Comparative Proteomic Analysis for a Putative Pyridoxal Phosphate-Dependent Aminotransferase Required for Virulence in Acidovorax citrulli

Acidovorax citrulli (Ac) is the causative agent of bacterial fruit blotch disease in watermelon. Since resistant cultivars have not yet been developed, the virulence factors/mechanisms of Ac need to be characterized. This study reports the functions of a putative pyridoxal phosphate-dependent aminotransferase (PpdaAc) that transfers amino groups to its substrates and uses pyridoxal phosphate as a coenzyme. It was observed that a ppdaAc knockout mutant had a significantly reduced virulence in watermelon when introduced via germinated-seed inoculation as well as leaf infiltration. Comparative proteomic analysis predicted the cellular mechanisms related to PpdaAc. Apart from causing virulence, the PpdaAc may have significant roles in energy production, cell membrane, motility, chemotaxis, post-translational modifications, and iron-related mechanisms. Therefore, it is postulated that PpdaAc may possess pleiotropic effects. These results provide new insights into the functions of a previously unidentified PpdaAc in Ac.     

Power Estimation for Two-Sample Tests Using Importance and Antithetic Resampling

Collings and Hamilton (1988), described a uniform bootstrap method that is applied on observed or pilot data in order to approximate the power of the two-sample Wilcoxon test for location shift alternatives. In this paper we demonstrate how importance and antithetic resampling can be used to substantially reduce the amount of computation needed to approximate the power of the two-sample tests for location shift and scale alternatives. Importance and antithetic bootstrap resampling methods are applied to simulated data of different sample sizes from a variety of distributions as well as to data from the Iowa 65+ Rural Health Study. Also, a suggestion is given for using a combination of importance and antithetic resampling for approximating the power of two-sample tests.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of fertilization and polyspermy in serotonin-spawned eggs of the zebra mussel,Dreissena polymorpha

Eggs isolated from animals spawned with 10⁻³ M serotonin were inseminated with sperm concentrations ranging from 10³–10⁶ sperm/ml. Multiple sperm attached to the surface of the egg and sperm incorporation occurred within 3 min postinsemination (PI). Sperm mitochondria, centrioles, and flagellum were also incorporated. Incorporation was essentially complete by 6 min PI. In the egg cortex, the sperm head rotated 180°, and a rapid translocation of the sperm through the cytoplasm towards the egg interior began by 5–6 min PI. In heavily polyspermic inseminations, translocations of the sperm were either minimal or nonexistent. In monospermic eggs, nuclear decondensation occurred after translocation was complete, beginning by 9–10 min PI. A male pronucleus began to develop in the cytoplasm by 21 min PI and enlarged to 20 μm before fusing with the female pronucleus. Oscillation of the egg cytoplasm and mitotic spindle apparatus was observed immediately prior to cleavage. Cleavage occurred at 60 min PI. Sperm incorporation and pronuclear formation were confirmed with fluorescent and confocal microscopy using the DNA-specific dyes Hoescht 33342 and 7-aminoactinomycin D. In sperm concentrations >10⁴ sperm/ml, 26–76% of the eggs exhibited polyspermy. The high incidence of polyspermy suggests that rapid, effective blocks to polyspermy were not present or were ineffective in a significant proportion of serotonin-spawned eggs. © 1996 Wiley-Liss, Inc.     

Circuit-Specific Coinfection of Neurons in the Rat Central Nervous System with Two Pseudorabies Virus Recombinants

Neurotropic alphaherpesviruses have become popular tools for transynaptic analysis of neural circuitry. It has also been demonstrated that coinfection with two viruses expressing unique reporters can be used to define more complicated circuitry. However, the coinfection studies reported to date have employed nonisogenic strains that differ in their invasive properties. In the present investigation we used two antigenically distinct recombinants of the swine pathogen pseudorabies virus (PRV) in single and double infections of the rat central nervous system. Both viruses are derivatives of PRV-Bartha, a strain with reduced virulence that is widely used for circuit analysis. PRV-BaBlu expresses beta-galactosidase, and PRV-D expresses the PRV membrane protein gI, the gene for which is deleted in PRV-BaBlu. Antibodies to beta-galactosidase identify neurons infected with PRV-BaBlu, and antibodies monospecific for PRV gI identify neurons infected with PRV-D. The ability of these strains to establish coinfections in neurons was evaluated in visual and autonomic circuitry in which the parental virus has previously been characterized. The following conclusions can be drawn from these experiments. First, PRV-D is significantly more neuroinvasive than PRV-Bartha or PRV-BaBlu in the same circuitry. Second, PRV-D is more virulent than either PRV-Bartha or PRV-BaBlu, and PRV-BaBlu is less virulent than PRV-Bartha. Third, in every model examined, PRV-D and PRV-BaBlu coinfect some neurons, but single infections predominate. Fourth, prior infection with one virus renders neurons less permissive to infection by another virus. Fifth, prior infection by PRV-D is more effective than PRV-BaBlu in reducing invasion and spread of the second virus. Collectively, the data define important variables that must be considered in coinfection experiments and suggest that the most successful application of this approach would be accomplished by using isogenic strains of virus with equivalent virulence.     

Construction and Transposon Mutagenesis in Escherichia coli of a Full-Length Infectious Clone of Pseudorabies Virus, an Alphaherpesvirus

A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 in E. coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.     

Detection of Bacteriocins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of bacteriocins was investigated. A 30-s water wash of the sample on the MALDI-TOF MS probe was effective in removing contaminants of the analyte. This method was used for rapid detection of nisin, pediocin, brochocin A and B, and enterocin A and B from culture supernatants and for detection of enterocin B throughout its purification.     

Linkage of a gene causing familial focal segmental glomerulosclerosis to chromosome 11 and further evidence of genetic heterogeneity.

Focal segmental glomerulosclerosis (FSGS) is a pathological entity characterized by proteinuria, nephrotic syndrome, and the progressive loss of renal function. It is a common cause of end-stage renal disease (ESRD). Recently, familial forms of FSGS have been identified. Two families with autosomal dominant FSGS were evaluated for linkage using 351 genomic microsatellite markers. Linkage, multipoint analysis, and tests for heterogeneity were performed on the subsequent results. In addition, three small families were used for haplotype analysis. Evidence for linkage was found on chromosome 11q21-q22 for the largest family, with a maximum lod score of 9.89. The gene is currently localized to an 18-cM area between flanking markers D11S2002 and D11S1986. The disease in a second family was not linked to this locus or to a previously described locus on chromosome 19q13. There were no shared haplotypes among affected individuals in the three smaller families. Our findings demonstrate that genetic heterogeneity is prevalent in FSGS in that at least three genes cause the FSGS phenotype. Identification of the genes that cause familial FSGS will provide valuable insights into the molecular basis and pathophysiology of FSGS.