of protein structure and the simulation of protein folding. However, the validity, scope and limitations of these potentials are still vigorously debated and disputed, and the optimal choice of the reference state--a necessary component of these potentials--is an unsolved problem. PMFs are loosely justified by analogy to the reversible work theorem in statistical physics, or by a statistical argument based on a likelihood function. Both justifications are insightful but leave many questions unanswered. Here, we show for the first time that PMFs can be seen as approximations to quantities that do have a rigorous probabilistic justification: they naturally arise when probability distributions over different features of proteins need to be combined. We call these quantities "reference ratio distributions" deriving from the application of the "reference ratio method." This new view is not only of theoretical relevance but leads to many insights that are of direct practical use: the reference state is uniquely defined and does not require external physical insights; the approach can be generalized beyond pairwise distances to arbitrary features of protein structure; and it becomes clear for which purposes the use of these quantities is justified. We illustrate these insights with two applications, involving the radius of gyration and hydrogen bonding. In the latter case, we also show how the reference ratio method can be iteratively applied to sculpt an energy funnel. Our results considerably increase the understanding and scope of energy functions derived from known biomolecular structures.     

Activation of a cryptic splice donor in human immunodeficiency virus type-1

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function ascis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5 splice donor of HIV-1 was mutated in the context of othergag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5 splice donor and thetat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.     

Alpha-tectorin involvement in hearing disabilities: one gene – two phenotypes

The human alpha-tectorin (TECTA) gene has recently been cloned and proposed to be involved in autosomal dominant non-syndromic hearing impairment (NSHI) in two families linked to the DFNA12 locus. We have studied a Swedish pedigree with autosomal dominant NSHI with possible digenic inheritance of the disease, involving locus DFNA12 in chromosome 11 and locus DFNA2 in chromosome 1. Mutation analysis of the TECTA gene in this family has identified eight nucleotide substitutions indicating that TECTA is highly polymorphic. One of the changes results in a cysteine to serine (C 1057 S) mutation, in the zonadhesin domain of TECTA; this segregates with the disease haplotype on chromosome 11 and is not present in a control population. The mutation results in the replacement of a cysteine in one of the repeats of the zonadhesin/Von Willebrand domain of the protein and might cause a change in the crosslinking of the polypeptide. These findings add support to the involvement of TECTA in hearing disabilities. However, the three families carrying different TECTA mutations also show phenotypic differences: the hearing loss ranges from prelingual to progressive with late onset. The explanation for the different phenotypes and some clues regarding the functions of TECTA may lie in the localization of the mutations in the different modules of the protein. Another possibility is that the phenotype in the Swedish family is the result of two defective genes.     

Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.