Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

Immunoglobulin A levels in southern elephant seal (Mirounga leonina) milk during the suckling period

Immunoglobulin A (IgA) levels in milk samples from southern elephant seals at King George Island, Antarctica are reported. IgA levels were determined throughout the suckling period (approximately 23 days). The IgA concentration in southern elephant seal milk was lower than in other mammals and, unlike most mammalian milk, was not high during early lactation. There was not a definite pattern in IgA levels, which fluctuated within narrow limits throughout the suckling period (mean +/- SD, 30.81 +/- 6.38 mg IgA/100 g milk). If IgG was present, its level was too low to be detected by the method used. This is the first evidence in Southern elephant seal of the possibility of transmission of passive immunity after birth involving secretion of IgA in the milk.     

Veterinary practice and occupational health. An epidemiological study of several professional groups of Dutch veterinarians. I. General physical examination and prevalence of allergy, lung function disorders, and bronchial hyperreactivity

The prevalence of allergy, lung function disorders, and bronchial hyperreactivity was studied in 102 Dutch veterinarians, subdivided into five professional groups (predominantly working with either swine, cattle, poultry, companion animals, or as a non-practitioner). The mean age of the participants was 43 years; 6 participants were females. Twenty-two per cent of the participants were overweight, and relatively more non-practitioners than practitioners were overweight. Approximately 23% of the vets reported complaints of prolonged fatigue. The data suggest a relationship between complaints of prolonged fatigue and a more than average number of daily working hours. Only a small proportion of vets were sensitized against several allergens. There were no significant differences in prevalence of distinct lung function disorders or bronchial hyperreactivity between professional groups. It is hypothesized that the respiratory complaints (chronic coughing, chronic phlegm production, stuffed nose, sneezing) reported by the vets predominantly working in swine and/or poultry practice could be caused by irritation and/or inflammation of the first part of the trachea-bronchial tree that has no measurable and permanent consequences for changes in lung function or increased bronchial hyperreactivity. The results of a skin test against allergens and determination of allergen-specific IgE in blood indicated that the respiratory complaints were probably not related to allergy against the panel of allergens tested.     

A comparison of breeding value predictors for longevity using a linear model and survival analysis

A comparison was made among breeding values of sires for longevity that were obtained by different methods: phenotypic averages of daughters using only uncensored records, BLUP using only uncensored records, survival analysis using only uncensored records, and survival analysis using both censored and uncensored records. Two data files were used: one contained data from small herds, and the other contained data from large herds. The results from both data files were similar. Different methods of predicting breeding values resulted in different rankings of sires. The results obtained using phenotypic averages were weakly correlated (< or = 0.46) with those results obtained using the other methods of prediction. The REML BLUP had strong correlations (< or = -0.91) with the survival analysis predictor if the same data were used, and correlations weakened (< or = -0.60) when censored records were included in the survival analysis. The correlations are negative because the linear method analyzed longevity, and survival analysis measured the risk of being culled, which has an antagonistic relationship with longevity. The results from REML BLUP and survival analysis methods differed mainly because of the different data that were used (uncensored only versus both censored and uncensored).     

Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

Healthy women and patients with endometriosis show high concentrations of inhibin A, inhibin B, and activin A in peritoneal fluid throughout the menstrual cycle

Inhibin A, inhibin B, and activin A are growth factors which play local autocrine/paracrine roles in reproductive tissues. Since peritoneal fluid hormone content may reflect in part ovarian and endometrial secretory activities, the present study aimed to evaluate: (i) whether inhibin alpha-, activin betaA- and betaB-subunits, and activin receptor type II and type IIB mRNA are expressed in peritoneal tissues; (ii) expression and secretion of inhibin A and B, and activin A in cultured endometriotic cells; and (iii) concentrations of inhibin A and B, and activin A in serum and in peritoneal fluid in healthy women and in patients with endometriosis throughout the menstrual cycle. A group of women (n = 72) was recruited at laparoscopy for infertility investigation and divided into two groups: (i) control healthy women (n = 35), (ii) women with endometriosis (n = 37). Both groups were subdivided according to the follicular and luteal phase of the menstrual cycle. At the time of laparoscopy, specimens of peritoneal tissues were collected from three healthy women, while endometriotic tissue samples were collected and cultured from three women with endometriosis. Peritoneal tissues and cultured endometriotic cells expressed inhibin alpha-, activin betaA-, and betaB-subunits, and activin receptors mRNAs; in addition, inhibin-related proteins were measurable in culture medium. In healthy women, inhibin A and B, and activin A concentrations in peritoneal fluid were significantly higher than in serum (P < 0.001), at both phases of the menstrual cycle. Peritoneal inhibin A and B, and activin A concentrations were not significantly different between healthy women and patients with endometriosis, either when evaluated according to the degree of the disease and/or to the phase of the menstrual cycle. In conclusion, the findings that high concentrations are present in peritoneal fluid and that menstrual cycle-related changes occur suggest that reproductive organs may contribute to inhibin-related proteins in peritoneal fluid.     

Efficacy of venlafaxine and placebo during long-term treatment of depression: a pooled analysis of relapse rates

An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spv A) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.     

Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A)

We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.