Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.     

Biochemical characterization of CD39L4

Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.     

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient.Bacteria capable of mineralizing 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide, are found in many different phylogenetic groups (2, 3, 7, 11, 22, 23). Evidence suggests that numerous variants of 2,4-D catabolic genes exist and that catabolic operons consist of a near-random mixing of these variants (7). Interspecies gene transfer is a well-documented phenomenon (13), and horizontal gene transfer of the 2,4-D-degrading plasmid pJP4 has been shown (3, 5). However, not all 2,4-D catabolic operons are found on plasmids (10, 11, 16, 20). The extent to which other 2,4-D genes have been exchanged in nature is unknown. The aim of this research was to assess the role of horizontal gene transfer in the evolution of 2,4-D-degrading strains. This article summarizes the results of two aspects of this work—the study of the transfer of the entire 2,4-D pathway by using standard mating experiments and a phylogenetic study of the tfdA gene. The tfdA gene codes for an α-ketoglutarate-dependent 2,4-D dioxygenase which converts 2,4-D into 2,4-dichlorophenol and glyoxylate (6). This 861-bp gene was first sequenced from Ralstonia eutropha JMP134 (19). Two more tfdA genes were cloned from chromosomal locations in Burkholderia strain RASC and Burkholderia strain TFD6 (16, 20). These proved to be identical to each other and 78.5% similar to the original. An alignment of the two variants allowed conserved areas to be identified and primers to be designed for the amplification of tfdA-like genes from other sources (24). Sequence analysis of putative tfdA fragments and the small-subunit ribosomal DNA (SSU rDNA) of the strains carrying them allowed us to construct phylogenies of the genes and their hosts and to look for congruency between them.

Mating experiments.

A collection of 2,4-D degraders containing 15 unique strains as determined by genomic fingerprinting (7) was used as a source of donors in a series of mating experiments (Table ​(Table1).1). Burkholderia cepacia D5, lacking the ability to grow on 2,4-D and not hybridizing to any tfd genes, was used as a recipient in mating experiments. Strain D5 contains neomycin phosphotransferase genes (nptII) carried on transposon Tn5 and is resistant to 50 μg each of kanamycin, carbenicillin, and bacitracin per ml. All of the 2,4-D strains used were sensitive to these antibiotics. Filter matings were performed with a donor-to-recipient ratio of 1:10. Colonies which grew on selective medium (500 ppm of 2,4-D in mineral salts agar [MMO] [23] including 50 μg of kanamycin, carbenicillin, and bacitracin per ml) were subjected to further tests. Their ability to catabolize 2,4-D was tested in liquid medium (same composition as that described above).

TABLE 1

2,4-D-degrading strains, geographic origins, and GenBank accession numbers

Acid-induced expression of an LPS-associated gene in Helicobacter pylori

To investigate urease-independent mechanisms by which Helicobacter pylori resists acid stress, subtractive RNA hybridization was used to identify H. pylori genes whose expression is induced after exposure to acid pH. This approach led to the isolation of a gene that encoded a predicted 34.8 kDa protein (WbcJ), which was homologous to known bacterial O-antigen biosynthesis proteins involved in the conversion of GDP-mannose to GDP-fucose. An isogenic wbcJ null mutant strain failed to express O-antigen and Lewis X or Lewis Y determinants and was more sensitive to acid stress than was the wild-type strain. Qualitative differences in LPS profiles were observed in H. pylori cells grown at pH 5 compared with pH 7, which suggests that H. pylori may alter its LPS structure in response to acidic pH. This may be an important adaptation facilitating H. pylori colonization of the acidic gastric environment.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.     

Bull selection and use in northern Australia. 4. Calf output and predictors of fertility of bulls in multiple-sire herds

On 10 northern Australian properties, the number of calves sired (calf output) by individual bulls in multiple-sire matings was measured by DNA typing for paternity. There were 235 bulls (92 Santa Gertrudis, 25 5/8 Brahman and 119 Brahman) from 37 multiple-sire mating groups. Number of bulls in groups ranged from 2 to 25 and ages of bulls ranged from 2 to 5 years. Mating periods were for 3-12 months and bull mating percentages were 2.5-6%. In all, there were 4251 calves tested and the resolution of paternity ranged from 92.5 to 100% and averaged 97.7% across all sites. This included 9.9% of calves with no potential sires in any of the mating groups. Of the 235 bulls mated, 58% sired 10% or less calves in each of their respective mating groups with 6% not siring any calves. In contrast, 14% sired over 30% of the calves in each of the respective mating groups. When bulls were mated in groups of 8-24, the maximum percent of calves sired by individual bulls was 26+/-7% (mean+/-S.D.) with a range 11-36%. However, when bulls were mated in groups of 2-7, the maximum percent of calves sired by individual bulls was 59+/-19% with a range 24-94%. Calf output of bulls was moderately repeatable across years at four of five sites. Multiple regression models relating pre-mating measures of physical, seminal and behavioural traits to calf output were developed for the three breed groups. In all, only 138 of the 235 bulls were included in the models (40 Santa Gertrudis, 24 5/8 Brahman and 74 Brahman). Sheath and testicular traits, such as scrotal circumference and testicular tone, were generally not related to calf output, the exceptions being sheath depth in Brahman bulls which was negatively related (P<0.05) and scrotal circumference in 5/8 Brahmans which was positively related (P=0.08) to calf output. Dominance was only included in the 5/8 Brahman model but there was no significant relationship between dominance hierarchy and calf output. Semen motility was only related (P<0.05) to calf output in 5/8 Brahmans. However, measures of semen quality based on spermatozoa morphology were important contributors to calf output in the Santa Gertrudis and Brahman models where percent normal spermatozoa was positively related (P<0.01) to calf output. In Santa Gertrudis and Brahman bulls, measures of sexual behaviour in the serving capacity test were related to calf output. In Santa Gertrudis, these were for the number of displays of sexual interest (P<0.05), and mounts (P<0.01), but not number of serves, whilst in Brahman bulls, libido score was positively related to calf output (P<0.05). The models only explained 35-57% of the variation in calf output.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.