Haemagglutinin production by enterotoxigenic strains of Clostridium perfringens type A

Thirty-nine enterotoxigenic cultures of Clostridium perfringens type A were studied for enterotoxin and haemagglutinin production. Enterotoxin was quantitated by sandwich ELISA and DOT-ELISA techniques and haemagglutinin titres were determined using sheep and human erythrocytes. Haemagglutinins from only six cultures reacted against both sheep and human erythrocytes; a further 13 reacted only against human erythrocytes, and another five only against sheep cells.The authors are with the Department of Veterinary Public Health and Epidemiology, Ranchi Veterinary College, Birsa Agricultural University, Ranchi-834007 (Bihar), India.     

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

Afflicted neurons in Alzheimer disease have been shown to display an imbalance in the expression of TrkA and p75^NTR at the cell surface, and administration of nerve growth factor (NGF) has been considered and attempted for treatment. However, wild-type NGF causes extensive elaboration of neurites while providing survival support. This study was aimed at developing recombinant NGF muteins that did not support neuritogenesis while maintaining the survival response. Critical residues were identified at the ligand-receptor interface by point mutagenesis that played a greater importance in neuritogenesis versus survival. By combining point mutations, two survival-selective recombinant NGF muteins, i.e./7-84-103 and KKE/7-84-103, were generated. Both muteins reduced neuritogenesis in PC12 (TrkA⁺/p75^NTR+) cells by >90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA⁺ only) and PCNA fibroblast (p75^NTR+-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC₅₀ for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.     

ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli

Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the ‘Min’ proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

MnSOD antisense treatment and exercise-induced protection against arrhythmias

Exercise provides protection against ischemia-reperfusion (I-R)-induced arrhythmias, myocardial stunning, and infarction. An exercise-induced increase in myocardial manganese superoxide dismutase (MnSOD) activity has been reported to be vital for protection against infarction. However, whether MnSOD is essential for exercise-induced protection against ventricular arrhythmias is unknown. We determined the effects of preventing the exercise-induced increase in MnSOD activity on arrhythmias during I-R resulting in myocardial stunning. Male rats remained sedentary or were subjected to successive bouts of endurance exercise. During in vivo myocardial I-R, the incidence of arrhythmias was significantly lower in the exercise-trained rats than in the sedentary rats as evidenced by the arrhythmia. When exercised rats were pretreated with antisense oligonucleotides directed against MnSOD, protection from arrhythmias was attenuated. Moreover, I-R resulted in significant increases in nitro-tyrosine (NT) in the sedentary group. Exercise abolished this I-R-induced NT formation but this protection was unchanged by antisense treatment. Protein carbonyls were increased by I-R, but neither exercise nor antisense treatment impacted carbonyl formation. These data demonstrate that an exercise-induced increase in MnSOD activity is important for protection against arrhythmias. The mechanism by which MnSOD provides protection does not appear to be linked to protein nitrosylation or oxidation.