Calcium-independent and dependent steps in action of Clostridium perfringens enterotoxin on HeLa and Vero cells.

Purified enterotoxin (20–200 ng/ml) of $\text{Clostridium}$$\text{perfringens}$ rapidly induced bled and balloon formation on HeLa and Vero cells in the presence, but not the absence, of Ca²⁺. The action of the toxin involved two, sequential, temperature-dependent steps: The first was Ca²⁺-independent and included binding of toxin and the bound toxin after 30–60 sec could no longer be removed by washing. The second step was Ca²⁺-dependent and eventually led to bled and balloon formation. On adding Ca²⁺ to cells pretreated with toxin in Ca²⁺-free medium, bled and balloon formation started immediately. The ionophore A23187 mimicked the action of toxin. The effects of sucrose (0.2 M), trypsin-treatment of the cells and various pretreatments of the toxin on the action of enterotoxin were studied.     

Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet–fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms of sublethal tubular cell injury.     

Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.     

Reversible Dissociation of Crystalline Yeast Phosphoglyceric Acid Mutase in Alkali

The structure of crystalline yeast phosphoglyceric acid mutase has been investigated by sedimentation-velocity and equilibrium measurements, optical rotatory dispersion measurements and viscometry. The data indicate that this enzyme is a globular, compact and highly organized protein with a low helix content. The native structure remains unchanged at pH 10.5. Dissociation of the enzyme into subunits has been observed at pH values of 11.5 and above. From optical rotatory dispersion measurements, it is found that the enzyme loses a large part of its organized conformation when it dissociates in alkaline solution. On neutralization, the alkali-treated enzyme regains its activity. The ability to regain the enzyme activity is gradually lowered with the increase of pH value to be incubated and with time of exposure. Inactivation at pH 13.0 is almost irreversible. However, the reversibility of the inactivation at pH 13.0 is appreciably enhanced by the presence of phosphate compounds in the reactivation system. Particulary, it is found that presence of substrates or the coenzyme is effective for considerable improvement of the reversibility. Molecular weight analyses by ultracentrifugation indicate that subunits have approximately equal molecular weights and that the native enzyme is consisted of four polypeptide chains.     

RPAP3 interacts with Reptin to regulate UV‐induced phosphorylation of H2AX and DNA damage

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor‐α and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II‐associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin‐remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV‐irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV‐induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV‐induced DNA damage by regulating H2AX phosphorylation. J. Cell. Biochem. 106: 920–928, 2009. © 2009 Wiley‐Liss, Inc.