Occurrence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals

Salmonella pathogenesis is a complex phenomenon and a Type III secretion system plays a central role in the development of Salmonella-induced enteritis. One such Type III secretion protein is Salmonella outer protein E (SopE). Prevalence of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enterica isolated from man and animals were investigated. Of 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE, 130 strains belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry sopE gene irrespective of their source of isolation when tested by PCR amplification technique using its specific primers. Of these 130 strains, 112 strains were found to express SopE protein phenotypically as detected by Dot-ELISA using SopE antibody. Among the different serovars tested only serovars Gallinarum, Enteritidis and Virchow expressed SopE protein phenotypically in vitro. Role of SopE protein in pathogenesis of salmonellosis has been discussed.     

Genotype-phenotype associations in Sotos syndrome: an analysis of 266 individuals with NSD1 aberrations

We identified 266 individuals with intragenic NSD1 mutations or 5q35 microdeletions encompassing NSD1 (referred to as "NSD1-positive individuals"), through analyses of 530 subjects with diverse phenotypes. Truncating NSD1 mutations occurred throughout the gene, but pathogenic missense mutations occurred only in functional domains (P < 2 x 10(-16)). Sotos syndrome was clinically diagnosed in 99% of NSD1-positive individuals, independent of the molecular analyses, indicating that NSD1 aberrations are essentially specific to this condition. Furthermore, our data suggest that 93% of patients who have been clinically diagnosed with Sotos syndrome have identifiable NSD1 abnormalities, of which 83% are intragenic mutations and 10% are 5q35 microdeletions. We reviewed the clinical phenotypes of 239 NSD1-positive individuals. Facial dysmorphism, learning disability, and childhood overgrowth were present in 90% of the individuals. However, both the height and head circumference of 10% of the individuals were within the normal range, indicating that overgrowth is not obligatory for the diagnosis of Sotos syndrome. A broad spectrum of associated clinical features was also present, the occurrence of which was largely independent of genotype, since individuals with identical mutations had different phenotypes. We compared the phenotypes of patients with intragenic NSD1 mutations with those of patients with 5q35 microdeletions. Patients with microdeletions had less-prominent overgrowth (P = .0003) and more-severe learning disability (P = 3 x 10(-9)) than patients with mutations. However, all features present in patients with microdeletions were also observed in patients with mutations, and there was no correlation between deletion size and the clinical phenotype, suggesting that the deletion of additional genes in patients with 5q35 microdeletions has little specific effect on phenotype. We identified only 13 familial cases. The reasons for the low vertical transmission rate are unclear, although familial cases were more likely than nonfamilial cases (P = .005) to carry missense mutations, suggesting that the underlying NSD1 mutational mechanism in Sotos syndrome may influence reproductive fitness.     

Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling

The forward and antegrade interactions that comprise the agonist receptor-G protein complex were studied in Chinese hamster fibroblasts transfected to express the beta(2)-adrenergic receptor (beta(2)AR), the beta(2)AR and the alpha-subunit of its cognate G protein (G(s)), and a protein consisting of the beta(2)AR fused at its carboxy terminus with G(alpha)(s) (beta(2)AR-G(s)). Expression levels were matched at approximately 600 fmol/mg. Basal adenylyl cyclase activities were increased with the fusion receptor membranes compared to coexpressed receptor plus G(alpha)(s), and to wild-type beta(2)AR (20.5 +/- 1.8 vs 9.0 +/- 0.88 vs 8.7 +/- 0.93 pmol min(-)(1) mg(-)(1)), confirming in mammalian cells that the fusion of beta(2)AR and G(alpha)(s) results in a state not attained by expression of unfused components. However, agonist-stimulated activities were not increased proportionally, such that the stimulation over basal of the beta(2)AR-G(s) fusion protein (1. 5-fold) was less than wild-type beta(2)AR (2.1-fold). Agonist competition studies performed in the absence of guanine nucleotide exhibited high-affinity binding sites with a lower K(H) (1.75 vs 8. 47 nM) and greater %R(H) (51% vs 44%) for beta(2)AR-G(s), but GppNHp failed to convert most of these to the low-affinity state. Functional studies with the inverse agonist ICI 118551 did not show enhanced efficacy or potency with the fusion protein. Adenylyl cyclase studies with three partial agonists with diverse structures (dobutamine, ritodrine, and phenylephrine) showed no enhancement of efficacy with beta(2)AR-G(s) and a minor trend toward enhanced potency. Taken together, these results indicate that the tethering of G(alpha)(s) to the beta(2)AR causes a conformational change in the receptor that stabilizes a species "trapped" between the non-guanine nucleotide-bound state and the GTP-bound form. Functionally the receptor is not characterized by a consistent pattern of properties ascribed to other states such as constitutive activation or precoupling, but rather represents a unique state in the transition from high- to low-affinity forms.     

