Molecular and immunological approach to hematological disease: detection and analysis of intracellular modified nucleosides by flow cytometry

Modified nucleosides are components of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). 1-methyladenosine and pseudouridine are members of those modified nucleosides. The urinary concentration of 1-methyladenosine and pseudouridine of cancer patients are higher than that of healthy controls, and those compounds were reduced after effective chemotherapy. Thus those compounds might be expected to use as tumor markers. In this study cellular origin of 1-methyladenosine and pseudouridine were analysed about two tumor cell lines (HUT-102, THP-1), peripheral blood lymphocytes (PBL) from healthy adult and PBL under the phytohemagglutinin stimulation, by flow cytometric analysis and immunofluorescent staining of cellular RNA using monoclonal antibodies specific for 1-methyladenosine (AMA) and pseudouridine (APU). Both 1-methyladenosine and pseudouridine were detected in more than 90% of tumor cells above the thresholds of flow cytometric detection (Spectrum III, Ortho). The PBL under the PHA stimulation also tended to take the same way of the tumor cell lines, whereas few of the PBL contained 1-methyladenosine above the thresholds. According to the DNA analysis of those cell lines, high contents of the modified nucleosides in the cell might follow DNA synthesis, this leads to one reason for high levels of the urinary excretion of the modified nucleosides in cancer patient.     

Detection of the putative E2 protein of hepatitis C virus in human liver

The question was asked whether a predicted envelope protein, considered to be processed from the polyprotein precursor encoded by the putative E2/NS1 region of the hepatitis C virus (HCV) genome, may be observed in HCV-infected humans. Two polyclonal antibodies against recombinant E2/NS1 proteins were prepared and their reactivity tested against liver extracts from HCV-infected patients by immunoblotting analysis. A band corresponding to a size of 44 kDa was detected in liver extracts from patients who were positive for the HCV-specific antibody anti-C100-3 but not in liver extracts from patients who did not have anti-C100-3 antibody. Additionally, no band was detected using preimmune sera or antisera which had been preabsorbed with recombinant E2/NS1 proteins. Deglycosylation studies demonstrated that the 44 kDa protein was a glycosylated form of a 38 kDa protein which corresponds to the predicted molecular weight of the putative E2/NS1 protein. These results suggest that the 44 kDa protein is a product of the E2/NS1 region. Frequent observation of the 44 kDa band in cases of chronic active hepatitis C suggests a correlation between the expression of this protein and the progression of hepatitis. © 1994 Wiley-Liss, Inc.     

Detection of mutations in the enhancer 2/core promoter region of hepatitis B virus in patients with chronic hepatitis B virus infection: comparison with mutations in precore and core regions in relation to clinical status

To investigate the meaning of the mutations in the enhancer 2/core promoter (Enh2/CP) region of hepatitis B virus (HBV) during the chronic HBV infection, mutations were examined in the Enh2/ CP region (carboxyl half of X region) and their correlation with mutations in the precore and core regions in relation to the presence of chronic liver disease. The entire nucleotide sequences of the Enh2/CP region were determined by direct sequencing of the amplified products derived from 30 cases with chronic HBV infection. The results were compared to the mutations in the precore and core regions. In the Enh2/CP region, 91 generally scattered nucleotide substitutions were detected. There were 11 substitutions in the 10 asymptomatic healthy carriers (mean, 1.1/case) and 80 in the 20 chronic liver disease patients (4.0/case). The most frequent substitutions from A to T at nucleotide 1764 and from G to A at nucleotide 1766 were seen in none of the 10 asymptomatic carriers and in 14 (70%) of the 20 chronic liver disease patients. Comparisons of mutations in the precore and core regions revealed that 14 of 16 patients with mutations in the core region had the mutations in the Enh2/CP region and/or a precore stop codon mutation. These data suggest that mutations in the Enh2/CP and precore regions may affect the expression of the core and HBeAg peptides and might be involved in the pathogenesis of chronic liver disease.     

Influence of the parameters of several pulse sequences on the image contrast: comparison between the two major contrast media

We compared the influences of the parameters of several pulse sequences using two major commercially available gadolinium (Gd) contrast media for MR imaging. The phantom of Gd solutions of various concentration (0.1 - 10mmol/L) was prepared, and was scanned with a 1.5T clinical MR unit, using a spin-echo T1-weighted sequence, 2DFLASH, 3DFLASH, and 3DVIBE. The signal intensity was measured and the contrast enhancement ratio (CER) was calculated and plotted as a function of Gd concentration. The results were compared between the pulse sequences, and between the contrast media as well. Both 3DFLASH and 3DVIBE showed higher CER than other two sequences, showing similar CER curve configuration. There was no significant difference both in CER value and CER curve configuration between the two contrast media for each pulse sequence.     

Development of enzyme-labeled oligonucleotide probe for detection of mecA gene in methicillin-resistant Staphylococcus aureus.

A DNA hybridization method with an enzyme-labeled oligonucleotide probe (mecA-ELONP) was developed to detect the methicillin-resistant gene (mecA) in methicillin-resistant Staphylococcus aureus. For rapid identification, bacterial colonies were transferred from agar plates directly onto nylon membranes. Lysis of cells, denaturation of DNA, and hybridization were performed on the membranes. These procedures required only 3 h for completion. The results obtained by this test closely corresponded with those obtained by determining the MICs of oxacillin against S. aureus. The results of the mecA-ELONP also correlated well with those of a commercially available PCR test. Thus, mecA-ELONP proved to be a reliable and convenient method for the rapid identification of methicillin-resistant S. aureus, which could be useful in clinical microbiology laboratories.     

Locations of spinothalamic tract axons in cervical and thoracic spinal cord white matter in monkeys

The spinothalamic tract (STT) is the primary pathway carrying nociceptive information from the spinal cord to the brain in humans. The aim of this study was to understand better the organization of STT axons within the spinal cord white matter of monkeys. The location of STT axons was determined using method of antidromic activation. Twenty-six lumbar STT cells were isolated. Nineteen were classified as wide dynamic range neurons and seven as high-threshold cells. Fifteen STT neurons were recorded in the deep dorsal horn (DDH) and 11 in superficial dorsal horn (SDH). The axons of 26 STT neurons were located at 73 low-threshold points (<30 microA) within the lateral funiculus from T(9) to C(6). STT neurons in the SDH were activated from 33 low-threshold points, neurons in the DDH from 40 low-threshold points. In lower thoracic segments, SDH neurons were antidromically activated from low-threshold points at the dorsal-ventral level of the denticulate ligament. Neurons in the DDH were activated from points located slightly ventral, within the ventral lateral funiculus. At higher segmental levels, axons from SDH neurons continued in a position dorsal to those of neurons in the DDH. However, axons from neurons in both areas of the gray matter were activated from points located in more ventral positions within the lateral funiculus. Unlike the suggestions in several previous reports, the present findings indicate that STT axons originating in the lumbar cord shift into increasingly ventral positions as they ascend the length of the spinal cord.     

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.     

Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios.

A thermolabile direct hemolysin from an El Tor cholera vibrio strain has been isolated and partially characterized as a simple protein of ca. 20,000 molecular weight. In addition to its hemolytic activity, the hemolysin is cytotoxic, cardiotoxic, and rapidly lethal. In these respects it resembles the thermostable direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial hemolysins, although there are other significant differences. Because identical diseases are produced by both hemolytic and nonhemolytic cholera vibrios, the El Tor hemolysin may be presumed to be pathogenetically irrelevant. These observations raise the question of "When is a toxic substance also a toxin?"