A new antioxidant with alkyl hydroperoxide defense properties in yeast

To isolate new antioxidant genes, we have searched for activities that would rescue the tert-butyl hydroperoxide (t-BOOH)-hypersensitive phenotype of a Saccharomyces cerevisiae strain deleted for the gene encoding the oxidative stress response regulator Skn7. We report the characterization of AHP1, which encodes a 19-kDa protein similar to the AhpC/TSA protein family within a small region encompassing Cys-62 of Ahp1p and the highly conserved N-terminal catalytic AhpC/TSA cysteine. Ahp1p contains a peroxisomal sorting signal, suggesting a peroxisomal localization. AHP1 exerts strong antioxidant protective functions, as demonstrated both by gene overexpression and deletion analyses, and is inducible by peroxides in an Yap1- and Skn7-dependent manner. Similar to yeast Tsa1p, Ahp1p forms a disulfide-linked homodimer upon oxidation and in vivo requires the presence of the thioredoxin system but not of glutathione to perform its antioxidant protective function. Furthermore, in contrast to Tsa1p, which is specific for H2O2, Ahp1p is specific for organic peroxides. Therefore, with respect to substrate specificity, Ahp1p differs from Tsa1p and is similar to prokaryotic alkyl hydroperoxide reductase AhpC. These data suggest that Ahp1p is a yeast orthologue of prokaryotic AhpC and justifies its name of yeast alkyl hydroperoxide reductase.     

Investigations of cellular defense mechanism against Pseudomonas aeruginosa in mucoviscidosis (author's transl)

In the bronchial secretion of patients with mucoviscidosis Pseudomonas aeruginosa besides Staphylococci can be isolated very often. The part of disturbances in the cellular defense mechanisms was investigated by the stimulation of the nitroblue-tetrazolium-reduction after in vitro-incubation with Pseudomonas aeruginosa. In mucoviscidosis no selective deficiency in the defense of phagocyting cells of the peripheral blood could be found by these examinations, representing only one step of the intracellular destroying of bacteria.     

Crystal structure of a murine alpha-class glutathione S-transferase involved in cellular defense against oxidative stress

STATEMENT OF PROBLEM: The electric alloy brazed joints of removable partial denture alloys have failed frequently after routine usage. PURPOSE: A technique providing higher joint strengths was investigated. This investigation compared the tensile strengths of electric-brazed and laser-welded joints for a cobalt-chromium removable partial denture alloy. MATERIAL AND METHODS: Twenty-four cobalt-chromium standard tensile testing rods were prepared and divided into three groups of eight. All specimens in the control group (group 1) were left in the as-cast condition. Groups 2 and 3 were the test specimens, which were sectioned at the center of the rod. Eight specimens were joined by using electric brazing, and the remaining specimens were joined by using laser welding. After joining, each joint was ground to a uniform diameter, then tested to tensile failure on an Instron universal testing machine. Failure loads were recorded and fracture stress calculated. Statistical analysis was applied. RESULTS: The student-Newman-Keuls test showed a highly significant difference between the joint strengths of the as-cast control specimens, the electric-brazed and laser-welded joints. CONCLUSIONS: The tensile strengths of the as-cast joints were higher than those for the laser-welded joints, and both were higher than the electric-brazed joint strengths.     

A live recombinant avirulent oral Salmonella vaccine expressing pneumococcal surface protein A induces protective responses against Streptococcus pneumoniae

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Deltacya Deltacrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Deltaasd mutation was complemented by a plasmid vector possessing the asd+ gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd+ vector. After oral immunization, the recombinant Salmonella-PspA vaccine strain colonized the Peyer's patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonella strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.     

Leptin is an endogenous protective protein against the toxicity exerted by tumor necrosis factor

Decrements in muscle strength as a result of prolonged bed rest are well defined, but little is known about potential countermeasures for preventing loss of strength under this condition. The purpose of this study was to determine whether testosterone administration would preserve protein balance and muscle strength during prolonged bed rest. Ten healthy men (age, 36 +/- 2 yr; height, 177.2 +/- 3.4 cm; weight, 80.5 +/- 3.9 kg; mean +/- SE) were admitted to our in-patient metabolic unit. After a 1-week ambulatory run-in period, each subject was confined to bed for 28 days at 6 degree head-down tilt while receiving a daily oral dose of T3 (50 microg/day). During the bed rest/T3 period, six of the men were randomized to receive testosterone enanthate by i.m. injection (T; 200 mg/week) while four received placebo in a double blind fashion. Nitrogen balance was determined throughout, and whole body [13C]leucine kinetics were assessed at baseline and on day 26 of bed rest. Before bed rest and on the third day of reambulation, the muscle strength of the knee extensors and flexors and shoulder extensors and flexors was determined at 60 degrees/s on a Cybex isokinetic dynamometer. Despite improved [13C]leucine kinetics and maintenance of nitrogen balance and lean body mass in T-treated subjects, little preservation of muscle strength, particularly in the knee extensors, was noted. Muscle strength [reported as the best work repetition in foot-pounds (FtLb)] for right knee extensors declined (P = 0.011) similarly in both groups; from 165 +/- 15 to 126 +/- 18 FtLb in T-treated men and from 179 +/- 22 to 149 +/- 13 FtLb in placebo-treated men. Overall, there was less of a decline in extension and flexion strength of the shoulder compared to the knee, with no benefit from T. These results suggest that in the absence of daily ambulatory activity, T administration will not increase or, in the case of this bed rest model, preserve muscle strength.     

