Concentrations of cadmium, lead and zinc in livestock feed and organs around a metal production centre in eastern Kazakhstan.

This paper presents results of analysis of animal feed and meat (cattle, horse and sheep) products from a metal processing region (Oskemen) in east Kazakhstan. Samples were collected from a range of districts of differing distances from the main source of anthropogenic pollution and with differing underlying metal-containing geologies. Analyses for cadmium, lead and zinc revealed high concentrations in many feed and meat samples. Horse (an important food animal) samples had higher levels of contamination than cattle, which were higher than sheep. For example, mean cadmium concentrations in horse kidneys in one district were found to be 128 mg/kg and lead concentrations for liver 2.2 mg/kg. These, and other, results are generally higher than reported in many other studies in contaminated regions of eastern Europe and they can exceed State Maximal Allowed Concentrations by many times. As such levels of contamination pose a significant potential risk to human health, these results have formed the basis for subsequent research on levels of metal contamination in human tissues from affected populations.

Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs.

Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin alpha. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively. BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin alpha in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.

BRCA1 is a 220-kDa nuclear phosphoprotein that is expressed and phosphorylated in a cell cycle-dependent manner.

Mouse polyclonal antibodies, raised against three regions of the human BRCA1 protein, were characterized and revealed BRCA1 as a 220-kDa nuclear phosphoprotein in normal cells. All three antisera recognize both in vitro-translated and recombinant, baculovirus-derived BRCA1, which co-migrate with BRCA1 from the human breast epithelia cell line, HBL100. BRCA1 expression and phosphorylation are shown to be cell cycle dependent, with greatest expression and phosphorylation occurring in S and M phases. Cyclin-dependent kinase 2 and other kinases associated with cyclins D and A are shown to bind to and phosphorylate BRCA1, suggesting that the biological activity of BRCA1 may be regulated by cyclin-dependent kinases.

Isolation and characterization of the QM promoter.

This report describes the isolation, sequencing and preliminary characterization of the first 1 kb of the 5'-regulatory region of the human QM gene. This region and the 5' -half of the transcribed region of the QM gene are enriched for C and G nucleotides with no bias against CpG dinucleotides--indicative of a CpG island. Several consensus GC boxes are present within the sequence. Most are clustered at the distal end, with one site present in the proximal 200 bp of the promoter. Electrophoretic mobility shift experiments and luciferase assays done in insect cells transfected with an Sp1 expression construct suggest that most of these sites can bind Sp1 or a closely related factor. In addition, the promoter is shown to be responsive to cAMP via a response element (CRE) in the proximal promoter. Studies with 5'-end and internal deletion mutants suggest that elements in the distal promoter exert their positive effect through interactions with a proximal element(s). Candidate proximal elements include the proximal GC box and a 43 bp region between a KpnI site (at -182) and a Smal site (at -139).

The molecular basis of E2F-1/DP-1-induced S-phase entry and apoptosis.

The transcription factor E2F plays a critical role in the G1 to S transition. E2F is a heterodimer formed by members of the E2F and DP families of DNA-binding proteins. Ectopic expression of E2F-1, the first member of the E2F family identified, is sufficient to cause quiescent cells to enter S phase. Thus, the biological significance of the interaction of E2F-1 with its DP protein partner, DP-1, was unclear. Here, we report on the role of DP-1 in the mediation of E2F-induced S-phase entry and apoptosis. Cells inducible for DP-1, E2F-1, or both were established and characterized. Ectopic expression of DP-1 alone fails to promote cell cycle entry, even when the potent transactivation domain of human papillomavirus-VP16 is fused to the DNA-binding domain of DP-1. In contrast, coexpression of DP-1 and E2F-1 results in greater loss of G1 regulation and significantly more apoptosis than does E2F-1 alone. Using clones co-inducible for DP-1 and E2F-1, expression of potential target genes of E2F activity that may account for its ability to induce S-phase entry was also examined. Induction of E2F-1/DP-1 resulted in increased expression and activity of cyclins A and E, as well as CDK2, prior to S-phase entry. Cyclin D and CDK4, however, were not induced. Phosphorylation of the retinoblastoma protein is also increased following induction of E2F-1/DP-1, suggesting that E2F can feed-back on the retinoblastoma protein, presumably through activation of cyclin A- and/or E-associated kinase activity.

Design of a novel bicistronic expression vector with demonstration of a p16INK4-induced G(1)-S block(1).

