Analysis of proteins in urinary tract stones and urine of urolithic patients

In order to investigate the mechanism of urinary tract stone formation, we analyzed protein components in urine and the stone. Urinary proteins of healthy subjects and urolithic patients as well as protein components urinary tract stone of the urolithic patients were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic patterns of urinary proteins of the patients differed from those of healthy subjects after separating protein patterns into those larger than 66kDa or smaller than 30kDa. Protein constituents of urinary tract stone were mainly separated into 18 bands ranging from 26.8 to 143 kDa. Major bands among these 18 bands differed among stones from different patients. On western blotting, the developed intensities of Tamm-Horsfall protein (THP) were fainter than those of healthy subjects. Whereas intensities of albumin (ALB) were stronger than those of healthy subjects. Moreover, blotting patterns of THP of the patients on non-reducing SDS-PAGE were obviously broad. Thus, we suggest that analysis of fractionated urinary proteins or protein components of urinary tract stone may provide a tool for monitoring the prognosis or relapse in the patients.     

The expression of neuronal nitric oxide synthase and dystrophin in rat regenerating muscles

We investigated the expression of neuronal nitric oxide synthase (nNOS) and dystrophin in the regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis by immunohistochemical studies and western blot analysis. In normal muscles, nNOS was moderately immunostained on type 2B fibers, but was faintly immunostained on type 2A or type 1 fibers. In immunohistochemical studies of regenerating muscles, nNOS was first observed at the sarcolemma of type 2B fibers on day 10, when the type discrimination between types 2A and 2B was first detected by ATP reactions. Subsequently, the immunostaining of nNOS grew progressively stronger in type 2B fibers, with faint staining in type 2A and type 1 fibers until day 28. Meanwhile, the immunostaining of dystrophin grew stronger equally in all three fibers until day 21. In western blot analysis of regenerating muscles, nNOS regenerated more slowly than dystrophin. The present data suggest that the expression of nNOS is related to the muscle fiber type differentiation, and that the role of nNOS is related to the function of the type 2B fibers of the muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel insertional mutation at exon VII of the myelin proteolipid protein gene in Pelizaeus -- Merzbacher disease

Pelizaeus — Merzbacher disease (PMD) is an X-linked neurologicaldisorder characterized by dysmyelination in the central nervoussystem (CNS). Recently mutations of the myelln proteollpid protein(PLP) gene which encodes both PLP and Its Isoform, DM-20 generatedby alternative spllcing, have been demonstrated In PMD patients.We analyzed the seven exons of the PLP gene of a Japanese boyaffected with PMD by direct sequencing and identified an Insertionevent In exon Vll of the PLP gene. This mutation was also presentIn his carrier mother, but was absent In ninety-five X chromosomesof normal Japanese. The frame-shift mutation leads to the productionof truncated PLP with altered carboxyl terminal amlno acid sequences,resulting In conslderable change of the structure of PLP andDM-20 necessary for functional purposes. This is the first reportof a mutation In exon Vll of the PLP gene associated with PMD.     

A pore-forming toxin produced by Aeromonas sobria activates Ca2+ dependent Cl- secretion

Bacteria produce many types of hemolysin that induce diarrhea by mechanisms that are not completely understood. Aeromonas sobria hemolysin (ASH) is a major virulence factor produced by A. sobria, a human pathogen that causes diarrhea. Since epithelial cells in the intestine are the primary targets of hemolysin, we investigated the effects of ASH on ion transport in human colonic epithelial (Caco-2) cells. ASH increased short-circuit currents (Isc) in a dose-dependent manner, and it also activated a 125I efflux from Caco-2 cells. ASH-induced Isc increases and 125I efflux activations were both suppressed by low Ca2+ levels in the extracellular solution or by pretreatment with the Ca2+ chlelator BAPTA-AM. Intracellular Ca2+ levels were increased by ASH in a biphasic fashion characterized by a rapid sharp increase (peak 1) followed by a sustained low plateau (peak 2). ASH-induced peak 1 was inhibited by pretreatment with pertussis toxin, indicating that Ca2+ was mobilized from intracellular stores, and peak 2 was induced by an influx of extracellular Ca2+. Peak 2 but not peak 1 was related to Cl- secretion. These results indicate that ASH activates Ca2+-dependent Cl- secretion.     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Purification and Characterization of Thermostable Direct Hemolysin of Vibrio parahaemolyticus

