Mechanism of histone deacetylase inhibitor Trichostatin A induced apoptosis in human osteosarcoma cells

Although histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in malignancy, how they exert their effect on osteosarcoama cells is as yet unclear. This study was undertaken to investigate the underlying mechanism of a HDAC inhibitor Trichostatin A (TSA)-induced apoptosis in a osteosarcoma cell line HOS. We observed that TSA treatment decreased the viability of the cells and prominently increased acetylation of histone H3. Evidence was obtained indicating that TSA induced apoptosis of HOS cells as follows: (1) Generation of DNA fragmentation; (2) activation of procaspase-3; (3) cleavage of PARP; and (4) increase of DNA hypoploidy. The reduction of MMP and the release of cytochrome c to cytosol were also shown, indicating that TSA induces apoptosis in HOS cells in a histone acetylation- and mitochondria-dependent fashions. We also examined whether TSA can sensitize HOS cells to the action of an antitumor agent genistein. The combination therapy of TSA and genistein showed synergistic anticancer effect indicating that TSA can be considered as a novel therapeutic strategy for osteosarcoma not only from its direct apoptosis-inducing activity but also from the possibility of sensitization to other antitumor agents.     

Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.     

Cutting edge: MHC class II-restricted peptides containing the inflammation-associated marker 3-nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune response

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-E(K)-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC(88-103)) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC(88-103) with nitrotyrosine abrogates recognition by the MCC(88-103)-specific T cell hybridoma 2B4. CBA (H2(K)) mice immunized with MCC(88-103) or nitrated MCC(88-103) peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC(88-103), but exhibited a robust immune response against nitrated MCC(88-103). Analysis of T cell hybridomas specific for nitrated-MCC(88-103) indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.     

Prevalence of antibodies to selected viruses in a long-term closed breeding colony of rhesus macaques (Macaca mulatta) in Brazil

The rhesus macaque breeding colony of the Oswaldo Cruz Foundation (FIOCRUZ) was established in 1932 from a founding stock of 100 animals. This population has remained closed to new animal introductions for almost 70 years. A serologic survey was performed to determine the prevalence of antibodies to selected viruses as a first approach to identifying viral pathogens endemic in this population. Banked serum samples were tested for antibodies to simian immunodeficiency virus (SIV), simian T-lymphotropic virus (STLV), simian type D retrovirus (SRV), cercopithecine herpesvirus type-1 (B virus), rhesus cytomegalovirus (RhCMV), measles virus (MV), and hepatitis A virus (HAV). All samples were negative for antibodies against the simian retroviruses. The overall prevalence of antibodies was 95% for RhCMV, 45% for B virus, 35% for HAV, and 1% for MV. Prevalence was found to vary by age group.     

Solution structure of the yeast ubiquitin-like modifier protein Hub1

abbreviationsUBL, ubiquitin-like modifier; Saccharomyces cerevisiae, S. cerevisiae; Eschericia coli, E. coli; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlated spectroscopy.     

Clustering gene-expression data with repeated measurements

Clustering is a common methodology for the analysis of array data, and many research laboratories are generating array data with repeated measurements. We evaluated several clustering algorithms that incorporate repeated measurements, and show that algorithms that take advantage of repeated measurements yield more accurate and more stable clusters. In particular, we show that the infinite mixture model-based approach with a built-in error model produces superior results.     

Apoptotic index from fine needle aspiration cytology as a criterion to predict histologic grade of non-Hodgkin's lymphoma

OBJECTIVE: To investigate whether the assessment of apoptotic index (AI) from fine needle aspiration (FNA) smears of non-Hodgkin's lymphomas (NHL) is reliable and has potential utility as a criterion to predict histologic grade. STUDY DESIGN: AI was independently determined by four cytopathologists as a percentage from routine FNA smears in 96 NHLs and 15 lymphoid hyperplasias. Working formulation (WF) grades from corresponding surgical biopsies were modified to include mantle zone-derived NHLs as intermediate grade and to make diffuse large cell NHL a separate category called "high" grade, whereas WF high grade NHLs were called "very high" grade. Histologic grades were also derived from the Revised European American Lymphoma (REAL) classification. AI was compared with histologic grade using the unpaired, two-tailed Student t test. These data were used to determine potential thresholds for AI that separate lower from higher grade NHLs. RESULTS: Measurements of AI strongly correlated between cytopathologists (median r = .93). Low and intermediate grade NHLs had indistinguishable AIs, whereas higher grade NHLs had significantly higher AIs. Appropriate potential AI thresholds between low or intermediate grade and higher grade NHLs were in the range of 1.5-2.5% (modified WF) and 1-2% (REAL). CONCLUSION: There is excellent interobserver reliability in the measurement of AI from FNAs of NHLs. Higher AIs distinguish higher from lower grade NHLs. Diffuse large cell NHLs had AIs that were similar to WF high grade NHLs.     

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.     

Characterization of a novel insertional mouse mutation, kkt: A closely linked modifier of Pax1

We describe a novel transgene insertional mouse mutant with skeletal abnormalities characterized by a kinked tail and severe curvature of the spine. The disrupted locus is designated kkt for "kyphoscoliosis kinked tail." Malformed vertebrae including bilateral ossification centers and premature fusion of the vertebral body to the pedicles are observed along the vertebral column, and the lower thoracic and lumbar vertebrae are the most affected. Some of the homozygous kkt neonates displayed two backward-pointing transverse processes in the sixth lumbar vertebra (L6) that resembled the first sacral vertebra, and some displayed one forward- and one backward-pointing transverse process in L6. The fourth and fifth sternebrae were also fused, and the acromion process of the scapula was missing in kkt mice. The skeletal abnormalities are similar to those observed in the mouse mutant undulated (un). The transgene is integrated at the distal end of chromosome 2 close to the Pax1 gene, as revealed by FISH analysis. However, mutation of the Pax1 gene is responsible for the un phenotype, but the Pax1 gene in the kkt mice is not rearranged or deleted. Pax1 is expressed normally in kkt embryos and in the thymus of mature animals, and there is no mutation in its coding sequence. Thus, the skeletal abnormalities observed in the kkt mutant are not due to a lack of functional Pax1. Mouse genomic sequences flanking the transgene and PAC clones spanning the wild-type kkt locus have been isolated, and reverse Northern analysis showed that the PACs contain transcribed sequence. Compound heterozygotes between un and kkt (un(+/-)/kkt(+/-)) display skeletal abnormalities similar to those of un or kkt homozygotes, but they have multiple lumbar vertebrae with a split vertebral body that is more severe than in homozygous un or kkt neonates. Furthermore, the sternebrae are not fused and no backward-pointing transverse processes are detected in L6. It is therefore apparent that these two mutations do not fully complement each other, and we propose that a gene in the kkt locus possesses a unique role that functions in concert with Pax1 during skeletal development.     

Simian immunodeficiency viruses of diverse origin can use CXCR4 as a coreceptor for entry into human cells

Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [n = 6]), stumptail (SIVstm [n = 1]), mandrill (SIVmnd [n = 1]), and African green (SIVagm [n = 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5(+/+) donor, and seven of eight isolates tested also infected CCR5(-/-) PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.