Copy Number Related Transgene Expression and Mosaic Somatic Expression in Hemizygous and Homozygous Transgenic Tilapia (Oreochromis Niloticus)

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7kb 5 regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these three lines which differed between linesbut were consistent within a line. We also observedthat expression of the reporter gene in homozygoustransgenic fish was approximately two-fold greater thanin the hemizygous transgenics. Analysis of expressionof the reporter gene on a tissue-to-tissue basisdemonstrated that lacZ expression of thereporter gene in stably transformed fish occured withvariable intensity in different organs and tissues andwas also sometimes variable in different cells of thesame tissue in G1and G2 generations of the transgenic lines.     

Roles of the intracellular regions of angiotensin II receptor AT2 in mediating reduction of intracellular cGMP levels

We have shown previously that the angiotensin II (Ang II) receptor AT2 reduces the intracellular levels of cGMP in Xenopus oocytes when activated by ligand binding, and the C-terminal cytoplasmic tail of the AT2 acts as a negative regulator of this function. Here we report the effects of mutations in the 2nd and 3rd intracellular loops of AT2 on AT2-mediated cGMP reduction. Mutating the highly conserved DRY motif (D141G-R142G-Y143A) of the 2nd ICL implicated in activating G(alpha) subunit of trimeric G-proteins did not affect AT2-mediated cGMP reduction. Moreover, anti-Gialpha antibody or phosphodiesterase inhibitor IBMX did not inhibit AT2-mediated cGMP reduction, suggesting that Gialpha activation and subsequent phosphodiesterase activation are not involved in this function. In contrast, mutations T250R-R251N and L255F-K256R located in the C-terminus of the 3rd ICL of AT2 retained ligand-binding properties of the wild-type AT2, and its ability to interact with the ErbB3 in yeast two-hybrid assay, but abolished AT2-mediated cGMP reduction. Similarities in the roles of ICLs of AT2 in AT2-mediated cGMP reduction in oocytes, and AT2-mediated SHP1 activation in COS-7 cells, (need of 3rd ICL for both functions and lack of involvement of DRY motif), suggest that the cascade of events in these two signaling mechanisms could be similar, and that an oocyte-specific SHP1-like protein may be involved in AT2-mediated cGMP reduction in these cells.     

Mechanisms predisposing to childhood overgrowth and cancer

Several overgrowth conditions are believed to be associated with elevated risks of cancer, particularly in childhood. Beckwith-Wiedemann syndrome and Sotos syndrome are the most common overgrowth conditions, and both carry increased risks of certain tumors. In recent years, the identification of both the gene causing Sotos syndrome and the epigenetic subgroups underlying Beckwith-Wiedemann syndrome have enabled clarification of the cancer types and risks associated with these conditions. This has revealed striking differences in the cancer phenotypes associated with different molecular abnormalities. Elucidation of the mechanisms underlying cancer in overgrowth syndromes might yield important insights into the molecular basis of childhood tumors.     

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid populations growth. Artificial diets were used to provide active (1, 10, 100 and 500 g ml^–1) and inactive (500 g ml^–1) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day populations doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.     

Cytotoxic and genotoxic effects of the azo-dye p-dimethylaminoazobenzene in mice: a time-course study

The cytotoxic and genotoxic effects of chronic feeding of the azo-dye p-dimethylaminoazobenzene (p-DAB) during 7, 15, 30, 60, 90 and 120 days have been assessed in mice. The endpoints used for genotoxic analysis were chromosome aberrations (CA), micronuclei (MN) and mitotic index (MI) in bone-marrow cells, and sperm-head abnormality (SHA) in male gonads. The activities of marker enzymes for toxicity, such as glutamate oxalo-acetate transaminase (GOT), glutamate pyruvate transaminase (GPT), acid phosphatase (ACP) and alkaline phosphatase (ALKP) were also assayed periodically, as was lipid peroxidation (LPO). Chronic feeding of p-DAB produced increased numbers of chromosome aberrations, nuclear anomalies and sperm-head abnormalities, as compared with normal untreated controls, generally in a time-dependent manner until 60 days, after which the anomalies persisted, but rather erratically. However, although there was some noticeable modulation in enzyme activities in the corresponding p-DAB-fed mice as well, these were not strictly time-dependent.     

Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions

To shed new light on gene involvement in plant cuticular-wax production, 11 eceriferum (cer) mutants of Arabidopsis having dramatic alterations in wax composition of inflorescence stems were used to create 14 double cer mutants each with two homozygous recessive cer loci. A comprehensive analysis of stem waxes on these double mutants revealed unexpected CER gene interactions and new ideas about individual CER gene functions. Five of the 14 double cer mutants produced significantly more total wax than one of their respective cer parents, indicating from a genetic standpoint a partial bypassing (or complementation) of one cer mutation by the other. Eight of the 14 double cer mutants had alkane amounts lower than both respective cer parents, suggesting that most of these CER gene products play a major additive role in alkane synthesis. Other results suggested that some CER genes function in more than one step of the wax pathway, including those associated with sequential steps in acyl-CoA elongation. Surprisingly, complete epistasis was not observed for any of the cer gene combinations tested. Significant overlap or redundancy of genetic operations thus appears to be a central feature of wax metabolism. Future studies of CER gene product function, as well as the utilization of CER genes for crop improvement, must now account for the complex gene interactions described here.