Relationship of dorsoventral eyeshine distributions to habitat depth and animal size in mesopelagic decapods

Eyeshine distribution patterns recorded from the eyes of 19 mesopelagic decapod species were examined and related to the depths at which the species are found. For most species examined, eyeshine was found to be brighter ventrally than dorsally. Deep-water decapod species that do not undergo diel vertical migrations had brighter dorsal eyeshine than migratory species. Eyeshine intensity increased with body size in five of the species examined and decreased in two. These changes in eyeshine intensity may be an adaptation to variations in depth distributions that occur with increasing body size. It is suggested that the depth and size-related changes reflect the importance of remaining camouflaged in the mesopelagic realm and are an example of ecologically functional development.     

Assessment of insecticide resistance after the outbreak of diamondback moth (Lepidoptera: Plutellidae) in California in 1997

During an outbreak of the diamondback moth, Plutella xylostella (L.), in California in 1997, nine populations were collected from the major broccoli areas throughout the state. Populations were assayed for their susceptibility to currently used materials (Bacillus thuringiensis subsp. kurstaki, permethrin, and methomyl) and to newer materials that had not yet been commercially used in California (spinosad, emamectin benzoate, and chlorfenapyr). For the currently used insecticides, elevated levels of resistance were seen only with permethrin and seven of the nine populations had tolerance ratios (TR) of > 100. With the newer chemistries, TR values were all < 15. To compare potential cross-tolerance, TR values of the currently used insecticides were compared with TR values of the newer insecticides. There were significant relationships found between: methomyl and emamectin benzoate, methomyl and spinosad, and permethrin and spinosad. Further biochemical studies are needed to confirm the actual mechanisms that lead to these relationships and field tests are needed to determine what impact, if any, such TR levels would have on control in the field. These data indicate that resistance to at least one of the commonly used insecticides (permethrin) may have played a role in the outbreak during 1997. However, other factors may have been at least equally important. The winter of 1996-1997 was warmer than normal, and during the period from February through August of 1997 the amount of rainfall was < 50% of normal. Hot and dry conditions are known to be conducive to outbreaks of P. xylostella. These data add to an overall knowledge about the geographic variation of resistance in P. xylostella populations within the United States. They also serve as a baseline for monitoring changes in susceptibility to these newer insecticides and can also help explain the occurrence of outbreaks caused by factors other than insecticide resistance.     

High frequency hearing loss correlated with mutations in the GJB2 gene

Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.     

Activation of peroxisome proliferator-activated receptor gamma does not inhibit IL-6 or TNF-alpha responses of macrophages to lipopolysaccharide in vitro or in vivo

We have investigated the potential use of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARgamma agonists. Although 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-alpha and IL-6, four other high affinity PPARgamma ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARgamma or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARgamma ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARgamma does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARgamma-independent mechanism. To examine the anti-inflammatory activity of PPARgamma agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-alpha and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARgamma in leukocytes will not be useful for the treatment of acute inflammation.     

Estimation of viable Escherichia coli O157 in surface waters using enrichment in conjunction with immunological detection

The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples. In principle, E. coli O157 populations (C(initial E. coli O157)) can be derived from enrichment data according to the equation: C(initial E. coli O157) = C(initial coliforms) x C(final E. coli O157)/C(final coliforms)), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining C(final E. coli O157) in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of C(initial coliforms). Estimates of C(final coliforms) were determined from turbidity data. Initial E. coli O157 populations (C(initial E. coli O157)) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of < 1 cell per 100 ml. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k= 0.47 +/- 0.13 h(-1), n= 72). There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique. Although variability in growth rates was expected to yield some low values, the fact that most E. coli O157 concentrations were < 1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of C(initial coliforms) and k(coliforms), MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 10(3) to 10(8) cells ml(-1) enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E. coli O157 cell per 100 ml surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli.     

The glycoprotein NOWA and minicollagens are part of a disulfide-linked polymer that forms the cnidarian nematocyst wall

The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.     

AICA-ribosiduria: a novel, neurologically devastating inborn error of purine biosynthesis caused by mutation of ATIC

In a female infant with dysmorphic features, severe neurological defects, and congenital blindness, a positive urinary Bratton-Marshall test led to identification of a massive excretion of 5-amino-4-imidazolecarboxamide (AICA)-riboside, the dephosphorylated counterpart of AICAR (also termed "ZMP"), an intermediate of de novo purine biosynthesis. ZMP and its di- and triphosphate accumulated in the patient's erythrocytes. Incubation of her fibroblasts with AICA-riboside led to accumulation of AICAR, not observed in control cells, suggesting impairment of the final steps of purine biosynthesis, catalyzed by the bifunctional enzyme AICAR transformylase/IMP cyclohydrolase (ATIC). AICAR transformylase was profoundly deficient, whereas the IMP cyclohydrolase level was 40% of normal. Sequencing of ATIC showed a K426R change in the transformylase region in one allele and a frameshift in the other. Recombinant protein carrying mutation K426R completely lacks AICAR transformylase activity.     

Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, Ca(2+)-binding, endofacial plasma membrane protein originally identified by its capacity to accelerate transbilayer movement of membrane phospholipids. We recently reported that when palmitoylation of PLSCR1 does not occur, it is localized to the nucleus rather than the plasma membrane. Nuclear localization of PLSCR1 was also observed upon induction of its de novo synthesis by cytokines such as interferon alpha that activate the PLSCR1 gene. Despite its capacity to enter the nucleus, its sequence does not predict a nuclear localization signal. To gain insight into the mechanism and potential significance of nuclear PLSCR1, we investigated the conditions required for its import and retention in the nucleus. We show that nuclear localization of PLSCR1 is dependent on cytosolic factors and energy. Furthermore, we show that PLSCR1 is specifically transported into the nucleus by the importin alpha/beta import pathway, and binds directly and with high affinity to importin alpha. Analysis of deletion mutants suggested that the NLS of PLSCR1 is between residues 242 and 290 and, furthermore, that a peptide within this region encompassing residues (257)GKISKHWTGI(266) is sufficient for nuclear import when conjugated to BSA. In addition, in intact cells, mutation of positively charged amino acids within this putative NLS in the full-length protein completely blocked its entry into the nucleus, consistent with its role in targeting PLSCR1 to the nucleus. Release of PLSCR1 from the nucleus was only observed after treatment of cells with both detergent and an elevated NaCl concentration, or following DNase treatment of the nucleus, suggesting ionic interactions of PLSCR1 with a nuclear component bound to genomic DNA or directly with genomic DNA. Purified PLSCR1 was also found to bind directly to a genomic DNA-cellulose conjugate, and its elution from DNA also required an elevated NaCl concentration. These data support a mechanism of receptor-mediated nuclear import of PLSCR1 and suggest a potential nuclear function for this plasma membrane protein.     

Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism

Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.