Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in Streptococcus pneumoniae

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.     

Effects of temperature and nitrate on phosphomonoesterase activities between carbon source and sink tissues in Zostera marina L.

Inorganic phosphorus (P_i) is important in the regulation of many carbon and nitrogen metabolic processes of plants. In this study, we examined alterations of phosphomonoesterase activity (PA; both alkaline and acid) in a submersed marine angiosperm, Zostera marina, grown in P_i non-limiting conditions under elevated temperature and/or nitrate enrichment. Control plants (ambient water-column NO₃⁻ < 2.5 μM, with weekly mean water temperatures between 26.5-27.0 °C based on a 20-yr data set in a local embayment) were compared to treated plants that were exposed to increased water-column nitrate (8 μM NO₃⁻ above ambient, pulsed daily at 0900 h), and/or increased temperature (ca. 3 °C above weekly means) over eight weeks in late summer-fall. Under both nitrate regimes, increased temperature resulted in periodic increased leaf and root-rhizome tissue carbon content, and increased acid and alkaline PA activities (AcPAs and AlPAs, respectively). There was a positive correlation between AlPA and AcPA activities and sucrose synthase activities in belowground structures, and a negative correlation between AlPA activities and sucrose concentrations. There were also periodic changes in PA partitioning between carbon source and sink tissues. In high-temperature and high-nitrate treatments, AcPAs significantly increased in leaves relative to activities in root-rhizome tissues (up to 12-fold higher in aboveground than belowground tissues in as little as 3 weeks after initiation of treatments). These responses were not observed in control plants, which maintained comparable AcPA activities in above- and belowground tissues. In addition, AlPA activity was significantly higher in leaf than in root-rhizome tissues of plants in high-temperature (weeks 3 and 6) and high temperature combined with high nitrate treatments (week 8), relative to AlPA activities in control plants. The observed changes in PAs were not related to P_i growth limitation, and may allow Z. marina to alter its carbon metabolism during periods of increased carbon demand/mobilization. This response would make it possible for Z. marina to meet short-term P requirements to maximize carbon production/allocation. Such a mechanism could help to explain the variability in PA activities that has been observed for many plant species during periods when environmental P_i exceeds requirements for optimal growth.     

THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; rather they presented evidence that they used non‐inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte‐like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non‐inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We, and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte‐like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte‐like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.     

ESTUARINE HETEROTROPHIC CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE): LIFE CYCLE AND CULTURE STUDIES1

Cryptoperidiniopsoids are an unclassified group of delicately thecate heterotrophic dinoflagellates known to be common in eastern U.S. estuarine waters. Over the past 10 years cryptoperidiniopsoids were isolated from different geographical regions and cultured with cryptophyte algal prey. In the seven clonal isolates examined, reproduction was strongly linked to the availability of prey cells. The dinoflagellates phagocytized the contents of prey cells through a tube‐like peduncle, similarly as close relatives of Pfiesteria spp. and several other heterotrophic species. Cell division occurred while encysted, most commonly yielding two biflagellated offspring. Abundant fusing gametes, phagotrophic planozygotes, and cysts with a pronounced nuclear cyclosis characterized persistent sexuality. Cysts with nuclear cyclosis produced two flagellated offspring cells. The resistance of reproductive cysts to antimicrobial treatments was examined, and a simple high‐yield technique was developed for population synchronization while ridding the dinoflagellates of most contaminating vacuolar prey DNA and external contaminants. The DNA content and population DNA profiles of synchronously excysted cryptoperidiniopsoids from different isolates were measured using flow cytometry and were related to the life history of these and other dinoflagellates. Cryptophyte‐fed cultures with versus without extracellular bacteria were compared, and bacteria apparently promoted cryptoperidiniopsoid feeding and growth. Externally bacteria‐free dinoflagellates were cultured in media enriched with dissolved organic nutrients, and nutritional benefit may have occurred in some treatments. The potential for mixotrophic nutrition from maintenance of cryptophyte chloroplasts was examined using flow cytometrically sorted cells, but evidence of kleptoplastidy was not found in these isolates under the conditions imposed.     

Rio Meta strain of Plasmodium vivax in New World monkeys and anopheline mosquitoes

An archived strain of Plasmodium vivax, isolated from Rio Meta, northern Colombia, in 1972 was adapted to grow in splenectomized Aotus lemurinus griseimembra and A. nancymai monkeys. Anopheles freeborni, An. maculatus, An. dirus, An. culicifacies, and An. albimanus were shown to be susceptible to infection by feeding on infected monkeys. Infections were more readily obtained by feeding on A. L. griseimembra than on A. nancymai. Transmission through sporozoites was obtained in an A. l. griseimembra monkey after a prepatent period of 24 days.     

