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Background: An association between childhood maltreatment and subsequent alcohol abuse and/or dependence (AAD) has been found in multiple studies of females. Less is known about the association between childhood maltreatment and AAD among males, and the mechanisms that underlie this association in either gender. One explanation is that childhood maltreatment increases risk for AAD. An alternative explanation is that the same genetic or environmental factors that increase a child's risk for being maltreated also contribute to risk for AAD in adulthood., Method: Lifetime diagnosis of AAD was assessed using structured clinical interviews in a sample of 3527 male participants aged 19-56 years from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. The sources of childhood maltreatment-AAD association were estimated using both a matched case-control analysis of twin pairs discordant for childhood maltreatment and bivariate twin modeling., Results: Approximately 9% of participants reported childhood maltreatment, defined as serious neglect, molestation, or physical abuse occurring before the age of 15 years. Those who experienced childhood maltreatment were 1.74 times as likely to meet AAD criteria compared with males who did not experience childhood maltreatment. The childhood maltreatment-AAD association largely reflected environmental factors in common to members of twin pairs. Additional exploratory analyses provided evidence that AAD risk associated with childhood maltreatment was significantly attenuated after adjusting for measured family-level risk factors., Conclusions: Males who experienced childhood maltreatment had an increased risk for AAD. Our results suggest that the childhood maltreatment-AAD association is attributable to broader environmental adversity shared between twins.

This study was conducted in prostate cancer patients in biochemical relapse after radical prostatectomy, to assess the feasibility, safety, and immunogenicity of therapeutic vaccination with autologous dendritic cells (DCs) pulsed with human recombinant prostate-specific antigen (PSA) (Dendritophage-rPSA). Twenty-four patients with histologically proven prostate carcinoma and an isolated postoperative rise of serum PSA (>1 ng/ml to 10 ng/ml) after radical prostatectomy were included. The patients received nine administrations of PSA-loaded DCs by combined intravenous, subcutaneous, and intradermal routes over 21 weeks. Postbaseline blood tests were performed at months 1, 3, 6, 9, and 12 (PSA levels), at months 6 and 12 (circulating prostate cancer cells), at month 6 (anti-PSA IgG and IgM antibodies), and at up to eight time points before, during, and after immunization (PSA-specific T cells). Circulating prostate cancer cells detected in six patients at baseline were undetectable at 6 months and remained undetectable at 12 months. Eleven patients had a postbaseline transient PSA decrease on one to three occasions, predominantly occurring at month 1 (7 patients) or month 3 (2 patients). Maximum PSA decrease ranged from 6% to 39%. PSA decrease on at least one occasion was more frequent in patients with low Gleason score ( p=0.016) at prostatectomy and with positive skin tests at study baseline ( p=0.04). PSA-specific T cells were detected ex vivo by ELISpot for IFN-gamma in 7 patients before vaccination and in 11 patients after vaccination. Of the latter 11 patients, 5 had detectable T cells both before and during the vaccination period, 4 only during the vaccination period, while 2 patients could for technical reasons not be assessed prevaccination. No induction of anti-PSA IgG or IgM antibodies was detected. There were no serious adverse events or otherwise severe toxicities observed during the trial. Immunization with Dendritophage-rPSA was feasible and safe in this cohort of patients. An immune response specific for PSA could be detected in some patients. A notable effect was the disappearance of circulating prostate cells in all patients who were RT-PCR positive before vaccination.

Subpopulations of human leukocyte antigen (HLA) class II molecules were studied in antigen presenting cells. We present evidence for double dimers or "superdimers" of HLA class II molecules that were stable in an SDS solution at room temperature but dissociated when heated to 50 degrees C into 60-kDa alphabeta heterodimers. Development of an immunofluorescence assay allowed us to quantify the expression of HLA antigens as reflected by the number of bound isotype-specific monoclonal antibodies per cell. The total expression of class II (DR, DQ, and DP) augmented 6-fold after a 36-h interferon-gamma (IFNgamma) treatment of freshly isolated monocytes. Next, we used a recombinant and fluorescein-conjugated form of the class II-associated invariant chain as a quantitative probe for empty peptide-binding sites. The fraction of empty class II molecules was 0.73-2.9% in resting monocytes but was reduced to 0. 12-0.5% of the total after IFNgamma treatment. The fraction of empty sites in B lymphocytes was 0.09-0.36%. The mean number of empty sites per cell were: 6.3 x 10(3) (monocytes), 7.2 x 10(3) (IFNgamma-activated monocytes), 5.2 x 10(2) (B lymphocytes), and 3.6 x 10(3) (Raji B cells). A minor population (4.3-7.4% of total cells), which expressed a much higher number of empty sites, was consistently present in all cell types studied.

