2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

Background  

Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.     

Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

Background  

The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors.     

p27Kip1 and p130 cooperate to regulate hematopoietic cell proliferation in vivo

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.     

Temperature-dependent kinetic variation among phosphoglucose isomerase allozymes from the wing-polymorphic water strider, Limnoporus canaliculatus

Phosphoglucose isomerase (PGI) allozymes were isolated from the wing- polymorphic water strider, Limnoporus canaliculatus, and were characterized biochemically with respect to temperature-dependent kinetic and thermostability properties. At higher temperatures, the allozymes exhibited significant differences in Michaelis constant (Km) values for substrates of both the forward and reverse reaction directions. Results were consistent with expectations of adaptive kinetic differentiation based on the latitudinal variation of PGI allele frequencies. PGI genotypes also differed with regard to maximal velocity (Vmax)/Km ratios at higher temperatures. These differences were due primarily, if not exclusively, to allozyme-dependent variation in Km values. The allozymes also exhibited dramatic differences in thermostability. However, no thermostability differences were observed when the substrate analogue 6-phosphogluconate was present in the incubation medium. The data from this study, together with data from Mytilus edulis and Metridium senile on temperature-dependent kinetic variation among PGI allozymes, form a consistent picture of natural selection influencing the clinal variation of alleles at this locus in these three phylogenetically distant organisms. More definitive support of this hypothesis, however, must await additional studies on the physiological effects of the allozymic variation as well as direct measurements of fitness differences among the enzyme genotypes.      

Structural conservation and variation in the mitochondrial control region of fringilline finches (Fringilla spp.) and the greenfinch (Carduelis chloris)

We sequenced the entire control region and portions of flanking genes (tRNA(Phe), tRNA(Glu), and ND6) in the common chaffinch (Fringilla coelebs), blue chaffinch (F. teydea), brambling (F. montifringilla), and greenfinch (Carduelis chloris). In these finches the control region is similar in length (1,223-1,237 bp) and has the same flanking gene order as in other birds, and contains a putative TAS element and the highly conserved CSB-1 and F, D, and C boxes recognizable in most vertebrates. Cloverleaf-like structures associated with the TAS element at the 5' end and CSB-1 at the 3' end of the control region may be involved with the stop and start of D-loop synthesis, respectively. The pattern of nucleotide and substitution bias is similar to that in other vertebrates, and consequently the finch control region can be subdivided into a central, conserved G-rich domain (domain II) flanked by hypervariable 5'-C-rich (domain I) and 3'-AT-rich (domain III) segments. In pairwise comparisons among finch species, the central domain has unusually low transition/transversion ratios, which suggests that increased G + T content is a functional constraint, possibly for DNA primase efficiency. In finches the relative rates of evolution vary among domains according to a ratio of 4.2 (domain III) to 2.2 (domain I) to 1 (domain II), and extensively among sites within domains I and II. Domain I and III sequences are extremely useful in recovering intraspecific phylogeographic splits between populations in Africa and Europe, Madeira, and a basal lineage in Nefza, Tunisia. Domain II sequences are highly conserved, and are therefore only useful in conjunction with sequences from domains I and III in phylogenetic studies of closely related species.      

Relocation of a Ca2+-dependent protein kinase activity during pollen tube reorientation

Pollen tube reorientation is a dynamic cellular event that is crucial for successful fertilization. We have shown previously that pollen tube orientation is regulated by cytosolic free calcium ([Ca2+]c). In this paper, we studied the activity of a Ca2+-dependent protein kinase during reorientation. The kinase activity was assayed in living cells by using confocal ratio imaging of BODIPY FL bisindolylmaleimide. We found that growing pollen tubes exhibited higher protein kinase activity in the apical region, whereas nongrowing cells showed uniform distribution. Modification of growth direction by diffusion of inhibitors/activators from a micropipette showed the spatial redistribution of kinase activity to predict the new growth orientation. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ that led to reorientation also increased kinase activity. Molecular and immunological assays suggest that this kinase may show some functional homology with protein kinase C. We suggest that the tip-localized gradient of kinase activity promotes Ca2+-mediated exocytosis and may act to regulate Ca2+ channel activity.     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.      

Evolutionary history of the COII/tRNALys intergenic 9 base pair deletion in human mitochondrial DNAs from the Pacific

Length changes in human mitochondrial DNA (mtDNA) are potentially useful markers for inferring the evolutionary history of populations. One such length change is a nine base pair (9-bp) deletion that is located in the intergenic region between the COII gene and the Lysine tRNA gene (COII/tRNALys intergenic region). This deletion has been used as a genetic marker to trace descent from peoples of East Asian origin. A geographic cline of the deletion frequency across modern Pacific Islander populations suggests that the deletion may be useful for tracing prehistoric Polynesian origins and affinities. Mitochondrial DNA sequence variation within two variable segments of the control region (CR) permits a number of inferences regarding the evolutionary history of the 9-bp deletion that cannot be determined from frequency data alone. We obtained CR sequences from 74 mtDNAs with the 9-bp deletion from Indonesia, coastal Papua New Guinea (PNG), and American Samoa. Phylogenetic and pairwise distribution analysis of these CR sequences pooled with previously published CR sequences reveals that the deletion arose independently in Africa and Asia and suggests possible multiple origins of the deletion in Asia. A clinal increase of the frequency of the 9-bp deletion across the three Pacific populations is associated with a decrease in CR sequence diversity, consistent with founder events. Furthermore, analysis of pairwise difference distributions indicates an expansion time of proto-Polynesians that began 5,500 yr ago from Southeast Asia. These results are consistent with the express train model of Polynesian origins.