Lymphocytes in host defense against Pneumocystis carinii

To determine the neuroendocrine (NE) features of thymic epithelial tumor, immunohistochemistry and electron microscopy studies were performed on eight NE tumors (thymic carcinoids) and 26 non-NE tumors (nine thymic carcinomas, five atypical thymomas, and 12 thymomas other than lymphocytic thymoma). Immunohistochemical studies were performed with antibodies against general markers for NE cells (synaptophysin, alpha subunit of a guanine nucleotide-binding protein, Go, and small-cell lung carcinoma cluster 1 antigen), and a broad panel of antibodies for hormonal substances. Thymic carcinoid showed synchronous diffuse immunoreactivity for the three NE markers and contained cells that were positive for a variety of hormonal products: human chorionic gonadotropin (hCG) alpha-subunit (eight of eight), hCG beta-subunit (three of eight), adrenocorticotropic hormone (ACTH) (three of eight), calcitonin (two of eight), calcitonin gene-related peptide (two of eight), and serotonin (one of eight). Conversely, although positivity for NE markers was neither synchronous nor diffuse in non-NE tumors, seven of nine thymic carcinomas, three of five atypical thymomas (focal or dispersed distribution), and none of the five thymomas were positive for at least two of these NE markers. A small number of neoplastic cells were positive for hCGalpha-subunit or ACTH in three thymic carcinomas and one atypical thymoma. Ultrastructurally, dense core granules (DCG) were much more frequent in thymic carcinoid, but several DCG-like granules were identified in 12 of 13 non-NE tumors with or without immunoexpression of NE markers. The presence of focal or dispersed NE cells in thymic carcinoma and atypical thymoma may reflect multidirectional differentiation within the tumor, which, like cytological atypia, epithelial CD5 expression, and lack of immature T cell infiltration, may be another feature of this group at thymic tumors.     

A nitrilase-like protein interacts with GCC box DNA-binding proteins involved in ethylene and defense responses

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.     

Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase

The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.     

A role for interleukin-6 in host defense against murine Chlamydia trachomatis infection

Interleukin-6-deficient (IL-6(-/-)) knockout mice had significantly increased Chlamydia trachomatis levels in lung tissue and increased mortality compared to B6129F2/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6(-/-) mice. IL-6 is needed for an optimal early host response to this infection.     

A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection

To evaluate the role of natural immunoglobulin (Ig)M in the immediate response against microbial infection, we tested mutant mice that are deficient in secreted (s)IgM in an acute peritonitis model induced by cecal ligation and puncture (CLP). 20% of wild-type mice died within 32 h of CLP, whereas 70% of sIgM-deficient mice died within the same time period. The increased susceptibility was associated with a reduced level of tumor necrosis factor (TNF)-alpha, a decreased neutrophil recruitment and an increased bacterial load in the peritoneum, and elevated levels of endotoxin and proinflammatory cytokines in the circulation. Resistance to CLP by sIgM-deficient mice was restored by reconstitution with polyclonal IgM from normal mouse serum. Reconstitution with a monoclonal IgM specific to phosphatidylcholine, a conserved cell membrane component, has a modest effect but a monoclonal IgM specific to phosphocholine is not protective. These findings demonstrate a critical role of natural IgM in the immediate defense against severe bacterial infection.     

A polyclonal antibody against synthetic peptide conserved in N-Myc protein reacts with water-soluble recombinant N-Myc protein