The human T-cell receptor Vbeta2-, D- and J-encoding domains were PCR-amplified from MOLT-4 total cDNA and subcloned in Escherichia coli. The V/D/J fragment was subsequently transferred to a prokaryotic expression vector in frame with a polyhistidine-encoding prosequence which enabled us to affinity-purify the fusion protein with IMAC (immobilized metal-ion affinity chromatography [correction of chromatorgraphy]). Since the recombinant (re-) human T-cell receptor Vbeta2 fusion protein (Vbeta2 sol) produced in E.coli was found to be insoluble, purification was carried out under denaturating conditions. The purified and renatured re-protein, Vbeta2 sol, was immunoreactive with an anti-Vbeta2 monoclonal antibody in an ELISA assay. The specificity of Vbeta2 sol was shown by its binding in vitro to the staphylococcal superantigen TSST-1, but not to the Staphylococcus aureus exotoxin-1 (SEA).

Stimulation of human B cells via HLA class II antigens leads to an increase of PKC activity as a consequence of a transcriptional upregulation of the PKC. Extending previous data, other known B-cell activators, which include anti-IgM, SAC, and TSST1, are shown here to increase the cytosolic PKC activity significantly. Human B cells express significant mRNA levels of the PKC alpha, beta, delta, epsilon, and zeta species while the gamma species is consistently absent. The levels of PKC alpha and epsilon mRNA are increased by exposure to a nonmitogenic anti-IgM antibody in a lymphoblastoid B-cell line while PKC beta and delta mRNA are instead downregulated by this agent. An anti-HLA class II antibody (D1.12) induced an increase of PKC alpha, beta, and delta mRNA. A time study of PKC mRNA levels in anti-IgM-treated cells showed that the accumulation of the PKC alpha mRNA precedes the increase of PKC enzymatic activity. Moreover, PKC beta mRNA decreased following treatment with SAC while, on the contrary, it increased following TPA, anti-HLA class II (1.35) mAb, or mitogenic anti-IgM treatment. Our results underline the complexity of signal transduction via the PKC pathway by revealing that the PKC isoforms are differentially regulated and are in keeping with the idea that they may have distinct physiologic roles in human B cells.

The invariant chain is a membrane protein associated with the major histocompatibility complex class II antigens both intra- and extracellularly. The extracellular portion of the human invariant chain (Ii) was expressed in Escherichia coli as a fusion protein with a polyhistidine tail and purified by metal affinity chromatography. The recombinant Ii was used as a ligand to probe binding to the cell surface of Chinese hamster ovary cells stably transfected with human class II alpha and beta genes of the DR4 isotype. We show that recombinant Ii inhibits peptide loading on class II polypeptides and also the converse; the presence of peptide in the antigen groove prevents binding of fluorescein-conjugated Ii. Moreover, blocking of Ii binding by peptide did not require a transition of the class II dimers to an SDS-stable state. A monoclonal antibody, L243, known to bind to (or close to) the peptide pocket of the class II molecule likewise blocked Ii-fluorescein binding. Further, we investigated whether or not the Ii, a variety of bacterial superantigens or the CD4 molecule, have overlapping binding sites on the class II heterodimer. Of the class II ligands tested, reduced binding was detected for the Staphylococcus superantigen type SEB on cells precincubated with soluble Ii while the binding of the other ligands was either unchanged or marginally changed. These data clarify by a direct biochemical approach the binding characteristics of Ii in comparison with other class II ligands.

HLA class II molecules are constitutively expressed on human B lymphocytes and are induced on human T lymphocytes after activation, through which signal transduction via these molecules has been extensively described. We have observed cell death of as many as 60% after stimulation of lymphocytes via HLA class II DR molecules, but not via DP or DQ. Propidium iodide fluorescence, DNA fragmentation and morphology of the dead cells were examined. The reported cell death was very rapid, and independent of Fc receptors and of complement. A morphologically distinct sub-population of activated B cells was sensitive to HLA-DR mediated death, while resting B lymphocytes from the same donor did not die as a result of HLA class II mediated stimulation. Death via HLA-DR was distinguishable from necrosis. Cytoskeletal integrity, serine/threonine phosphatase activity and endonucleases were required for the pathway leading to HLA-DR mediated death. The 'ladder' pattern of DNA fragmentation which typically characterizes apoptosis was not observed, despite the observation of cell and nuclear shrinkage normally associated with apoptosis. These data suggest that HLA class II mediated death is a means of rapidly removing either T or B lymphocytes which have already served their role in the immune response, thereby avoiding the inflammatory responses associated with necrosis and concentrating the ligands for new TCR and/or CD4 interactions.

