Osteosarcoma. Ultrastructural and immunohistochemical studies on alkaline phosphatase-positive tumor cells constituting a variety of histologic types

The osteosarcomas were subclassified into osteoblastic, fibroblastic, chondroblastic and telangiectatic types and examined by electron microscopy. Their immunohistochemical reactions were also studied. In an overall survey of the above types, fibroblast-like cells revealed poorly developed cytoplasmic organelles with rather short, branching rough endoplasmic reticulum, mixed with osteoblast-like cells that were hardly distinguishable from the former. They appeared to be an early stage of an osteoblastic cell lineage from the distribution and development of their cell organelles and highly positive vimentin activity. The tumor cells in malignant cartilage varied in appearance from chondroblast-like to osteoblast-like cells. All types of tumor cells expressed alkaline phosphatase activity to a significant degree. Immunohistochemical staining showed a mixture of procollagen type I-positive cells among the cells positive for both procollagen type II and S-100 protein in the malignant cartilage. Irrespective of any ultrastructural differences between these various tumor cell types, they all revealed a significant degree of ALPase activity unlike other types of bone tumors, suggesting that the tumor cells which constitute the various types of osteosarcoma are derived from a common precursor cell.     

CDK4 is a probable target gene in a novel amplicon at 12q13.3-q14.1 in lung cancer

Several chromosomal regions are recurrently amplified or deleted in lung tumors, but little is known about the underlying genes, which could be important mediators in tumor formation or progression. In lung cancer, the RB1-CCND1-CDKN2A pathway, involved in the G1-S transition, is damaged in nearly all tumors. In the present study, we localized a novel amplicon in lung tumors to a fragment of less than 0.5 Mb at 12q13.3-q14.1 by using comparative genomic hybridization (CGH) on cDNA microarrays. This approach enabled us to identify 10-15 genes with the most consistent amplifications. Semiquantitative RT-PCR analyses of 13 genes in this region showed that four of them (CDK4, CYP27B1, METTL1, and TSFM) were also highly up-regulated. Immunohistochemical (IHC) analysis of 141 tumor samples on a tissue microarray showed that CDK4 was expressed at a high level in 23% of lung tumors. Six (21.4%) of the tumors with high CDK4 expression (n = 28) were shown by fluorescence in situ hybridization (FISH) to contain the 12q13.3-q14.1 amplification. For CDK4, a positive correlation was found between gene copy number (FISH and CGH array), mRNA expression (RT-PCR), and level of protein expression (IHC). CDK4 expression did not correlate with CDKN2A methylation status. Amplification of CDK4 has been described in other tumor types, but its role in lung cancer remains to be elucidated. Although CDK4 amplification seems to be a relatively rare event (4.3%) in lung tumors, it indicates the significance of the RB1-CCND1 pathway in lung tumorigenesis.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Comparison of mutagenic potentials and mutation spectra of benzene metabolites using supF shuttle vectors in human cells

Benzene is a human leukemogen and the metabolites are thought to be deeply involved in benzene leukemogenesis. In a previous study we reported the molecular analysis of p-benzoquinone (p-BQ) mutagenesis by using a supF shuttle vector plasmid and here we report the mutagenesis of the other metabolites, hydroquinone (HQ) and trans, trans-muconaldehyde (MUC). HQ is a precursor of p-BQ and MUC is produced by a ring-opening metabolic pathway. We found that the HQ redox cycle produced an oxidative lesion in plasmid DNA and significant differences among the mutagenic potentials of MUC, HQ and p-BQ. HQ has stronger mutagenicity than the others. It is about 20 and 600 times stronger than p-BQ and MUC, respectively. Furthermore, we found notable differences in each mutational feature. The MUC mutational type was characterized by a high frequency of tandem base substitutions that could be due to crosslinks produced by its aldehyde moieties, while HQ was characterized by frequent deletion. This HQ feature is the same as in vivo benezene mutagenesis of Big Blue mice reported by Provost et al. in 1996 and is also quite similar to a hydrogen peroxide mutational feature. Therefore, we presume that HQ and reactive oxygen species may play an important role in benzene carcinogenesis.     

Pseudotype hepatitis C virus enters immature myeloid dendritic cells through the interaction with lectin

Dendritic cells (DC) are the most potent antigen-presenting cells that regulate immune responses. One of the mechanisms for hepatitis C virus (HCV) persistence is the ability of HCV to suppress DC function. Direct HCV infection to blood DC has been implicated for DC dysfunction. To clarify the susceptibility of each DC subset to HCV, we used pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins (E1 and E2). We demonstrate that pseudotype VSV enters myeloid DC (MDC) but not plasmacytoid DC (PDC). The highest efficiency of pseudotype VSV entry to MDC was observed when MDC were cultured with GM-CSF. Such efficiency decreased when MDC are matured with the treatment of IL-4, CpG oligodeoxynucleotide, or CD40 ligand. Mannan inhibited pseudotype VSV entry to MDC, but Ca(2+) chelators failed to do so. These results show that pseudotype VSV possessing HCV-E1 and E2 enters immature MDC through the interaction with lectins in a Ca(2+)-independent manner.     

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.