Pattern of segmental recombination in the distal inversion of mouse t haplotypes

To examine genetic exchange between t haplotypes and their wild-type homologs, four previously identified mouse Chromosome (Chr) 17 variants termed mosaic haplotypes were analyzed in detail. Three of these haplotypes-one from a Mus musculus population in Bulgaria, one from a Mus domesticus population in Chile, and one from a M. domesticus population in Germany-display properties indicative of the t complex. All four haplotypes are exceptional because they are characterized by the presence of a few wild-type DNA markers in the distal inversion [In(17)4] of a t haplotype chromosome: thus, they are classified as mosaic t haplotypes. The mosaic pattern for each haplotype is distinct, however. We compared the mosaic haplotypes with each other, and with several well-characterized laboratory t haplotypes, by analyzing several DNA markers in the In(17)4 region of the t complex, where all of the mosaicism occurs. We used a combination of high-resolution restriction mapping, DNA sequencing, and analysis of new DNA markers to classify the haplotypes. This analysis shows that segmental exchange, either by gene conversion or double crossing-over, has occurred at molecular markers in the vicinity of a gene, Dnahc8, that is a candidate for the t complex distorter locus Tcd2. While it is unclear whether segmental exchanges have included the Tcd2 gene, it is apparent that several independent recombination events have occurred in In(17)4 during the recent evolution of t haplotypes.     

Gravitropic moss cells default to spiral growth on the clinostat and in microgravity during spaceflight

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.     

Uptake of CdSe and CdSe/ZnS quantum dots into bacteria via purine-dependent mechanisms

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.     

Comparison of Campylobacter isolates from poultry and humans: association between in vitro virulence properties, biotypes, and pulsed-field gel electrophoresis clusters

The in vitro virulence properties of 197 temporally and geographically related Campylobacter isolates from chicken broilers and humans were compared. Comparisons of the virulence properties associated with genotypes and biotypes were made. All isolates adhered to, and 63% invaded, INT-407 cells, whereas 13% were cytotoxic for CHO cells. CHO cell-cytotoxic extracts were also cytotoxic for INT-407 cells, but the sensitivity for Vero cells was variable. The proportion of isolates demonstrating a high invasiveness potential (>1,000 CFU ml(-1)) or Vero cell cytotoxicity was significantly higher for human than for poultry isolates. Invasiveness was associated with Campylobacter jejuni isolates of biotypes 1 and 2, whereas CHO and INT-407 cell cytotoxicity was associated with C. jejuni isolates of biotypes 3 and 4. Cytotoxic isolates were also clustered according to pulsed-field gel electrophoresis profiles.     

The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.     

Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation

Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 microm) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations.     

Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine,IL-6

The host response to Salmonella typhimurium involves movement of polymorphonuclear leukocytes (PMN) across the epithelium and into the intestinal lumen. Following their arrival in the lumen, the PMN attempt to combat bacterial infection by activating antimicrobial defenses such as granule release, oxidative burst, phagocytosis, and cell signaling. We sought to examine PMN-S. typhimurium interaction following PMN arrival in the lumenal compartment. Here, for the first time, we demonstrate that PMN that have transmigrated across model intestinal epithelia have an enhanced ability to kill S. typhimurium. Our data provide evidence to indicate that the extracellular release of the primary and secondary granules of PMN, myeloperoxidase and lactoferrin, respectively, is correlated with enhanced bacterial killing. Furthermore, epithelial cells, during PMN transmigration, release the cytokine IL-6. IL-6 is known to increase intracellular stores of Ca(2+), and we have determined that this epithelial released cytokine is not only responsible for priming the PMN to release their granules, but also stimulating the PMN to kill S. typhimurium. These results substantiate the pathway in which PMN transmigration activates the epithelial release of IL-6, which in turn increases intracellular Ca(2+) storage. Our results, herein, extend this pathway to include an enhanced PMN granule release and an enhanced killing of S. typhimurium.     

Enhancers and suppressors of testicular cancer susceptibility in single- and double-mutant mice

Susceptibility to spontaneous testicular germ cell tumors (TGCTs), a common cancer affecting young men, shows unusual genetic complexity. Despite remarkable progress in the genetics analysis of susceptibility to many cancers, TGCT susceptibility genes have not yet been identified. Various mutations that are inherited as Mendelian traits in laboratory mice affect susceptibility to spontaneous TGCTs on the 129/Sv inbred genetic background. We compared the frequency of spontaneous TGCTs in single- and double-mutant mice to identify combinations that show evidence of enhancer or suppressor effects. The lower-than-expected TGCT frequencies in mice with partial deficiencies of TRP53 and MGF-SLJ and in 129.MOLF-Chr19 (M19) consomic mice that were heterozygous for the A(y) mutation suggest that either these genes complement each other to restore normal functionality in TGCT stem cells or together these genes activate mechanisms that suppress incipient TGCTs. By contrast, the higher-than-expected TGCT frequencies in Mgf(Sl-J)-M19 compound heterozygous mice suggest that these mutations exacerbate each other's effects. Together, these results provide clues to the genetic and molecular basis for susceptibility to TGCTs in mice and perhaps in humans.