Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified.     

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions

The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.     

Reproductive trade-offs in a specialized plant/pollinator system involving Asplundia uncinata Harling (Cyclanthaceae) and a derelomine flower weevil (Coleoptera: Curculionidae)

The reproductive interactions of a specialized plant/pollinator system involving Asplundia uncinata Harling (Cyclanthaceae) and a derelomine flower weevil (Coleoptera: Curculionidae: Derelomini) were studied at La Selva, Costa Rica. The inflorescences of A. uncinata exhibit a suite of cantharophilous characters including a precisely synchronized anthesis, protogyny, thermogenesis, olfactory attraction of visitors via modified staminodes, and narrow interfloral entrances. The weevil pollinators use the inflorescences for feeding, mating, and oviposition. The larvae are detritivorous and develop either in the detaching staminate flowers or, at a more favorable rate, in the rotting infructescences. The rate of infructescence abortion was high and caused by low levels of pollination. Manual pollination treatments yielded significantly higher seed counts than obtained under natural conditions, and furthermore demonstrated the inflorescences' ability to reproduce via geitonogamy. In the longer term, the reproductive benefits of maintaining low levels of pollination may shift away from the weevils and towards the plants via an increase in the size of the pollinator population.     

The expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase gene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.     

Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)

To unravel the relationship between the European wild apple, Malus sylvestris (L.) Mill., and its domesticated relative M. domestica Borkh., we studied chloroplast DNA variation in 634 wild and 422 domesticated accessions originating from different regions. Hybridization between M. sylvestris and M. domestica was checked using 10 nuclear microsatellites and a Bayesian assignment approach. This allowed us to identify hybrids and feral plants escaped from cultivation. Sixty-eight genotypes belonging to 12 other wild Malus species, including 20 M. sieversii (Ledeb.) Roem. accessions were also included in the analysis of chloroplast diversity. Marker techniques were developed to type a formerly described duplication and a newly detected transversion in the matK gene. Chloroplast DNA variation was further investigated using PCR-RFLP (Polymerase Chain Reaction-Random Fragment Length Polymorphism), and haplotypes were constructed based on all mutational combinations. A closer relationship than presently accepted between M. sylvestris and M. domestica was established at the cytoplasmic level, with the detection of eight chloroplast haplotypes shared by both species. Hybridization between M. sylvestris and M. domestica was also apparent at the local level with sharing of rare haplotypes among local cultivars and sympatric wild trees. Indications of the use of wild Malus genotypes in the (local) cultivation process of M. domestica and cytoplasmic introgression of chloroplast haplotypes into M. sylvestris from the domesticated apple were found. Only one of the M. sieversii trees studied displayed one of the three main chloroplast haplotypes shared by M. sylvestris and M. domestica. This is surprising as M. sieversii has formerly been described as the main maternal progenitor of the domesticated apple. This study hereby reopens the exciting discussion on the origin of M. domestica.     

Factors affecting mosquito production from stormwater drains and catch basins in two Florida cities.

Mosquito production from drains and catch basins in the cities of Vero Beach and Key West, FL were monitored from May 2004 through August 2005. A total of 48,787 mosquitoes were captured during the study. Of these, over 99% belonged to two species, Culex nigripalpus Theobald (2,630) and Culex quinquefasciatus Say (45,946). Other species collected included Culex restuans Theobald, Culex salinarius Coquillett, Aedes aegypti (L.), Aedes albopictus (Skuse), and Uranotaenia lowii Theobald. Significantly greater numbers of Cx. nigripalpus were collected at Vero Beach than at Key West, but no significant differences in numbers of Cx. quinquefasciatus and in total numbers of mosquitoes between the two cities were evident. Rainfall, salinity, type of structure, structure setting, and presence or absence of predators or competitors influenced the numbers of mosquitoes collected and/or the frequency of positive, negative, dry, or flooded samples.     

Mechanism of flavin reduction in class 2 dihydroorotate dehydrogenases

Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (Ser for Class 2 DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concerted or stepwise was addressed for two Class 2 enzymes, those from Escherichia coli and Homo sapiens, by determining kinetic isotope effects on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined for the E. coli enzyme at two pH values below a previously reported pKa controlling reduction [Palfey, B. A., Bj?rnberg, O., and Jensen K. F. (2001) Biochemistry 40, 4381-4390] and were about 3-fold for DHO labeled at the 5-position, about 4-fold for DHO labeled at the 6-position, and about 6-7-fold for DHO labeled at both the 5- and 6-positions. These isotope effects are consistent with either a stepwise oxidation of DHO or a concerted mechanism with significant quantum mechanical tunneling. At a pH value above the pKa controlling reduction, no isotope effect was observed in E. coli DHOD for DHO deuterated at the 5-position (the proton donor in the reaction). This is consistent with a stepwise reaction; above the (kinetic) pKa, the deprotonation of C5 is fast enough that it does not contribute to the observed rate constant and, therefore, is not isotopically sensitive. All available information points to Ser acting as a component in a proton relay network which allows its transient deprotonation. The H. sapiens DHOD also appears to have a pKa near 9.4 controlling reduction, similar to that previously reported for the E. coli enzyme. Similar KIEs were obtained with the H. sapiens enzyme at a pH value below the pKa.     

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

AIMS: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrP(Sc)). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease. METHODS AND RESULTS: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high-degradation activity against a scrapie PrP(Sc). Sequence analysis indicated that this serine-protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9-10 and 60-70 degrees C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy-infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrP(Sc) that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD-1, a previously reported keratinase. CONCLUSIONS: These results indicate that the isolated protease exhibited higher activity for PrP(Sc) degradation compared with other proteases examined. SIGNIFICANCE AND IMPACT OF THE STUDY: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrP(Sc) contamination.