Like in adults, normal values of maximal inspiratory pressure (PImax) and maximal expiratory pressure (PEmax) span a large range in children, making interpretation of low values difficult. Recently, sniff nasal inspiratory pressure (Pnsn) was developed as a new noninvasive test of inspiratory muscle strength. In healthy adults, Pnsn is most often higher than PImax. The aim of this study was to establish reference values of Pnsn in children and to compare them with PImax. A group of 180 unselected healthy children age 6 to 17 yr was studied in a school setting. All had a forced vital capacity (FVC) > 80% of predicted and a ratio of forced expiratory volume in one second/forced vital capacity (FEV1/ FVC) > 85% of predicted. All maneuvers were performed in the sitting position. The Pnsn was measured using a catheter occluding one nostril during maximal sniffs performed through the contralateral nostril from FRC. The PImax was measured from FRC and residual volume, and PEmax from FRC and total lung capacity. All children were able to perform the Pnsn maneuver easily. Pnsn was 104 +/- 26 cm H2O in boys and 93 +/- 23 cm H2O in girls (p < 0.005). These values were similar to those previously measured in healthy adults. Pnsn correlated with age, weight, and height in boys, but not in girls. In both sexes, Pnsn was higher than PImax measured at the same lung volume (FRC) (p < 0. 0001). Pnsn was >= PImaxFRC in 73 of 93 boys and 79 of 87 girls. We conclude that Pnsn can be easily used to assess inspiratory muscle strength in children age 6 yr or more, providing values higher than PImax. Normal values are independent of age in girls, and can be predicted from age by a first-degree equation in boys. Being easy and noninvasive, Pnsn may prove useful to assess inspiratory muscle strength in children with neuromuscular disorders.     

Taurine: protective properties against ethanol-induced hepatic steatosis and lipid peroxidation during chronic ethanol consumption in rats

Alcohol was administered chronically to female Sprague Dawley rats in a nutritionally adequate totally liquid diet for 28 days. This resulted in hepatic steatosis and lipid peroxidation. Taurine, when co-administered with alcohol, reduced the hepatic steatosis and completely prevented lipid peroxidation. The protective properties of taurine in preventing fatty liver were also demonstrated histologically. Although alcohol was found not to affect the urinary excretion of taurine (a non-invasive marker of liver damage), levels of serum and liver taurine were markedly raised in animals receiving alcohol + taurine compared to animals given taurine alone. The ethanol-inducible form of cytochrome P-450 (CYP2E1) was significantly induced by alcohol; the activity was significantly lower than controls and barely detectable in animals fed the liquid alcohol diet containing taurine. In addition, alcohol significantly increased homocysteine excretion into urine throughout the 28 day period of ethanol administration; however, taurine did not prevent this increase. There was evidence of slight cholestasis in animals treated with alcohol and alcohol + taurine, as indicated by raised serum bile acids and alkaline phosphatase (ALP). The protective effects of taurine were attributed to the potential of bile acids, especially taurine conjugated bile acids (taurocholic acid) to inhibit the activity of some microsomal enzymes (CYP2E1). These in vivo findings demonstrate for the first time that hepatic steatosis and lipid peroxidation, occurring as a result of chronic alcohol consumption, can be ameliorated by administration of taurine to rats.     

Vaccination against Haemonchus contortus with denatured forms of the protective antigen H11

As part of a systematic examination of the protective epitopes on H11, groups of sheep were vaccinated with preparations of purified H11 used untreated (group A), or progressively denatured (linearized) by incubation with sodium dodecyl sulphate (SDS) (group B) or by boiling with SDS in the presence of dithiothreitol (group C). All the sheep developed antibodies which bound to the untreated H11. When challenged with 10,000 infective larvae of Haemonchus contortus the mean levels of protection relative to the mean values for adjuvant controls were 99.8%, 85% and 79% for faecal egg counts and 95%, 79% and 54% for worm burden at post-mortem for groups A, B and C respectively. The H11-specific antibodies inhibited the microsomal aminopeptidase activity of H11 in vitro up to 80%. The levels of inhibition by sera from individual animals correlated with levels of protection with r2, of 0.69-0.87.     

Preconditioning with bright light evokes a protective response against light damage in the rat retina

Constant exposure to bright light induces photoreceptor degeneration and at the same time upregulates the expression of several neurotrophic factors in the retina. At issue is whether the induced neurotrophic factors protect photoreceptors. We used a preconditioning paradigm to show that animals preconditioned with bright light became resistant to subsequent light damage. The preconditioning consisted of a 12-48 hr preexposure, followed by a 48 hr "rest phase" of normal cyclic lighting. The greatest protection was achieved by a 12 hr preexposure. Preconditioning induces a prolonged increase in two endogenous neurotrophic factors: basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). It also stimulates the phosphorylation of extracellular signal-regulated protein kinases (Erks) in both photoreceptors and Müller cells. These findings indicate that exposure to bright light initiates two opposing processes: a fast degenerative process that kills photoreceptors and a relatively slower process that leads to the protection of photoreceptors. The extent of light damage, therefore, depends on the interaction of the two processes. These results also suggest a role of endogenous bFGF and CNTF in photoreceptor protection and the importance of Erk activation in photoreceptor survival.     

