A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells

BACKGROUND: This study reports relationships between suicidal behavior and its risk factors in prepubertal children and whole blood and platelet serotonin-related measures. METHODS: Seventy-five prepubertal psychiatric inpatients including 23 (30.7%) nonsuicidal, 32 (42.7%) with suicidal ideation, and 20 (26.6%) with a suicide attempt were compared to 35 normal prepubertal controls with regard to platelet serotonin content, serotonin-amplified platelet aggregation, and whole blood tryptophan. RESULTS: Mean whole blood tryptophan content was significantly lower among inpatient children with a recent suicide attempt than among normal controls or inpatients with suicidal ideation (F = 3.94, df = 3.54, p < or = .01). Inpatient children with a mood disorder had significantly higher platelet serotonin content than inpatients without a mood disorder (F = 3.86, df = 2.80, p < or = .03). Racial/ethnic differences were also observed for inpatients and normal controls, with whites having significantly lower levels of platelet serotonin (expressed as ng/mL blood or ng/10(9) platelets) than blacks or Latinos. Blacks had significantly higher levels of whole blood tryptophan than other racial/ethnic groups. CONCLUSIONS: The results suggest that whole blood tryptophan and platelet serotonin content should be studied for their predictive validity as risk factors for suicidal behavior in youth while controlling for racial/ethnic differences.     

Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.     

DNA topoisomerase II alpha expression is associated with alkylating agent resistance

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.     

Seroepidemiologic studies of hantavirus infection among wild rodents in California

To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli. The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor. The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes. Further analyses by slot blot revealed high affinity of the immobilized protein with significant reaction at 10 pmol. As the immobilized receptor protein also reacted with structurally related hormones (125I-labeled LH/125I-labeled human chorionic gonadotropin), we confirmed that interaction most probably occurred via the common alpha-subunit of these glycoprotein hormones. Our results reveal that this N-terminal portion of the FSH receptor contain(s) major site(s) for hormone recognition that could be mediated via the alpha-subunit. A rabbit antibody to the receptor inhibited FSH action in receptor bearing cells, revealing the utility of such recombinant FSH receptor protein(s) for modulation of hormone action.     

The participation of the genetic apparatus in the mechanisms of memory trace formation: the role of the calcium-regulatory system of the neurons in rats

Historically, formalin fixed (FF) tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction (PCR) technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study evaluates the potential use of DNA from FF and formalin fixed paraffin embedded (FFPE) tissues in 13 PCR systems; HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, GC, D1S80, vWA31, THO1, F13A1, FES/FPS, TPOX, and CSF1PO. The first six, HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, and GC are reverse dot blot systems, D1S80 is an amplified fragment length polymorphism (AmpFlp) system and the others are short tandem repeats (STRs). This study shows that FFPE tissue which has not been fixed in formalin for more than three days is a useful source of DNA for 12 of the 13 PCR systems. In contrast, FF tissue did not prove to be a reliable source of DNA for the PCR techniques examined here.     

Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/dA adduct: glycosidic rotation from syn to anti at the modified deoxyadenosine

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.     

The efficacy, safety and tolerability of antidepressants in late life depression: a meta-analysis

BACKGROUND: To determine the efficacy, safety and tolerability of antidepressants in depressed elderly patients. METHODS: Search for randomized controlled double-blind studies evaluating atypical antidepressants (ATYPs), reversible inhibitors of monoamine oxidase-A, selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants in moderate/severe depressed patients > or = 60 years for > or = four weeks. The random effects model (single-arm; comparative) was used to aggregate efficacy, safety and dropout. RESULTS: No difference in single-arm aggregation of outcomes for four antidepressant classes. Comparative analyses showed no statistical difference between outcomes, except SSRIs had a higher response rate than ATYPs. CONCLUSION: Elderly show no differences in antidepressant class outcomes. LIMITATIONS: Heterogeneity and lack of power. CLINICAL RELEVANCE: There is little advantage for antidepressant classes over another in the aged.     

Three-dimensional segregation of supramolecular activation clusters in T cells

Activation of T cells by antigen-presenting cells (APCs) depends on the complex integration of signals that are delivered by multiple antigen receptors. Most receptor-proximal activation events in T cells were identified using multivalent anti-receptor antibodies, eliminating the need to use the more complex APCs. As the physiological membrane-associated ligands on the APC and the activating antibodies probably trigger the same biochemical pathways, it is unknown why the antibodies, even at saturating concentrations, fail to trigger some of the physiological T-cell responses. Here we study, at the level of the single cell, the responses of T cells to native ligands. We used a digital imaging system and analysed the three-dimensional distribution of receptors and intracellular proteins that cluster at the contacts between T cells and APCs during antigen-specific interactions. Surprisingly, instead of showing uniform oligomerization, these proteins clustered into segregated three-dimensional domains within the cell contacts. The antigen-specific formation of these new, spatially segregated supramolecular activation clusters may generate appropriate physiological responses and may explain the high sensitivity of the T cells to antigen.     

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.