Enzymatic determination of a molar ratio of free branched-chain amino acids to tyrosine (BTR) and its clinical significance in plasma of patients with various liver diseases

A molar ratio of free branched-chain amino acids to tyrosine (BTR) was determined in the plasma of patients with liver diseases using a new enzymatic method. In addition, clinical significance of BTR was studied by comparing particularly with that of Fischer's ratio (a molar ratio of branched-chain amino acids to aromatic amino acids (tyrosine+phenylalanine], which was obtained by conventional HPLC (Amino acid autoanalyzer, Hitachi 835). Following results were obtained: 1) Enzymatically determined branched-chain amino acids and tyrosine showed significant correlations with respective results obtained by HPLC (r = 0.937, 0.972). 2) Significant correlation was also found between enzymatically determined BTR and Fischer's ratio obtained by HPLC. Changes of BTR in clinical courses were found to be in parallel with those of Fischer's ratio. 3) BTR as well as Fischer's ratio correlated significantly with ICG R15, KICG, prothrombin time (%) and serum albumin level. 4) BTRs in patients with decompensated liver cirrhosis or with fulminant hepatitis were significantly lower than those in patients with acute or chronic hepatitis. In conclusion, new enzymatic assay of branched-chain amino acids and tyrosine as described here is quite simple method, and is also considered to be very useful parameter of the clinical conditions of patients with liver diseases, particularly representing the severity of liver diseases and the protein nutritional status.     

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.     

Role of p27Kip1 and Cyclin-Dependent Kinase 2 in the Proliferation of Non-Small Cell Lung Cancer

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27^Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.     

Phylogenetic Classification of Trichophyton mentagrophytes Complex Strains Based on DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1 Regions

Using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences from 37 stock strains and clinical isolates provisionally termed Trichophyton mentagrophytes complex in Japan, we demonstrated the mutual phylogenetic relationships of these strains. Members of this complex were classified into 3 ITS1-homologous groups and 13 ITS1-identical groups by their sequences. ITS1-homologous group I consists of Arthroderma vanbreuseghemii, T. mentagrophytes human isolates, and several strains of T. mentagrophytes animal isolates. Five strains of Arthroderma simii form a cluster comprising ITS1-homologous group II. The Americano-European and African races of Arthroderma benhamiae, T. mentagrophytes var. erinacei, and one strain of a T. mentagrophytes animal isolate constitute ITS1-homologous group III. According to the phylogenetic tree constructed with Trichophyton rubrum as an outgroup, ITS1-homologous groups I and II comprised a monophyletic cluster and ITS1-homologous group III constituted another cluster which was rather distant from the others in the complex. This system was applicable to the phylogenetic analysis of closely related strains. Using this technique, human and animal isolates of T. mentagrophytes were also clearly distinguishable from each other.Dermatophytes have the capacity to invade keratinized tissues, that is, the skin, hair, and nails, of humans and other animals to produce an infection, dermatophytosis, referred to as ringworm or tinea. Trichophyton mentagrophytes (8) is known as a complex species (22) and is one of the major pathogens causing this infection (23). Using mating tests and microscopic observation of ascospores, three perfect fungal states of T. mentagrophytes have been identified in this imperfect or conidial “species.” They are Arthroderma vanbreuseghemii, Arthroderma simii, and Arthroderma benhamiae (1, 20, 22), the latter being classified into two races, American-European and African (21). The phylogeny of T. mentagrophytes, however, remains unclear because the phenotypic features of members of the T. mentagrophytes complex are poor and many isolates from medical and veterinary samples have lost their sexual activity (22). From a clinical point of view, because the T. mentagrophytes complex includes both anthrophilic and zoophilic species (23), it is important to have a reliable method of identifying the human-pathogenic species of the complex. Establishment of the phylogenetic classification of this complex has been achieved by molecular biological studies on the phylogeny of pathogenic fungi, primarily using the G+C content of chromosomal DNA (5), total DNA homology (6), restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) (7, 13, 17, 18), and the base sequence of the 18S (11) or 28S (14) rRNA or rRNA gene (rDNA). However, for dermatophytes, including T. mentagrophytes, the phylogenic relationship or species-specific sequences cannot be defined by these methods, because the members of this group of fungi are phylogenetically and taxonomically very closely related. Specific DNA sequences of the internal transcribed spacer 1 region (ITS1) of the rDNA in the T. mentagrophytes complex, mainly of strains stocked in Japan, were therefore determined and phylogenetically analyzed. ITS1 is located between the 18S and 5.8S rDNAs. As reported previously, the variable ITS regions have proven useful in resolving relationships between close taxonomic relatives (2, 3, 15). We were able to successfully differentiate between members of the T. mentagrophytes complex and a related species, Trichophyton rubrum, and to demonstrate their phylogenetic relationship by base pair comparisons of ITS1 regions.     

