Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis DSM10140(T) and characterization of the enzyme expressed in Escherichia coli

Bifidobacterium lactis is a moderately oxygen-tolerant, saccharolytic bacterium often used in combination with fructooligosaccharides (FOS) as a probiotic supplement in diverse dairy products. This is the first report describing the gene structure and enzymatic properties of a beta-fructofuranosidase [EC 3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 10140(T) and heterologously expressed in Escherichia coli. The G+C content was identical with the G+C content as determined for the total genomic DNA (61.9 mol %). The gene codes for a 532-aa residue polypeptide of 59.4 kDa. Surprisingly, the deduced aa sequence revealed only minor similarity to other fructofuranosidases (18% to E. coli cscA). The enzyme was purified to homogeneity after incorporation of a C-terminal 6 x HIS affinity tag. It hydrolased sucrose, 1-kestose, Raftilose, Actilight, inulin, and raffinose (100%, 91%, 84%, 80%, 37%, 4%). Fructose moieties were released in an exo-type fashion. Substrates with alpha-glycosidic linkages or residues other than fructose were not attacked. The kinetic parameters K(m) and V(max) for sucrose hydrolysis were 10.3 m M and 0.031 microM/min (pH 7.6; 37 degrees C). The activity was abolished by Zn(2+) (1 m M) and significantly inhibited by Fe(2+) and Ni(2+) (10 m M). The enzyme showed its maximal activity at 40 degrees C.     

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens isolated at an Irish poultry processing plant

AIMS: The antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens were determined in order to evaluate the level of antibiotic resistance of Campylobacter species in the Irish poultry industry. METHODS AND RESULTS: Seventy-eight Camp. jejuni and 22 Camp. coli strains were examined for susceptibility to eight antibiotics using the disc diffusion assay. The highest level of resistance of the Camp. jejuni isolates was recorded to ampicillin (35.9%), followed by 20.5% to tetracycline, 20.5% to naladixic acid, 17.9% to ciprofloxacin, 10.2% to erythromycin, 2.5% to streptomycin and 1.2% to kanamycin. Multidrug resistance to two or more antibiotics was seen for 30.7% of Camp. jejuni strains. Resistance of the Camp. coli isolates was shown to ampicillin (9%) and tetracycline (18.2%). CONCLUSIONS: The majority of Camp. jejuni strains were susceptible to antibiotics commonly used for human therapy. Camp. coli strains showed very low resistance levels and were susceptible to six of the eight antimicrobial agents studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Levels of Camp. jejuni and Camp. coli antimicrobial resistance in Irish poultry production was assessed to determine the current situation in Ireland. The prevalence of antibiotic resistance of Campylobacter strains isolated from broiler chickens was low.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

ST7 is a novel low-density lipoprotein receptor-related protein (LRP) with a cytoplasmic tail that interacts with proteins related to signal transduction pathways

In 1997, McCormick and co-workers identified a novel putative tumor suppressor gene, designated ST7, encoding a unique protein with transmembrane receptor characteristics [Qing et al. (1999) Oncogene 18, 335-342]. Using degenerate primers corresponding to the highly conserved region of the ligand-binding domains of members of the low-density lipoprotein receptor (LDLR) superfamily, Ishii et al. [Genomics (1998) 51, 132-135] discovered a low-density lipoprotein receptor-related protein (LRP) that closely resembles ST7. Later, another LRP closely resembling ST7 and LRP3 was found (murine LRP9) [Sugiyama et al. (2000) Biochemistry 39, 15817-15825]. These results strongly suggested that ST7 was also a novel member of the low-density lipoprotein receptor superfamily. Proteins of this superfamily have been shown to function in endocytosis and/or signal transduction. To evaluate the relationship of ST7 to the LDLR superfamily proteins and to determine whether ST7 may function in endocytosis and/or signal transduction, we used proteomic tools to analyze the functional motifs present in the protein. Our results indicate that ST7 is a member of a subfamily of the LDLR superfamily and that its cytoplasmic domain contains several motifs implicated in endocytosis and signal transduction. Use of the yeast two-hybrid system to identify proteins that associate with ST7's cytoplasmic domain revealed that this domain interacts with three proteins involved in signal transduction and/or endocytosis, viz., receptor for activated protein C kinase 1 (RACK1), muscle integrin binding protein (MIBP), and SMAD anchor for receptor activation (SARA), suggesting that ST7, like other proteins in the LDLR superfamily, functions in these two pathways. Clearly, ST7 is an LRP, and therefore, it should now be referred to as LRP12.     

Effects of resistance versus aerobic training on coronary artery disease risk factors

Individuals exhibiting "the metabolic syndrome" have multiple coronary artery disease risk factors, including insulin resistance, hyperlipidemia, hypertension, and android obesity. We performed a randomized trial to compare the effects of aerobic and resistance training regimens on coronary risk factors. Twenty-six volunteers who exhibited android obesity and at least one other risk factor for coronary artery disease were randomized to aerobic or resistance training groups. Body mass index, waist-to-hip ratio, glucose, insulin, body composition, 24-hr urinary albumin, fibrinogen, blood pressure, and lipid profile were measured at baseline and after 10 weeks of exercise training. Both groups showed a significant reduction in waist-to-hip ratio and the resistance training group also showed a reduction in total body fat. There was no significant change in mean arterial blood pressure in either group. Fasting plasma glucose, insulin, total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides were unchanged in both groups. High-density lipoprotein (HDL) cholesterol increased (13%) with aerobic training only. Plasma fibrinogen was increased (28% and 34%, P < 0.02) in both groups and both groups showed a significant decrease (34% and 28%, P < 0.03) in microalbuminuria after their respective training regimen. In conclusion, resistance training was effective in improving body composition of middle-aged obese sedentary males. Only aerobic training was effective in raising HDL cholesterol. More studies are warranted to assess the effects of exercise on plasma fibrinogen and microalbuminuria.     

hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (±)-7β,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.     

The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.     

Tyrphostins protect neuronal cells from oxidative stress

Tyrphostins are a family of tyrosine kinase inhibitors originally synthesized as potential anticarcinogenic compounds. Because tyrphostins have chemical structures similar to those of the phenolic antioxidants, we decided to test the protective efficacy of tyrphostins against oxidative stress-induced nerve cell death (oxytosis). Many commercially available tyrphostins, at concentrations ranging from 0.5 to 200 microm, protect both HT-22 hippocampal cells and rat primary neurons from oxytosis brought about by treatment with glutamate, as well as by treatment with homocysteic acid and buthionine sulfoximine. The tyrphostins protect nerve cells by three distinct mechanisms. Some tyrphostins, such as A25, act as antioxidants and eliminate the reactive oxygen species that accumulate as a result of glutamate treatment. These tyrphostins also protect cells from hydrogen peroxide and act as antioxidants in an in vitro assay. In contrast, tyrphostins A9 and AG126 act as mitochondrial uncouplers, collapsing the mitochondrial membrane potential and thereby reducing the generation of reactive oxygen species from mitochondria during glutamate toxicity. Finally, the third group of tyrphostins does not appear to be effective as antioxidants but rather protects cells by increasing the basal level of cellular glutathione. Therefore, the effects of tyrphostins on cells are not limited to their ability to inhibit tyrosine kinases.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.