Traditional eukaryotic gene expression systems have many shortcomings that make them unsuitable for the analysis of cytotoxic and growth-arrest genes. An intestinal alkaline phosphatase bicistronic expression vector was specifically designed to facilitate such studies. Intestinal alkaline phosphatase serves as a marker of cells that have been transfected and, therefore, must also be co-expressing the gene under study. Using flow cytometry, a trivariate analysis was performed on p16INK4-transfected U87 glioblastoma cells. An average G1-S block of 77.5% was demonstrated compared to controls despite a 1% transfection efficiency. This vector has universal applications, including: (a) analysis of cytotoxic and growth-inhibitory genes in transient assays; (b) 100% enrichement in gene expression studies, especially in low transfection efficiency experiments; and (c) facilitation of the study of cell cycle kinetics.

HMGI(Y) and Sp1 in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene.

Expression of the chemokine MGSA/GRO is upregulated as melanocytes progress to melanoma cells. We demonstrate that constitutive and cytokine induced MGSA/GRO alpha expression requires multiple DNA regulatory regions between positions -143 to -62. We have previously shown that the NF-kappa B element at -83 to -65 is essential for basal and cytokine induced MGSA/GRO alpha promoter activity in the Hs294T melanoma and normal retinal pigment epithelial (RPE) cells, respectively. Here, we have determined that the Sp1 binding element located approximately 42 base pairs upstream from the NF-kappa B element binds Sp1 and Sp3 constitutively and this element is necessary for basal MGSA/GRO alpha promoter activity. We demonstrate that the high mobility group proteins HMGI(Y) recognize the AT-rich motif nested within the NF-kappa B element in the MGSA/GRO alpha promoter. Loss of either NF-kappa B or HMGI(Y) complex binding by selected point mutations in the NF-kappa B element results in decreased basal and cytokine induced MGSA/GRO alpha promoter activity. Thus, these results indicate that transcriptional regulation of the chemokine MGSA/GRO alpha requires at least three transcription factors: Sp1, NF-kappa B and HMGI(Y).

Extreme evolutionary conservation of QM, a novel c-Jun associated transcription factor.

QM is a 214 amino acid polypeptide, encoded by a gene (DXS648) in Xq28, that contains a high percentage of charged amino acids and has been found to bind c-Jun and DNA. Searches of the GenBank database revealed no matches between QM and any other known transcription factors. However, we and others have isolated QM homologs from a diverse array of eukaryotes. Alignment of these sequences indicated a high degree of conservation throughout the first 175 residues of the protein and revealed several interesting features. Most notable is the considerable conservation of charged amino acids within specific regions of the protein. Secondary structure analysis suggests that two of these regions form amphipathic alpha-helices, one basic and one acidic. A third conserved charged domain, comprising the N-terminal 30 amino acids, is both basic and proline rich. The rate of sequence divergence of the various homologs was found to be slow (of the order of 1% change every 22 million years), consistent with a critical role for QM in eukaryotic cells. A role for QM as a novel class of transcription regulatory protein is suggested.

BWTG3 hepatoma cells can acquire phenylalanine hydroxylase, cystathionine synthase and CPS-I without genetic manipulation, but activation of the silent OTC gene requires cell fusion with hepatocytes.

The mouse hepatoma BWTG3 has been tested for its ability to grow in three different media that select for traits normally expressed in adult liver: homocysteine medium to select for cystathionine synthase (CS), tyrosine-free medium for phenylalanine hydroxylase (PH), and ornithine medium for carbamylphosphate synthetase-I (CPS-I) and ornithine transcarbamylase (OTC). In no case were the cells immediately capable of bulk growth, showing that all these traits were in some degree deficient. However, the cultures in homocysteine medium and in tyrosine-free medium both gave rise, spontaneously, to growing clones with frequencies of approximately 10(-3) and 10(-5), respectively. The deficiencies of CS and PH were accordingly excluded from further study, in view of their inherent instability. In contrast, no colonies ever formed in ornithine medium. Though neither CPS-I nor OTC were detectable in stock BWTG3 cells, it was found that CPS-I was readily inducible by hormones. The deficiency of OTC, however, appeared to be totally stable showing no reversion in response either to hormones or to azacytidine treatment. This deficiency was investigated by fusing the hepatoma to OTC+ liver cells prepared from normal or sparse-fur (spf) mice. Sparse-fur mice were used because their OTC is mutant and has a distinctive pH-dependence. OTC+ hybrids were readily produced, without the need for any specific selection for OTC, and, in one case at least, with only minimal chromosome segregation. In all the OTC+ hybrids made with spf cells, there was clear reactivation of the wild-type, hepatoma-derived OTC gene.(ABSTRACT TRUNCATED AT 250 WORDS)