Discontinuation of infliximab in rheumatoid arthritis patients in clinical remission

Biologic drugs are effective but are also expensive, and it is difficult to evaluate the duration of treatment. Infliximab, an anti-TNFα antibody, suppresses arthritic activity and inhibits bone destruction in patients with rheumatoid arthritis (RA). Here, we document that infliximab could be discontinued after clinical remission in RA patients. Among 172 patients with RA who reached clinical remission following infliximab (3 mg/kg) and methotrexate (MTX, >6 mg/w), nine patients with sustained remission discontinued it. Clinical assessment was based on a disease activity score (DAS) that included a 28-joint count/erythrocyte sedimentation rate (DAS28-ESR). The disease was assessed before and after the start of infliximab treatment, and concomitant drug treatment—in the order of corticosteroid, nonsteroidal anti-inflammatory drugs (NSAIDs), and disease-modifying anti-rheumatic drugs (DMARDs) other than MTX—was gradually discontinued. We considered patients for discontinuation of infliximab treatment after remission (DAS28-ESR < 2.6) had been sustained for more than 24 weeks. The nine patients able to discontinue treatment were all females, with a mean age of 53.8 years; eight patients were at stage I or II. The mean duration of disease was 28.7 months, and these patients were on corticosteroid treatment equivalent to a mean of 2.28 mg prednisolone (PSL). These nine patients all met the remission standard—that DAS28-ESR < 2.6 for ≥24 weeks) —and so their treatment with concomitant drugs was discontinued. After the discontinuation of infliximab, the mean period of sustained remission was 14.2 months and the longest period was 29 months. The duration of disease was significantly shorter and the points from Steinbrocker’s stage-classification were significantly lower in the infliximab-discontinued group than in the infliximab-continued group. Strategic reductions and/or discontinuations of concomitant treatment were performed in RA patients who attained clinical remission (DAS28 < 2.6) through treatment with infliximab and MTX. Nine patients successfully discontinued infliximab after maintaining clinical remission for more than 24 weeks. After infliximab was discontinued, clinical remission and suppression of joint destruction were maintained with MTX alone, especially in early RA patients.     

Narrow-band imaging optical chromocolonoscopy： Advantages and limitations

Narrow-band imaging （NBI） is an innovative optical technology that modifies the center wavelength and bandwidth of an endoscope＇s light into narrow-band illumination of 415 ：1： 30 nm. NBI markedly improves capillary pattern contrast and is an in vivo method for visualizing microvessel morphological changes in superficial neoplastic lesions. The scientific basis for NBI is that short wavelength light falls within the hemoglobin absorption band, thereby facilitating clearer visualization of vascular structures. Several studies have reported advantages and limitations of NBI colonoscopy in the colorectum. One difficulty in evaluating results, however, has been nonstandardization of NBI systems （Sequential and nonsequential）. Utilization of NBI technology has been increasing worldwide, but accurate pit pattern analysis and sufficient skill in magnifying colonoscopy are basic fundamentals required for proficiency in NBI diagnosis of colorectal lesions. Modern optical technology without proper image interpretation wastes resources, confuses untrained endoscopists and delays interinstitutional validation studies. Training in the principles of ＂optical image-enhanced endoscopy＂ is needed to close the gap between technological advancements and their clinical usefulness. Currently available evidence indicates that NBI constitutes an effective and reliable alternative to chromocolonoscopy for in vivo visualization of vascular structures, but further study assessing reproducibility and effectiveness in the colorectum is ongoing at various medical centers.     

Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12

Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage λ in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the λ c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.     

Evaluation of segregation ratio in Wilson''s disease.

Two problems relating to segregation analysis for Wilson's disease are discussed and a practical solution is presented. A problem in the ascertainment of families with Wilson's disease is illustrated by comparing segregation ratios calculated by the single selection, complete truncate, and multiple incomplete selection methods. The effect on the segregation ratio of exclusion from the analysis of those sibs who had died of other diseases at a young age is also discussed and a method of adjustment of the number of the affected using the data on age at onset is proposed. The segregation ratio by multiple incomplete selection (Weinberg proband method) after adjustment for those sibs who had died of other diseases was 0.243, consistent with the theoretical value for autosomal recessive inheritance. The segregation ratio calculated by the single selection method tended to give a lower value, while that calculated by the complete truncate method was greater than the theoretical value. Recessive inheritance is, however, supported. The actual effect of exclusion of those sibs who had died of other diseases on gene frequency estimation is shown to be very small.     

Olanzapine potentiates neuronal survival and neural stem cell differentiation: regulation of endoplasmic reticulum stress response proteins

Summary Recent clinical neuroimaging studies have suggested that morphological brain changes occur and progress in the course of schizophrenia. Although the neurogenetic and neurotrophic effects of antipsychotics are considered to contribute to the prevention of reduction in brain volume, the cellular molecular mechanisms of action of antipsychotics have not yet been elucidated. We examined the effects of antipsychotics on the endoplasmic reticulum (ER) stress-induced damages of neurons and neural stem cells (NSCs) using cultured cells. In the neuronal cultures, the atypical antipsychotic olanzapine protected neurons from thapsigargin (1 μM)-induced injury. It was observed that a low concentration of thapsigargin (10 nM) that did not affect the neuronal survival could reduce neuronal differentiation of cultured NSCs, suggesting a role of ER stress in the differentiation function of NSCs. Treatment with olanzapine increased the neuronal differentiation suppressed by the exposure to thapsigargin (10 nM). The thapsigargin-induced ER chaperones, GRP78, which indicate the ER stress condition of the cell, were decreased by the treatment with the atypical antipsychotics olanzapine and quetiapine but not by the typical antipsychotic haloperidol. These results indicate that the amelioration of ER-stress might be involved in the cellular mechanisms of atypical antipsychotics to produce neuroprotective and neurogenetic actions in neurons and NSCs, suggesting potential roles of these drugs for treatment of schizophrenia.