Malignancy may adversely influence the quality and behaviour of oocytes

A case series of eight cycles of in-vitro fertilization (IVF) in five women diagnosed with malignant disorders is presented. These patients chose to defer definitive treatment for a chance for preservation of potential fertility. The response of these patients to ovarian stimulation, and the outcome, was compared with 17 IVF cycles in 12 age- matched patients with isolated tubal infertility. An apparent adverse influence of malignant disease on the quality and behaviour of oocytes was observed. Despite a comparable total number of oocytes per cycle in the two groups, a significantly reduced percentage of mature oocytes was retrieved per cycle from patients with malignant diseases. The oocytes from patients with malignant disorders were of a poorer quality and exhibited a significantly impaired fertilization rate compared to the controls. We propose that neoplastic processes, irrespective of the site or cell of origin, may have a detrimental impact on the biology of oocytes, an effect akin to that seen on spermatozoa in men with certain malignancies.      

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.      

Inhibition of Respiratory syncytial, parainfluenza 3 and measles viruses by 2-deoxy-D-glucose.

2-Deoxy-d-glucose was found to inhibit the replication and cytopathic effects of respiratory syncytial, parainfluenza 3 and measles viruses. The effects of the drug were completely reversible and appeared directed against viral functions occurring in the second half of the viral growth cycle. At least some viral functions appeared to be unaffected by the agent. Electron microscopic studies with a temperature-sensitive mutant of respiratory syncytial virus indicated that 2-deoxy-d-glucose produced a morphological change. Surface projections were greatly reduced in virions grown in the presence of the drug.     

Molecular markers in acute and chronic phases of human toxoplasmosis: determination of immunoglobulin G avidity by Western blotting

We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.     

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.     

Immunoglobulin G and immunoglobulin M enzyme-linked immunosorbent assays and defined toxoplasmosis serological patterns.

Enzyme-linked immunosorbent assay (ELISA) for toxoplasmosis was evaluated in serum samples presenting defined toxoplasmosis serological patterns, as determined by results in immunoglobulin (Ig)G and IgM immunofluorescence (IgG-IF, IgM-IF), hemagglutination, and complement fixation tests. ELISA was carried out with alkaline phosphatase-labeled anti-IgG and anti-IgM antibodies. Serum titer was expressed as the serum dilution end point determined by mere observation of color development in the test. A straight agreement was found between IgG-ELISA and IgG-IF titers, both in group A sera of ancient infections (patterns II and III) and group B sera of recent infections (pattern I). A similar agreement was found between IgG-ELISA and hemagglutination titers in group A sera, for which coincident IgG-IF and hemagglutination titers are also frequent. However, in group B sera, in spite of the same toxoplasma extract being used to sensitize both plastic surfaces and erythrocytes, IgG-ELISA titers were much higher than hemagglutination titers, in a way similar to that observed for IgG-IF titers. IgM-ELISA was positive in every group B serum, with higher titers than corresponding IgM-IF titers. Occasional low-titered positive IgM-ELISA results were seen for group A sera, sometimes due to IgM-antiglobulin antibodies. An easy test to perform, ELISA seems to be an adequate substitute for toxoplasmosis IF tests for routine purposes.     

Interaction between the septal area and the subfornical organ in the control of water intake induced by thirst-eliciting procedures

Rats bearing lesions in the septal area followed by lesions in the subfornical organ were submitted to various thirst-eliciting procedures. The rats with hyperdipsia induced by lesions of the septal area drank more water than either during the control period or after lesion of the subfornical organ under the same thirst-eliciting or angiotensin-liberating stimuli (polyethyleneglycol, isoproterenol, water deprivation and ligation of the inferior vena cava). The overdrinking elicited by lesions in the septal area was blocked after lesion of the subfornical organ. Neither hypovolemia, nor hypotension or water deprivation could elicit increased water intake in animals whose subfornical organ had been destroyed. Animals with lesions in the subfornical organ showed decreased water intake after cellular dehydration. The results obtained suggest that the subfornical organ acts as a more important structure than the septal area in the regulation of water intake elicited by angiotensin, with two opposite effects: a direct one facilitating water intake, and an indirect one inhibiting the septal area. The septal area has an inhibitory effect on the subfornical organ and on water intake.