Characterization of the human jumonji gene

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.      

How to use Chlamydia antibody testing in subfertility patients

Screening for tubal factor subfertility by means of Chlamydia antibody testing (CAT) was introduced into the initial work-up of subfertile couples several years ago. The results reported, however, are heterogeneous, and no uniformity exists in cut-off levels of titres, or in definitions of tubal factor subfertility. We performed a prospective cohort study to evaluate the implications of varying the definitions of tubal pathology and of modifying the cut-off levels on the clinical impact of CAT in predicting tubal factor subfertility. In 227 consecutive patients who attended our fertility clinic, the Chlamydia IgG antibody titre was determined and related to tuboperitoneal abnormalities at laparoscopy as a reference standard. According to received operating characteristic (ROC) curve analysis, a titre of 16 is the optimum cut-off level. Increasing the cut-off level improves specificity and positive likelihood ratio (LR+), at the expense of sensitivity and negative LR (LR-). Changing the definition of tubal factor subfertility from unspecified tuboperitoneal abnormalities into extensive adhesions and/or bilateral distal tubal occlusion improves LR+, LR- and kappa significantly. We conclude that CAT is more accurate in predicting severe distal tubal pathology than unspecified tuboperitoneal abnormalities. Although from a statistical point of view a titre of 16 is the optimum cut-off level, from a clinical point of view 32 or 64 may be preferable, depending on the aim of screening and the inception cohort.      

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.      

Potentiation of FMD vaccines with polycationic-nucleic acid complexes

Summary The effect of various polycations on the immune response potentiated with poly I:C was studied. It was found that low molecular weight polycations had no potentiating effect. Polylysine was ineffective whereas protamine was superior to lysozyme, poly-arginine, poly-histidine, DEAE-Dextran and histone.A foot-and-mouth disease trivalent vaccine composed of strains A₂₄ Cruzeiro, O₁ Caseros and C₂ Resende elicited no immune response in swine when adjuvanted with aluminium hydroxide but was effective when emulsified in oil. In general, the immune response was potentiated ten-fold when the emulsion contained poly I:C. The antibody production was in most cases further potentiated by a factor of ten when the nucleic acid double-strand was complexed with 1:10 (w/w) DEAE-Dextran. Protamine was as effective, or perhaps even more, than DEAE-Dextran to this effect.Guinea pigs vaccinated with a water-in-oil emulsion type monovalent C₃ vaccine showed an increase in antibody production when the vaccine contained poly I:C or poly I:C complexed with 1:10 (w/w) protamine.With 4 Figures     

Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.     

Flow cytometric analysis of cell proliferation dynamics in the B cell compartment of the mouse

Using a method which allows simultaneous flow cytometric detectionof cell surface markers and 5-bromo-2'-deoxyuridine (BrdU) incorporation,the distribution and proliferative behavior of B lineage subpopulationswas studied in intact adult mice. In the bone marrow we coulddefine two subsets of B cells on the basis of differential expressionof the pan-B cell marker B220 and of membrane-associated µand immunoglobulin heavy chains. B220^dullµ^+– Bcells were found to emerge from rapidly dividing cells and probablyrepresent B cells recently generated from B220^dullµ^–pie-B cells. In contrast, oniy few, If any, of the B220^brightµ⁺⁺B cells were labeled with BrdU after a period of 8 days, suggestingthat these cells represent long-lived B cells residing in thebone marrow. Analysis of BrdU-Incorporation into splenic B cellsshowed that only 20% of these cells had gone through cell divisionduring the preceding 8 days. Almost none of the B cells in theperitoneum, a large fraction of which belongs to the Ly1 B subset,were labeled with BrdU over a period of 7 days in 8-month-oldanimals.     

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.      

The association of socioeconomic status and use of crack/cocaine with unprotected anal sex in a cohort of men who have sex with men in Rio de Janeiro, Brazil.

To evaluate the relation between illicit drug use, sexual practices, and socioeconomic status, we analyzed data from the baseline interview of a cohort of 675 men who have sex with men conducted from 1994 to 1999 in Rio de Janeiro, Brazil. Bivariate analyses of factors associated with crack/cocaine use with sex revealed that men who reported crack/cocaine use were significantly ( p <.05) more likely than men who did not report drug use to be unemployed (42.7% vs. 29.1%), to have an income of <$250 per month (70.7% vs. 60.9%), to have <8 years of education (69.5% vs. 50.9%), to report bisexual activity (81.7% vs. 41.7%), and to engage in commercial sex (72.0% vs. 37.9%). Multivariate analysis of factors associated with unprotected anal sex with casual male partners in the last 6 months demonstrated that the following variables were associated with this outcome: an income <$250 per month (adjusted odds ratio [AOR] = 1.73, 95% confidence interval [CI]: 1.04-2.87), less than 8 years of education (AOR = 2.21, CI: 1.38-3.53), a greater sense of vulnerability (AOR = 2.58, CI: 1.54-4.33), a willingness to participate in vaccine trials (AOR = 1.91, CI: 1.20-3.05), and use of crack/cocaine (AOR = 1.91, CI: 1.05-3.46). Our findings suggest that HIV prevention programs for these men need to address drug use and how drug use may influence sexual behaviors.