An algorithm for constraint-based structural template matching: application to 3D templates with statistical analysis

MOTIVATION: Structural templates consisting of a few atoms in a specific geometric conformation provide a powerful tool for studying the relationship between protein structure and function. Current methods for template searching constrain template syntax and semantics by their design. Hence there is a need for a more flexible core algorithm upon which to build more sophisticated tools. Statistical analysis of structural similarity is still in its infancy when compared with its analogue in sequence alignment. In the context of template matching, there is an urgent need for normalization of scores so that results from templates with differing sensitivity may be compared directly. RESULTS: We introduce Jess, a fast and flexible algorithm for searching protein structures for small groups of atoms under arbitrary constraints on geometry and chemistry. We apply the algorithm to a set of manually derived enzyme active site templates, and derive an empirical measure for estimating the relative significance of hits encountered using differing templates.     

PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P₂], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P₂ production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P₂ is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.     

Novel protein vaccine candidates against Group B streptococcal infection identified using alkaline phosphatase fusions

Using an alkaline phosphatase-based genetic screening method, we identified a number of proteins that are potentially located on the outer surface of Group B streptococcus (Streptococcus agalactiae). In an enzyme-linked immunosorbent assay, antisera raised against two of the proteins, the streptococcal yutD homologue and a subunit of an ABC transporter, recognised clinically important serotypes of Group B streptococcus. In a neonatal rat model, purified IgG from the sera conferred significant levels of protection against a lethal challenge infection. The proteins identified show potential as protein subunit candidates for vaccines against Group B streptococcal disease in neonates.     

Rate of Helicobacter pylori infection in children and clonality of Taiwan strains

The infection rate of Helicobacter pylori in children from < 1 to 17 years old was investigated. Three techniques, namely culture, CLO test, and PCR, were employed to check the presence or absence of the organism in the antrum of the stomach. Several PCR positives without viable cultures were observed in babies of less than one year old. On the other hand, only two viable cultures were obtained from toddlers of less than two years old. The percentage of positive cultures steadily increased from 8% (3 of 42 cases) in the 0-4 years old age group to 32% (32 of 99 cases) in the 13-17 years old age group. A steady increase also was observed in the result of the CLO test. In PCR, the percentage of positives was greatly higher than that seen with the culture or CLO test. The rate of PCR positives also showed an increase with age but of a much slower rate. The overall infection rate in 295 children was 22% (64 of 295 cases) positive with culture and 76% (225 of 295 cases) with PCR, in contrast to 85% (40 of 49 cases) and 92% (43 of 47 cases), respectively, in adults. The urease activity of the H. pylori derived from children was much lower than that derived from adults (P < 0.001). Taken together, these results suggest that a child might be repeatedly infected and some infecting strains eventually might obtain a steady infection, perhaps by a strain of higher virulence such as higher urease activity. The base variations in the nucleotide sequences did not correlate to the varied urease activities or to the age of the child. The sequences, however, indicated that there were two types of strains. The strains in Taiwan appeared to be derived from the French type strain and not the English type strain. The amino acid sequences of the ureA and the phylogenetic relationship of the 29 strains indicated that the strains in Taiwan are rapidly evolving into a unique clone.     

NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature-sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD(+)-dependent DNA ligases.     

Zinc efficiency is correlated with enhanced expression and activity of zinc-requiring enzymes in wheat

Zinc (Zn) is an essential micronutrient for plants. The ability of plants to maintain significant yields under low Zn is termed Zn efficiency (ZE) and its genetic and mechanistic basis is still not well understood. Previously, we showed that root Zn uptake did not play a role in ZE. In the current study, Zn-efficient and -inefficient wheat (Triticum aestivum) genotypes were grown for 13 d in chelate buffer nutrient solutions at low (0.1 pM), sufficient (150 pM), and high (1 microM) Zn(2+) activities and analyzed for root-to-shoot translocation of Zn, subcellular leaf Zn distribution, and activity and expression of the Zn-requiring enzymes in leaves. No correlation between ZE and Zn translocation to the shoot was found. Furthermore, total and water-soluble concentrations of leaf Zn were not associated with ZE, and no differences in subcellular Zn compartmentation were found between Zn-efficient and -inefficient genotypes. However, the expression and activity of the Zn-requiring enzymes copper (Cu)/Zn superoxide dismutase (SOD) and carbonic anhydrase did correlate with differences in ZE. Northern analysis suggested that Cu/ZnSOD gene expression was up-regulated in the Zn-efficient genotype, Kirgiz, but not in inefficient BDME. Under Zn deficiency stress, the very Zn-efficient genotype Kirgiz and moderately Zn-efficient Dagdas exhibited an increased activity of Cu/ZnSOD and carbonic anhydrase when compared with Zn-inefficient BDME. These results suggest that Zn-efficient genotypes may be able to maintain the functioning of Zn-requiring enzymes under low Zn conditions; thus, biochemical Zn utilization may be an important component of ZE in wheat.     

Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce

Terpenoids are characteristic constitutive and inducible defense chemicals of conifers. The biochemical regulation of terpene formation, accumulation, and release from conifer needles was studied in Norway spruce [Picea abies L. (Karst)] saplings using methyl jasmonate (MeJA) to induce defensive responses without inflicting physical damage to terpene storage structures. MeJA treatment caused a 2-fold increase in monoterpene and sesquiterpene accumulation in needles without changes in terpene composition, much less than the 10- and 40-fold increases in monoterpenes and diterpenes, respectively, observed in wood tissue after MeJA treatment (D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann [2002] Plant Physiol 129: 1003-1018). At the same time, MeJA triggered a 5-fold increase in total terpene emission from foliage, with a shift in composition to a blend dominated by oxygenated monoterpenes (e.g. linalool) and sesquiterpenes [e.g. (E)-beta-farnesene] that also included methyl salicylate. The rate of linalool emission increased more than 100-fold and that of sesquiterpenes increased more than 30-fold. Emission of these compounds followed a pronounced diurnal rhythm with the maximum amount released during the light period. The major MeJA-induced volatile terpenes appear to be synthesized de novo after treatment, rather than being released from stored terpene pools, because they are almost completely absent from needle oleoresin and are the major products of terpene synthase activity measured after MeJA treatment. Based on precedents in other species, the induced emission of terpenes from Norway spruce foliage may have ecological and physiological significance.     

Parent-offspring conflict theory,signaling of need,and weight gain in early life

Human growth in early life has major implications for fitness. During this period, the mother regulates the growth of her offspring through placental nutrition and lactation. However, parent-offspring conflict theory predicts that offspring are selected to demand more resources than the mother is selected to provide. This general issue has prompted the development of begging theory, which attempts to find the optimal levels of offspring demand and parental provisioning. Several models have been proposed to account for begging behavior, whether by biochemical or behavioral pathways, including: (1) blackmail of parents; (2) scramble competition between multiple offspring; (3) honest signaling of nutritional need; and (4) honest signaling of offspring worth. These models are all supported by data from nonhuman animals, with species varying according to which model is relevant. This paper examines the evidence that human suckling and crying signal nutritional demand, need, and worth to the mother. While suckling provides hormonal stimulation of breast milk production and signals hunger, crying fulfills a different role, with evidence suggesting that it signals both worth and need for resources (nutrition and thermoregulation). The role of signaling in nutritional demand is examined in the context of three common health problems that have traditionally been assumed to have physiological rather than behavioral causes: excess weight gain, failure to thrive, and colic. The value of such an evolutionary approach lies in its potential to enhance behavioral management of these conditions.     

Epidemiologic determinants of aural abscessation in free-living eastern box turtles (Terrapene carolina) in Virginia

Epidemiologic determinants of 46 cases of aural abscessation in free-living eastern box turtles (Terrapene carolina) admitted to the Wildlife Center of Virginia (Virginia, USA) from 1991 to 2000 were evaluated. County human population density, year and season of admission, weight, and sex did not affect the risk for box turtles to develop aural abscessation. Counties with cases of aural abscessation were not randomly distributed, but rather were clustered into two multi-county regions. Geographic location was the only risk factor associated with aural abscessation in box turtles found in this study. Possible etiologies could include chronic infectious disease, malnutrition, or chronic exposure to environmental contamination with organochlorine compounds.     

Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv. syringae

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.