A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

Proinflammatory cytokines in bovine colostrum potentiate the mitogenic response of peripheral blood mononuclear cells from newborn calves through IL-2 and CD25 expression

Abstract: Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist, IL-1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL-2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL-1beta, TNF-alpha or IFN-gamma significantly enhanced the ConA response, whereas IL-1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL-2 mRNA expression in ConA-stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA-stimulated controls, the expression levels became comparable after pretreatment with IL-1beta, TNF-alpha or IFN-gamma. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL-1beta, TNF-beta and IFN-gamma. These results suggest that pretreatment of neonatal PBMC with IL-1beta, TNF-alpha or IFN-gamma promotes mitogenic response to ConA through up-regulating the production of IL-2 and the expression of the mature IL-2 receptor.     

The size of cagA based on repeat sequence has the responsibility of the location of Helicobacter pylori in the gastric mucus and the degree of gastric mucosal inflammation

The aim of this study was to examine whether there is a relationship between cagA size of Japanese Helicobacter pylori strains and the location of these strains in the mucous layer, the degree of gastric inflammation and acid survival. Upper gastrointestinal endoscopies were done to 144 patients with dyspeptic symptom with informed consent, sera, biopsy specimens and H. pylori strains were obtained, and gastric histology and susceptibility to pH 3 of the strains were evaluated. To determine cagA size of Japanese strains using PCR, cagA of strain CPY3401 was sequenced. 74 H. pylori samples (72 cagA+) were obtained from the body and 56 samples (56 cagA +) obtained from the antrum. cagA size of 72 H. pylori strains from the body was mainly classified into 3 groups (short (48), middle (8), long (9), and others (7)) by PCR and all of that of 56 strains from the antrum except 2 was short. The size of cagA of isolated strains from the body is associated with enhanced gastritis, acid survival, and the location in the mucus. The long size cagA of which strain is acid sensitive, may be a strong selective pressure on strain that colonizes close to the host, which enhanced gastritis.     

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti-cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC-3, and DU-145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC-3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC-3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC-3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU-145 cells (0.5% serum). The presence of 10% serum activated Akt in DU-145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC-3 but not in DU-145 cells.     

Survival of nonspecific porin-deficient mutants of Escherichia coli in black sea water

AIMS: The aim of this study was to investigate the links between survival of Escherichia coli in sea water microcosms in the laboratory and the presence of porins in the outer membrane. The E. coli strains studied were a wild-type strain and a series of outer membrane protein (omp) mutants. METHODS AND RESULTS: Bacteria were suspended in natural or filtered-autoclaved sea water microcosms and numbers determined over an incubation period by plate count and by count of cells capable of respiration. CONCLUSIONS: The type of omp mutation has a significant impact in bacterial survival. The double OmpC-OmpF mutant and the OmpR mutant (which was incapable of synthesizing OmpC and OmpF) survived poorly compared with single omp mutants and the wild-type strain. This suggests that these proteins are important in determining the entry of E. coli into the survival mode. The EnvZ mutant, which lacks the protein by which the cell senses some changes in the environment, survived as well as the wild-type strain when compared by plate counts and by respiring cell count. The loss of the EnvZ protein has no effect on survival but it could prevent the organism sensing the changes in the environment through which entry into the survival state is triggered. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is another piece in the puzzle as to how bacteria survive stress conditions.     

Mother–young distance in Japanese Black cattle at pasture

Distance between dam and offspring (1–121 days old) in a herd of Japanese Black cattle (Bos taurus) grazing a tropical grass (Paspalum notatum) pasture (1.5 ha) was investigated during 7-h grazing periods over grazing seasons from May (spring) to October (autumn). The mother–young distance was not constant throughout the grazing period, repeatedly increasing and decreasing. Although significant periodicity was always detected in the mother–young distance, there was no consistent dominant cycle, indicating the complexity of the within-day pattern of mother–young distance. The mean mother–young distance over the grazing period increased as a calf aged, reaching a plateau at an age of about 33 days. The mean distance of a calf from its mother was usually shorter than that from a non-mother cow, with the difference between the mean distances decreasing sharply until a calf became about 35 days old. The results and literature show that mutual independence of mother and young rapidly develops in the first 30–50 days after parturition. Electronic Publication     

Effects of temperature and larval diet on development rates and survival of the dengue vector Aedes aegypti in north Queensland, Australia

Immature development times, survival rates and adult size (wing-lengths) of the mosquito Aedes aegypti (L.) (Diptera: Culicidae) were studied in the laboratory at temperatures of 10-40 degrees C. The duration of development from egg eclosion (hatching of the first instar) to adult was inversely related to temperature, ranging from 7.2 +/- 0.2 days at 35 degrees C to 39.7 +/- 2.3 days at 15 degrees C. The minimum temperature threshold for development (t) was determined as 8.3 +/- 3.6 degrees C and the thermal constant (K) was 181.2 +/- 36.1 day-degrees above the threshold. Maximum survival rates of 88-93% were obtained between 20 and 30 degrees C. Wing-length was inversely related to temperature. The sex ratio (female:male) was 1:1 at all temperatures tested (15, 20, 25 and 35 degrees C) except 30 degrees C (4:3). Under field conditions at Townsville and Charters Towers, north Queensland, the duration of immature development varied according to the container position (i.e. shaded or exposed) and the availability of food resources, as well as inversely with temperature. These data indicate that containers with an abundance of organic matter (e.g. those used for striking plant cuttings) or those amongst foliage or under trees (e.g. discarded plastic tubs and tyres) tended to produce the largest adult Ae. aegypti, which had faster development and better immature survival. As such progeny have been linked to a greater risk of dengue transmission, it would seem important to focus on control of such containers.     

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.     

Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase

Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilić et al. 1998. J. Cell Biol. 143:547–560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK–p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3′-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.