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection

The effect of the muscarinic antagonist, scopolamine, was examined for a change in the increase in extracellular dopamine, dihydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), induced by haloperidol or clozapine in the striatum and nucleus accumbens of anaesthetised and awake rats, monitored using in vivo cerebral microdialysis. Rats received scopolamine (1 mg kg(-1); s.c.) or vehicle followed by haloperidol (1 mg kg(-1); s.c.) or clozapine (20 mg kg(-1); s.c.). Dopamine, DOPAC, HVA and 5-HIAA overflow into striatal or accumbens perfusates was determined using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Scopolamine failed to modify the clozapine- or haloperidol-induced efflux of dopamine or its metabolites in either the striatum or nucleus accumbens following systemic administration in anaesthetised or awake rats. Although pretreatment with scopolamine tended to produce a smaller increase in the clozapine-induced efflux of DOPAC in striatal perfusates than following clozapine treatment alone, this was not statistically significant. Furthermore, local infusion of scopolamine (100 microM) with clozapine (1 mM) via the microdialysis probe did not attenuate the elevated efflux of dopamine observed following clozapine alone, in either the striatum or nucleus accumbens, in anaesthetised rats. This treatment did prevent the clozapine-induced increase in DOPAC and HVA in the striatum but not the nucleus accumbens. Carbachol (50 microM) infused into the dorsolateral striatum or nucleus accumbens raised extracellular dopamine levels 200% and 150%, respectively above baseline. Our data suggest that the increased efflux of dopamine and its metabolites in the rat basal ganglia following clozapine administration is not significantly dependent upon the interaction of clozapine with muscarinic receptors.     

Epithelial barriers of the intestine. Significance and function in defense against infection

The epithelial surface of the intestinal tract provides for an important barrier between the organism and its environment. However, it permits the passage into the tissues of limited amounts of macromolecules and/or particles, especially bacteria. These functions are age- and species-dependent, and they are closely related to nonspecific and specific immune reactions. Continuous and intimate contact between antigenic material from the gut lumen and immunocompetent cells takes place in gut-associated lymphoid tissues.     

A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid

Degradation is one of several factors that may affect the level of accumulation of transgene products in plants. In plants engineered to secrete antimicrobial proteins to the intercellular compartment of leaves, the degenerative activity of proteases residing in leaf intercellular fluid (IF) could be critical to achieving the expected transgene function. We synthesized a structural analogue (MB39) of the antibacterial protein cecropin B and compared the susceptibility of both proteins to degradation in vitro by IF extracted from leaves of various crops. The half-life of the two proteins in the various IF extracts ranged from 3 min to 25.5 h, with the analogue MB39 displaying the longer half-life in IF from nine of 10 species. Overall, the half-life of MB39 averaged 2.9 times greater than that of cecropin B. Analysis of the peptides produced by endopeptidase activity in potato iF indicated that the 5.7-fold lower degradation rate of MB39 was associated with the substitution of valine for methionine at residue 11 of cecropin B. These findings point to the possibility of tailoring antimicrobial protein genes to reduce the rate of protein degradation in a particular target crop.     

Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice

1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolated perfused rat liver. Livers from fed rats were perfused in an open system with Krebs/Henseleit-bicarbonate buffer (pH 7.4). Glucose release, lactate plus pyruvate production (glycolysis) and oxygen uptake were measured. The following results were obtained: 2. In livers perfused with Ca(2+)-free Krebs/Henseleit-bicarbonate buffer and after depletion of the intracellular pools, the initial and transient stimulation of glycolysis, which is normally observed shortly after the onset of glucagon infusion, was more pronounced when compared to livers perfused with normal perfusion fluid (2.5 mM Ca2+) and without previous depletion of the intracellular pools (controls); the subsequent inhibition of glycolysis was delayed in Ca(2+)-free perfused livers and was less pronounced in comparison with the controls at the end of the glucagon infusion period (20 min). 3. Perfusion with a Ca(2+)-free medium supplemented with EDTA, without previous depletion of the intracellular pools, also produced a substantial reduction in the effects of glucagon on glycolysis. 4. Ca(2+)-free perfusion did not affect the stimulative action of glucagon on glucose release (glycogenolysis) and oxygen uptake. 5. Glycolysis inhibition by cAMP also was abolished in Ca(2+)-free perfused livers, and the initial stimulation was enhanced. 6. Mn2+, a metal ion known as a competitor of Ca2+, considerably reduced the action of glucagon on glycolysis; Mn2+ did not affect the basal rates of glycolysis. 7. Sr2+, a metal ion that is often recognized as Ca2+ by several biological structures and processes, increased the inhibitory action of glucagon on glycolysis. 8. Several organic compounds, which directly or indirectly take part in Ca2+ fluxes, were also able to diminish (e.g., verapamil) or even to abolish (carbenoxolone) the inhibitory action of glucagon on glycolysis. 9. It was concluded that, under the conditions of the living cell, Ca2+ is important for glycolysis inhibition by glucagon. In principle at least, the results can be explained in terms of the known Ca2+ dependencies of several protein kinases and protein phosphatases.