A tyrosine-based sorting signal regulates intracellular trafficking of protease-activated receptor-1: multiple regulatory mechanisms for agonist-induced G protein-coupled receptor internalization

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated, internalized, and then sorted to lysosomes and degraded. Internalization and lysosomal sorting of activated PAR1 is critical for termination of receptor signaling. We previously demonstrated that activated PAR1 is rapidly phosphorylated and internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. Toward understanding the mechanisms responsible for activated PAR1 internalization through clathrin-coated pits we examined the function of a highly conserved tyrosine-based motif, YXXL, localized in the cytoplasmic carboxyl tail of the receptor. A mutant PAR1 in which tyrosine 383 and leucine 386 were replaced with alanines (Y383A/L386A) was significantly impaired in agonist-triggered internalization and degradation compared with wild-type receptor. In contrast, constitutive internalization, and recycling of unactivated PAR1 Y383A/L386A mutant was not affected, suggesting that tonic cycling of the mutant receptor remained intact. Strikingly, a PAR1 C387Z truncation mutant in which the YXXL motif was exposed at the C terminus constitutively internalized and degraded in an agonist-independent manner, whereas C387Z truncation mutant in which the critical tyrosine and leucine were mutated to alanine (C387Z-Y383A/L386A) failed to internalize. Inhibition of PAR1 C387Z mutant constitutive internalization with dominant-negative K44A dynamin blocked agonist-independent degradation of the mutant receptor. Together these findings strongly suggest that internalization of activated PAR1 is controlled by multiple regulatory mechanisms involving phosphorylation and a highly conserved tyrosine-based motif, YXXL. This study is the first to describe a function for a tyrosine-based motif, YXX, in GPCR internalization and reveal novel complexities in the regulation of GPCR trafficking.     

Are variants in the CAPN10 gene related to risk of type 2 diabetes? A quantitative assessment of population and family-based association studies

The calpain-10 gene (CAPN10) on chromosome 2q37.3 was the first candidate gene for type 2 diabetes (T2D) identified through a genomewide screen and positional cloning. One polymorphism (UCSNP-43: G-->A) and a specific haplotype combination defined by three polymorphisms (UCSNP-43, -19, and -63) were linked to an increased risk of T2D in several populations. To quantitatively assess the collective evidence for the effects of CAPN10 on risk of T2D, we conducted a meta-analysis of both population-based and family-based association studies. We retrieved data from the MEDLINE, PubMed, and Online Mendelian Inheritance in Man databases, as well as from other relevant reports and abstracts published up to July 2003. From a total of 26 studies with primary data (21 population-based studies: 5,013 cases and 5,876 controls; 5 family-based studies: 487 parent-offspring trios), we developed a summary database that contains variables of study design, study population/ethnicity, specific polymorphisms and haplotype combinations in CAPN10, and diabetes-related metabolic phenotypes. For population-based studies, we used both fixed-effects and random-effects models to calculate the pooled odds ratio (OR) and 95% confidence interval (CI) for the associations of CAPN10 genotypes with the risk of T2D. We also calculated weighted mean differences for the associations between CAPN10 and diabetes-related quantitative traits. Under either an additive or a dominant effect model, we found no statistically significant relation between CAPN10 genotypes in the UCSNP-43 locus and T2D risk. However, under a recessive model, individuals homozygous for the common G allele had a statistically significant 19% higher risk of T2D than carriers of the A allele (OR 1.19; 95% CI 1.07-1.33). The association between the 112/121 haplotype combination and T2D risk appeared to be overestimated by several initial small studies with positive findings (OR 1.38; 95% CI 1.04-1.84). After we removed these initial studies, this association became nonsignificant (OR 1.11; 95% CI 0.91-1.35). Moreover, we found no evidence for the associations between the UCSNP-43 G/G genotype and the 112/121 haplotype combination and metabolic phenotypes. Our meta-analysis of family-based studies showed only an overtransmission of the rare allele C in UCSNP-44 from heterozygous parents to their affected offspring with T2D. Our analysis indicates that inadequate statistical power, racial/ethnic differences in frequencies of alleles, haplotypes and haplotype combinations, potential gene-gene or gene-environment interactions, publication bias, and multiple hypothesis testing may contribute to the significant heterogeneity in previous studies of CAPN10 and T2D. Our findings also suggest that both large-scale, well-designed association studies and functional studies are warranted to either reliably confirm or conclusively refute the initial hypothesis regarding the role of CAPN10 in T2D risk.