OF4949, new inhibitors of aminopeptidase B. IV. Effects of OF4949 and its derivatives on enzyme systems

OF4949-I and II inhibited aminopeptidase B from Ehrlich ascites carcinoma in a competitive way and the Ki value for both against L-arginine-beta-naphthylamide was 8 X 10(-9) M. Inhibition by I and II of various exopeptidases and endopeptidases was examined. OF4949-I and II both strongly inhibited leucine aminopeptidase and enkephalin-degrading aminopeptidase; I also inhibited enkephalinase B. The inhibitory effects of various derivatives of I and II on aminopeptidase B activity, showed that the terminal amino and carboxamide groups are essential for activity.     

Effect of apolipoprotein E (apoE) phenotype on the apoE content of CSF-HDL in children

BACKGROUND: The majority of the lipoprotein in cerebrospinal fluid (CSF) is apolipoprotein E (apoE)-containing HDL. Since neuronal cells express lipoprotein receptors which recognize apoE, apoE in CSF-HDL is believed to be important for the development of central nervous system (CNS) in children. In adults, the apoE phenotype affects the plasma apoE concentration and the epsilon 4 allele is a risk factor for Alzheimer's disease. Due to the requirement for CNS development, we examined whether the apoE phenotype affects the composition and concentration of CSF-HDL in children. METHODS: We determined the apoE phenotype in 107 neurologically normal subjects, including 67 children (<20 years), by isoelectronic focusing. We also measured apoE, total cholesterol (TC), and phospholipid (PL) concentrations in the CSF. RESULTS: The respective frequencies of apoE4/3, E3/3 and E3/2 were 16.4%, 77.6%, and 6.0%. The allele frequencies of epsilon 4, epsilon 3, and epsilon 2 were 0.082, 0.888, and 0.030, respectively. There were no significant differences in the CSF-apoE, TC, or PL concentrations or the apoE/PL ratio among the apoE phenotypes. However, the CSF-apoE/PL ratio was significantly higher in children than in adults. CONCLUSION: The apoE phenotype does not affect the composition or concentration of CSF-HDL in children. We speculate that an apoE4 carrier is prevented in childhood from the impaired development of central nervous system by CSF-HDL enriched with apoE.     

Cerivastatin induces type-I fiber-, not type-II fiber-, predominant muscular toxicity in the young male F344 rats

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are associated with adverse skeletal muscle toxicity, but the underlying mechanism remains unclear. To investigate the pathological mechanism of statin-induced myotoxicity, cerivastatin (20 ppm; corresponding to 2 mg/kg/day) was dietarily administered to young male F344 rats for 10 days, and time-course clinical observations, measurement of plasma creatine kinase activity, and light and electron microscopy of type I fiber-predominant skeletal muscle (soleus) or type II fiber-predominant skeletal muscles (extensor digitorum longus and tibialis anterior), were performed. Clinical symptoms including weakness of hind limbs, staggering gait and body weight loss, accompanied by marked plasma creatinine kinase elevation in rats fed cerivastatin at around Day 6 to 8. Interestingly, microscopic examination revealed that cerivastatin-induced muscle damages characterized by hypercontraction (opaque) and necrosis of the fibers were of particular abundance in the soleus muscle at Day 8, whereas these histological lesions in the extensor digitorum longus and tibialis anterior were negligible, even at Day 9. Prior to manifestation of muscle damage, swollen mitochondria and autophagic vacuoles in the soleus were observed as the earliest ultra structural changes at Day 6; then activated lysosomes, disarray of myofibril and dilated sarcoplasmic reticulum vesicles became ubiquitous at Day 8. These results demonstrate that cerivastatin induces type I fiber-predominant muscles injury, which is associated with mitochondrial damage, in young male F344 rats. Since the rat exhibiting type I fiber-targeted injury is a unique animal model for statin-induced myotoxicity, it will be useful for gaining insight into mechanisms of statin-induced myotoxicity.     

Disinfection methods for spores of Bacillus atrophaeus, B. anthracis, Clostridium tetani, C. botulinum and C. difficile

To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.     

Rapid typing of influenza viruses using super high-speed quantitative real-time PCR

The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18s/cycle; 40 cycles in less than 20min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(-1) plaque-forming unit/reaction for viruses in culture supernatants during 20min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.     

Phosphorylation of podocalyxin (Ser415) Prevents RhoA and ezrin activation and disrupts its interaction with the actin cytoskeleton

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.     

Live Birth Rate According to Maternal Age and Previous Number of Recurrent Miscarriages

Problem  In Japan, marital age and women's age at the first pregnancy are continuing to increase year by year. However, information concerning subsequent live birth rate according to maternal age and number of previous recurrent miscarriages is limited.
Method of study  We studied a total of 1250 unexplained patients suffering two or more consecutive miscarriages. We examined the live birth rate at the first pregnancy and the cumulative success rate for birth of at least one child after examination.
Results  The live birth rate of women in their 40s was 58.1%, which was similar to that of women who were 35–39 years old (58.4%) at the first pregnancy, as found after examination. From logistic regression, women's age and the number of previous miscarriages independently decreased the live birth rate in subsequent pregnancies ( p _s) as well as cumulative pregnancies ( p _c), as follows:
Conclusion  The information concerning the live birth rate can be given to each patient before subsequent pregnancy.