Antimicrobial susceptibilities of Coagulase-Negative Staphylococci (CNS) and Streptococci from bovine subclinical mastitis cases

The prevalence and antimicrobial susceptibilities of Staphylococci and Streptococci were assessed from subclinical mastitis cases. One hundred Coagulase-Negative Staphylococci (CNS) and 34 Streptoccocci were identified. The most frequently isolated species were Staphylococcus haemolyticus (27%) and Staphylococcus simulans (24%). Susceptible CNS species revealed the highest resistance to penicillin G (58%), ampicillin (48%), neomycin (20%), and oleandomycin (14%). CNS methicillin resistance rates within 82 isolates were 21.95% and 1.22% by disk diffusion and PCR methods, respectively. These results suggested the disk diffusion method was more prone to yield false positives. Partial sequencing of the 16S rRNA region from the mecA carrying isolate (S. haemolyticus) was homologous with S. haemolyticus sequences/accessions obtained from GenBank. However, the mecA gene sequence from this isolate was more closely allied with the S. aureus mecA gene of human origins. Identical sequence data was acquired from the National Center for Biotechnology Information (NCBI) database, suggesting horizontal gene transfer between the two species. CNS β-lactamase activity within 81 isolates was 29.63%. The most frequently isolated Streptococcus species were S. uberis (52%) and S. agalactiae (15%). Oleandomycin was the least effective antimicrobial agent on these isolates with 59% susceptibility. Results indicated that CNS and Streptococci exhibited various antimicrobial resistance responses. Consequently, isolation and identification of udder pathogens in herds suffering from subclinical agents is essential to select the most effective antimicrobial agent. Moreover, multiple resistance features of methicillin resistant (MR) isolates should be considered during antimicrobial susceptibility tests.     

Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities

Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O(2)(-)) or hydrogen peroxide (H(2)O(2)). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes.     

Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells

Up-regulation of the antioxidant system provides protection against NaCl-induced oxidative damage in plants. Antioxidants and activity of enzymes involved in the ascorbate-glutathione (ASC-GSH) cycle in tobacco Bright Yellow-2 (BY-2) were investigated to assess the antioxidant protection offered by exogenous proline and glycinebetaine (betaine from now on) against salt stress using cells grown in suspension culture. Reduced ascorbate (ASC) was detected in BY-2 cells but dehydroascorbate (DHA) was not. Large quantities of a reduced form of glutathione (GSH) and smaller quantities of an oxidized form of glutathione (GSSG) were detected in BY-2 cells. Salt stress significantly reduced the contents of ASC and GSH as well as activities of ASC-GSH cycle enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Exogenous proline or betaine increased the activities of all enzymes except MDHAR involved in NaCl-induced ASC-GSH cycle. Levels of ASC and GSH in BY-2 cells under salt stress were lower in the presence of proline or betaine than in the absence of proline or betaine whereas there was no difference in redox status. Proline proved more effective than betaine in maintaining the activity of enzymes involved in NaCl-induced ASC-GSH cycle. Neither proline nor betaine had any direct protective effect on NaCl-induced enzyme activity involved in the antioxidant system; however, both improved salt tolerance by increasing enzyme activity. The present study, together with our earlier findings [Hoque MA, Okuma E, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y. Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. J Plant Physiol 2006;164:553-61.], suggests that proline offered greater protection against salt stress than betaine did because proline was more effective in increasing the activity of enzymes involved in the antioxidant system.     

Opposite regulation of endothelial NO synthase by HSP90 and caveolin in liver and lungs of rats with hepatopulmonary syndrome

The hepatopulmonary syndrome is a complication of cirrhosis that associates an overproduction of nitric oxide (NO) in lungs and a NO defect in the liver. Because endothelial NO synthase (eNOS) is regulated by caveolin that decreases and heat shock protein 90 (HSP90) that increases NO production, we hypothesized that an opposite regulation of eNOS by caveolin and HSP90 might explain the opposite NO production in both organs. Cirrhosis was induced by a chronic bile duct ligation (CBDL) performed 15, 30, and 60 days before sample collection and pharmacological tests. eNOS, caveolin, and HSP90 expression were measured in hepatic and lung tissues. Pharmacological tests to assess NO released by shear stress and by acetylcholine were performed in livers (n = 28) and lungs (n = 28) isolated from normal and CBDL rats. In lungs from CBDL rats, indirect evidence of high NO production induced by shear stress was associated with a high binding of HSP90 and a low binding of caveolin to eNOS. Opposite results were observed in livers from CBDL rats. Our study shows an opposite posttranslational regulation of eNOS by HSP90 and caveolin in lungs and liver from rats with CBDL. Such opposite posttranslational regulation of eNOS by regulatory proteins may explain in part the pulmonary overproduction of NO and the hepatic NO defect in rats with hepatopulmonary syndrome.     

Conjugated linoleic acid protects against age-associated bone loss in C57BL/6 female mice

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age-related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Hence, this study was undertaken to examine the effect of CLA on bone mineral density (BMD) in middle-aged C57BL/6 female mice. After 10 weeks on diet, CLA-fed mice (14 months) maintained a higher BMD in different bone regions than corn oil (CO)-fed mice. The increased BMD was accompanied by a decreased activity of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and the receptor activator of NF-kappaB ligand) and decreased osteoclast function. Furthermore, a significant decrease in fat mass and an increase in muscle mass were also observed in CLA-fed mice compared to CO-fed mice. In conclusion, these findings suggest that CLA may prevent the loss of bone and muscle mass by modulating markers of inflammation and osteoclastogenic factors.     