A thermostable direct hemolysin was purified from culture filtrates of Vibrio parahaemolyticus. The purified hemolysin gave one precipitation line with the antihemolysin antiserum on agar-gel diffusion test and a single band on polyacrylamide gel electrophoresis. The hemolysin was not inactivated by heating at 70 to 100 C for 10 min. The hemolytic activity was not enhanced by the addition of lecithin. It was demonstrated that the hemolysin was a protein with a molecular weight of approximately 118,000. Amino acid analysis revealed that 43% of total amino acids were acidic amino acids, whereas 11% were basic amino acids.     

Dreaming during non-rapid eye movement sleep in the absence of prior rapid eye movement sleep

STUDY OBJECTIVES: There is a long-standing controversy surrounding the existence of dream experiences during non-rapid eye movement (NREM) sleep. Previous studies have not answered the question whether this "NREM dream" originates from the NREM sleep mechanism because the subject might simply be recalling experiences from the preceding rapid eye movement (REM) sleep. METHODS: We scheduled 11 healthy men to repeat 20-minute nap trials separated by 40-minute periods of enforced wakefulness across a period of 3 days. At the end of the nap trial, each participant answered questions regarding the formal aspects of his dream experiences during the nap trial, using the structured interviews. RESULTS: We obtained a total of 172 dream reports after naps containing REM sleep (REM naps) and 563 after naps consisting of only NREM sleep (NREM naps). Dream reports from NREM naps were less remarkable in quantity, vividness, and emotion than those from REM naps and were obtained more frequently during the morning hours when the occurrences of REM sleep were highest. CONCLUSIONS: These results suggest that the polysomnographic manifestations of REM sleep are not required for dream experiences but that the mechanisms driving REM sleep alter experiences during NREM sleep in the morning. A subcortical activation similar to REM sleep may occur in human NREM sleep during the morning when REM sleep is most likely to occur, resulting in dream experiences during NREM sleep.     

Proliferation Kinetics of Rat Kupffer Cells after Partial Hepatectomy Immunohistochemical and UItrastructural Analysis

In an effort to settle the conflicting views on the proliferation kinetics of Kupffer cells (Kc), we performed 2/3 partial hepatectomy on rats injected with Pelikan ink. Using an anti-rat macrophage monoclonal antibody, ED 2, we evaluated the numerical changes in total, carbon-positive ED 2⁺ cells and carbon-negative ED 2⁺ cells in the portal and central area. We also analyzed the ultrastructure and peroxidase cytochemistry of various types of cells observed during regeneration. The total numbers of ED 2⁺ cells in the remaining liver increased rapidly from day 2 to 5, and the number of dividing ED 2⁺ cells reached a maximum on day 2. Thus, the numerical increase in ED 2⁺ cells corresponded to the division phase. In contrast, the carbon-labeling experiment showed a continuous increase of carbon negative ED 2⁺ cells from day 2 to 7. In the central area where division was less frequent, the proportion of carbon-positive cells decreased markedly to 50% on day 7, as against 97% in control rats. These findings suggest the possibility of an influx of carbon-negative Kc in addition to cell division. Ultrastructurally, the presence of carbon-negative "small Kc" and "immature Kc" with morphological features different from those of carbon-positive Kc was demonstrated. Such carbon-negative Kc with a high nucleus-to-cytoplasm ratio and rather few phagosomes, were not observed in control rats. Furthermore, we demonstrated two types of possible precursor cell, i.e. "transitional" forms between monocytes and Kc, and "immature macrophages". The former showed peroxidase activity in some lysosomes as well as in the rough endoplasmic reticulum and nuclear envelope. Our result indicated that the proliferation kinetics of Kc depend upon both local proliferation and influx.