Mutational and biochemical analyses of the endolysin Lys(gaY) encoded by the Lactobacillus gasseri JCM 1131T phage phi gaY

The Lys(gaY) of Lactobacillus gasseri JCM 1131(T) phage phigaY endolysin was purified to homogeneity using the Escherichia coli/His.Tag system. Zymographic and spectrophotometric assays showed that Lys(gaY) lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37 degrees C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys(gaY) were involved in cell-lytic activity: in the beta/alpha(gaY) domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b(gaY) domain, Y272 and W284. In addition, deletion analysis demonstrated that the beta/alpha(gaY) domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys(gaY). These mutational experiments suggested that beta/alpha(gaY) (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b(gaY) promotes beta/alpha(gaY) possibly through cell-wall binding function. The purified His-tagged SH3b(gaY) domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys(gaY), (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131(T), L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys(gaY), and (iv) impeded autolysis of L. gasseri JCM 1131(T) in buffer systems. A truncated protein HDeltaSH3b(gaY) lacking in N-terminal 21 residues (from S217 to E237) of SH3b(gaY) and an amino-acid substituted protein HSH3b(gaY)G (W284G) lost the activities of HSH3b(gaY), showing that the N-terminal region and W284 probably play important roles in the SH3b(gaY) function(s).     

Temperature-dependent sugar accumulation in interspecific Capsicum F1 plants showing hybrid weakness

Journal of Plant Research - In plants, F1 hybrids showing hybrid weakness exhibit weaker growth than their parents. The phenotypes of hybrid weakness are often suppressed at certain temperatures....     

Oviposition and refractory periods of the small water strider Microvelia horvathi (Heteroptera: Veliidae) after a single mating event

Multiple mating can be costly, but also beneficial for females. When the costs outweigh the benefits, mated females can be reluctant to remate for some time. Conversely, females will be likely to remate when they need to replenish sperm and/or receive nutrients contained in the accessory ejaculate substances. Water striders have been commonly used in mating studies as many show a vigorous struggle between the sexes. Generally, water strider females frequently mate with many males and lay many eggs during mating period. However, little is known about their oviposition and refractory periods after an initial mating event. In this study, we revealed that single-mated females of the small water strider, Microvelia horvathi, could lay eggs for 40 days, reflecting approximately half of their potential oviposition periods. It suggested that M. horvathi females do not need to mate to replenish sperm for some time once they mate. Our results also showed that no female remated after 1 or 3 days of an initial mating event. The proportion of female remating increased with time after the initial mating, however, remating levels reached 26.7% even after 14 days. In this sense, any significant cost of mating outweighs the benefit, which might be one of the factors lowering their remating levels.     

Periodontal Disease Bacteria Specific to Tonsil in IgA Nephropathy Patients Predicts the Remission by the Treatment

Background

Immunoglobulin (Ig)A nephropathy (IgAN) is the most common form of primary glomerulonephritis in the world. Some bacteria were reported to be the candidate of the antigen or the pathogenesis of IgAN, but systematic analysis of bacterial flora in tonsil with IgAN has not been reported. Moreover, these bacteria specific to IgAN might be candidate for the indicator which can predict the remission of IgAN treated by the combination of tonsillectomy and steroid pulse.

Methods and Findings

We made a comprehensive analysis of tonsil flora in 68 IgAN patients and 28 control patients using Denaturing gradient gel electrophoresis methods. We also analyzed the relationship between several bacteria specific to the IgAN and the prognosis of the IgAN. Treponema sp. were identified in 24% IgAN patients, while in 7% control patients (P = 0.062). Haemophilus segnis were detected in 53% IgAN patients, while in 25% control patients (P = 0.012). Campylobacter rectus were identified in 49% IgAN patients, while in 14% control patients (P = 0.002). Multiple Cox proportional-hazards model revealed that Treponema sp. or Campylobactor rectus are significant for the remission of proteinuria (Hazard ratio 2.35, p = 0.019). There was significant difference in remission rates between IgAN patients with Treponema sp. and those without the bacterium (p = 0.046), and in remission rates between IgAN patients with Campylobacter rectus and those without the bacterium (p = 0.037) by Kaplan-Meier analysis. Those bacteria are well known to be related with the periodontal disease. Periodontal bacteria has known to cause immune reaction and many diseases, and also might cause IgA nephropathy.

Conclusion

This insight into IgAN might be useful for diagnosis of the IgAN patients and the decision of treatment of IgAN.     

Estimation of handgrip force using frequency-band technique during fatiguing muscle contraction

In this paper, we propose a force estimation model to compute the handgrip force from SEMG signal during fatiguing muscle contraction tasks. The appropriate frequency range was analyzed using various combinations of a wavelet scale, and the highest accuracy was achieved at a range from 242 to 365 Hz. After that, eight healthy individuals performed a series of static (70%, 50%, 30%, and 20% MVC) and dynamic (0–50% MVC) muscle contraction tasks to evaluate the performance of this technique in comparison with that of former method using the Root Mean Square of the SEMG signal. Both methods had comparable results at the beginning of the experiments, before the onset of muscle fatigue. However, differences were clearly observed as the degree of muscle fatigue began to increase toward the endurance time. Under this condition, the estimated handgrip force using the proposed method improved from 17% to 134% for static contraction tasks and 40% for dynamic contraction tasks. This study overcomes the limitation of the former method during fatiguing muscle contraction tasks and, therefore, unlocks the potential of utilizing the SEMG signal as an indirect force estimation method for various applications.     

Partial renal ischemia elicits heterogeneous control of renal sympathetic nerve activity to ischemic and nonischemic regions of the kidney

We tested the hypothesis that renal sympathetic nerve activity (RSNA) to the ischemic and nonischemic regions responded differently during partial ischemia of the kidney in pentobarbital-anesthetized cats. The renal artery divides into two branches at the front of the renal hilus: one branch perfuses predominantly the dorsal half of the kidney, and the other perfuses its ventral half. We identified the innervated area of a renal nerve bundle by supramaximal electrical stimulation and subsequently determined the changes in RSNA in response to occlusion of either renal arterial branch for 3 min. RSNA to the nonischemic region of the kidney gradually decreased by 23 +/- 4% during partial renal ischemia, whereas RSNA to the ischemic region of the same kidney showed no significant change. Crushing either all renal nerve bundles or only the renal nerve bundles terminated to the ischemic region abolished the decrease in RSNA to the nonischemic region. Furthermore, intra-arterial administration of a prostaglandin synthesis inhibitor (meclofenamate, 4 mg/kg) abolished the decrease in RSNA to the nonischemic region of the kidney. Following spinal transection at the level of T7, the inhibitory response in RSNA to the nonischemic region disappeared, whereas the RSNA to the ischemic region was markedly augmented by 47 +/- 17%. Thus it is likely that renal chemoreceptors activated during renal partial ischemia elicit heterogeneous control of renal sympathetic outflows to the ischemic and nonischemic regions of the same kidney, which may be determined by a net output between the supraspinal inhibitory and spinal excitatory reflexes.     

Molecular properties of the glucosaminidase AcmA from Lactococcus lactis MG1363: Mutational and biochemical analyses

The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu_acma as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu_acma (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu_atlwm from the Staphylococcus warneri M autolysin Atl_WM, these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu_atlwm were purified from E. coli recombinant cells, and their enzymatic properties were studied.     

Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype

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