Purification and some properties of galectin-1 derived from water buffalo (Bubalus bubalis) brain

An increasing number of galectins have been found in various animal species, the most abundant of which is galectin-1. The purpose of the present study was to purify and characterize galectin-1 from buffalo brain. We purified the galectin using a combination of ammonium sulphate fractionation and affinity chromatography and the homogeneity was determined by both native polyacrylamide gel electrophoresis (PAGE) and denaturing SDS-PAGE. The molecular weight of the galectin as determined by SDS-PAGE under reducing conditions and by gel filtration column under native conditions was 13.8 and 24.5 kDa, respectively, suggesting a dimeric form of galectin. The most potent inhibitor of the galectin activity was lactose, giving complete inhibition of hemagglutination at 0.8 mM. Galectin showed higher specificity towards human blood group A. Free thiol groups were estimated at a molar ratio of 2.9. The effects of alkylating reagents (iodoacetate and iodoacetamide) on saccharide binding of the galectin were studied. Both alkylating reagents significantly inactivated the activity of the galectin within 20 min. The temperature and pH stability of the galectin were determined. Our findings based on physico-chemical properties, carbohydrate and blood group specificities of the galectin may have future implications in biological and clinical applications.     

A recombinant allosteric lectin antagonist of HIV-1 envelope gp120 interactions

The first, critical stage of HIV-1 infection is fusion of viral and host cellular membranes initiated by a viral envelope glycoprotein gp120. We evaluated the potential to form a chimeric protein entry inhibitor that combines the action of two gp120-targeting molecules, an allosteric peptide inhibitor 12p1 and a higher affinity carbohydrate-binding protein cyanovirin (CVN). In initial mixing experiments, we demonstrated that the inhibitors do not interfere with each other and instead show functional synergy in inhibiting viral cell infection. Based on this, we created a chimera, termed L5, with 12p1 fused to the C-terminal domain of CVN through a linker of five penta-peptide repeats. L5 revealed the same broad specificity as CVN for gp120 from a variety of clades and tropisms. By comparison to CVN, the L5 chimera exhibited substantially increased inhibition of gp120 binding to receptor CD4, coreceptor surrogate mAb 17b and gp120 antibody F105. These binding inhibition effects by the chimera reflected both the high affinity of the CVN domain and the allosteric action of the 12p1 domain. The results open up the possibility to form high potency chimeras, as well as noncovalent mixtures, as leads for HIV-1 envelope antagonism that can overcome potency limits and potential virus mutational resistance for either 12p1 or CVN alone.     

Temporal transmission studies of mouse parvovirus 1 in BALB/c and C.B-17/Icr-Prkdc(scid) mice

Fecal shedding and transmission of mouse parvovirus 1 (MPV) to naive sentinels, breeding mates, and progeny were assessed. Neonatal SCID and BALB/c mice inoculated with MPV were evaluated over 24 wk; several mice from each strain were mated once during this period. Fecal MPV loads for each cage were determined weekly by quantitative polymerase chain reaction (PCR) analysis, and all mice were evaluated by quantitative PCR analysis of lymphoid tissues and seroconversion to MPV antigens in immunocompetent mice. Results indicated persistently high fecal shedding of MPV in SCID mice throughout the evaluation period sufficient to allow transmission to sentinels, naive breeding partners, and the progeny of infected male mice and naive partners. Lymphoid tissue viral loads in the progeny of infected female SCID mice were high at weaning but low at 6 wk of age. Infected BALB/c mice shed high levels of MPV in feces for 3 wk postinoculation, with seroconversion only in sentinels exposed during the first 2 wk postinoculation. Thereafter the feces of infected BALB/c mice and the lymphoid tissues of sentinels, naive breeding partners, and progeny intermittently contained extremely low levels of MPV DNA. Although pregnancy and lactation did not increase viral shedding in BALB/c mice, MPV exposure levels were sufficient to induce productive infection in some BALB/c progeny. These data indicate that the adaptive immune response suppresses, but does not eliminate, MPV shedding; this suppression is sufficient to inhibit infection of weanling and adult mice but allows productive infection of some progeny.     

Conformational dynamics of nonsynonymous variants at protein interfaces reveals disease association

Recent studies have shown that the protein interface sites between individual monomeric units in biological assemblies are enriched in disease‐associated non‐synonymous single nucleotide variants (nsSNVs). To elucidate the mechanistic underpinning of this observation, we investigated the conformational dynamic properties of protein interface sites through a site‐specific structural dynamic flexibility metric (dfi) for 333 multimeric protein assemblies. dfi measures the dynamic resilience of a single residue to perturbations that occurred in the rest of the protein structure and identifies sites contributing the most to functionally critical dynamics. Analysis of dfi profiles of over a thousand positions harboring variation revealed that amino acid residues at interfaces have lower average dfi (31%) than those present at non‐interfaces (50%), which means that protein interfaces have less dynamic flexibility. Interestingly, interface sites with disease‐associated nsSNVs have significantly lower average dfi (23%) as compared to those of neutral nsSNVs (42%), which directly relates structural dynamics to functional importance. We found that less conserved interface positions show much lower dfi for disease nsSNVs as compared to neutral nsSNVs. In this case, dfi is better as compared to the accessible surface area metric, which is based on the static protein structure. Overall, our proteome‐wide conformational dynamic analysis indicates that certain interface sites play a critical role in functionally related dynamics (i.e., those with low dfi values), therefore mutations at those sites are more likely to be associated with disease. Proteins 2015; 83:428–435. © 2014 Wiley Periodicals, Inc.     

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), β-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.