A human thioredoxin cDNA was modified to optimize Escherichia coli expression and subcloned into the plasmid pACA, a vector for T7 RNA polymerase-directed expression. The substitution of structural (noncatalytic) half-cystines in human thioredoxin (hTrx) was made by site-directed mutagenesis. The recombinant wild-type (wt) hTrx and its mutant C61S, C72S, and C61S/C72S were expressed and purified to homogeneity. Characterization of the wt and mutant hTrx was done with respect to redox activity with thioredoxin reductase (TR), tryptophan fluorescence, and effects of incubation with GS-Se-SG, which is believed to be the major metabolite of inorganic selenium compounds in mammalian tissues. The Km and kcat of wild-type hTrx for human placenta thioredoxin reductase (HP-TR) at pH 7.0 were 2.0 microM and 2800 min-1, respectively. The mutant proteins C61S, C72S, and C61S/C72S had Km and kcat values similar to those of the wt thioredoxin. Tryptophan fluorescence measurements showed that the wt and mutant proteins had similar stability to a denaturing agent. Incubation of fully reduced thioredoxin with 0.1 molar equivalent of GS-Se-SG resulted in continued oxidation of SH groups. After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin remained. With the oxidized protein, a pronounced lag phase in thioredoxin reductase-dependent insulin disulfide reduction was present. Disulfide-linked dimers of the protein were present. The results clearly showed that noncatalytic cysteine residues in hTrx were oxidized accompanied by dimerization and inactivation. The activities of the mutant proteins C72S and C61S/C72S were unchanged after 3 h of incubation with GS-Se-SG. No dimer appeared of the C72S thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The redox-active enzyme thioredoxin (Trx) is secreted by various virus-transformed cell lines of B- and T-cell origin and has been considered to play an autoregulatory role as a cofactor during cellular growth processes. We show in this paper that exposure of B lymphocytes from normal, healthy donors and B cells from B-type chronic lymphocytic leukemia (B-CLL) to Staphylococcus aureus Cowan I (SAC) induced expression of Trx mRNA. By combining SAC, or the phorbol ester TPA, with IL-2 and the conditioned medium of a T-cell hybridoma (BSF-MP6), we could strongly enhance the Trx expression. After [35S]methionine labeling of stimulated B-CLL cells in vitro, Trx was immunoprecipitated both from cell extracts and from the medium with antibodies against human placenta Trx. Secretion of newly synthesized Trx was also confirmed by a quantitative radioimmunoassay for human Trx. During 24 h cultivation experiments, treatment with SAC induced a 5-fold increase of the Trx content of normal B lymphocytes as well as in B-CLL cells. Approximately two-thirds of the total amount of the enzyme was released into the medium.

Napin is a seed storage protein from Brassica napus (rape) that is encoded by a gene family. We have isolated and characterized a novel napin gene, napB. Comparisons of the 5'-upstream region of napB to the promoter regions of previously published napin genes reveal that certain sequence motives are evolutionary conserved and may be implicated in gene regulation. These consensus motives, that overlap with purine/pyrimidine stretches, are TACACAT and CATGCA both of which frequently occur as overlapping, direct repeats. Related or identical sequences are also found in the upstream regions of the homologous genes of Arabidopsis thaliana. One copy of the CATGCA motif occurs in close proximity to the TATA box in all the above genes. In this case it overlaps with an octamer sequence (ATGCAAAT) which is a sequence element common in many eukaryotic promoters and enhancers. The TACACAT sequence, as part of a longer purine/pyrimidine stretch, was found to interact with a protein present in crude nuclear extracts from developing B. napus seeds. Napin genes appear to be methylated to almost equal extents whether present in expressing or non-expressing tissue.

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.

Napin (1.7 S protein) is a basic, low molecular weight storage protein synthesized in rapeseed (Brassica napus) embryos during seed development. Napin is composed of two polypeptide chains with molecular weights of 9000 and 4000 that are held together by disulfide bonds. Comparison of the deduced amino acid sequence of a napin cDNA clone with that of napin peptide fragments established that napin is initially synthesized as a precursor of 178 residues. This polypeptide is subsequently processed through several proteolytic events, which ultimately generate the two mature napin chains, of 86 and 29 residues, respectively. Protein biosynthesis in vitro showed that the initial translation product (Mr 20,000) contains a signal sequence which is removed during transfer of the protein into the endoplasmic reticulum. Two additional peptides, of 22 and 19 residues, as well as the COOH-terminal residue, are also removed during maturation of napin, as deduced from the sequence comparison. Comparisons of the napin sequence with other known protein sequences established that there is a significant homology between napin and two other small seed proteins, the castor bean storage protein and a trypsin inhibitor from barley.

A rapeseed chromosomal region containing a gene (napA), which encodes the 1.7 S seed storage protein (napin), was isolated in several overlapping recombinant clones from a phage lambda genomic library. Following restriction enzyme mapping of the genomic region, a subclone containing the napA coding region as well as some 1.1 and 1.4 kilobases of DNA from the 5' and 3' regions, respectively, was mapped and sequenced. The gene turned out to lack introns. Southern blotting analyses utilizing a napin cDNA clone as a probe revealed the presence of on the order of 10 napin genes in the rapeseed genome. The major polyadenylated transcript encoded by these genes was shown to be an 850-nucleotide species, the initiation site of which was mapped onto the napA gene. The major initiation site for transcription is located some 33 nucleotides downstream from a sequence perfectly conforming to the consensus sequence of a TATA box. Further analyses of the sequence revealed several features that may be of relevance for the expression of the napin genes.