Effect of Zn(II) on the reduction and accumulation of Cr(VI) by Arthrobacter species.

Natural habitats are often characterized by the coexistence of Zn and Cr. This study assessed the potential of two Gram-positive, Cr(VI)-reducing, aerobic bacterial strains belonging to Arthrobacter genera, which were isolated from basalt samples taken from the most polluted region of the Republic of Georgia, to remediate Cr(VI) in environments in the presence of Zn(II). Our batch experiments revealed that the addition of Zn(II) to the tested bacterial cells significantly enhanced the accumulation of Cr. According to electron spin resonance (ESR) measurements, the presence of Zn(II) ions did not change the nature of Cr(V) and Cr(III) complexes generated during the microbial reduction of Cr(VI). The efficiency of Cr(VI) reduction also remained unchanged after the addition of 50 mg/l of Zn(II) to the bacterial cells. However, at high concentrations of Zn(II) (higher than 200 mg/l), the transformation of Cr(VI) to Cr(V) and Cr(III) complexes decreases significantly. In addition, it was shown that the accumulation pattern of Zn in the tested bacterial species in the presence of 100 mg/l of Cr(VI) fits the Langmuir-Freundlich model well. The two tested bacterial strains exhibited different characteristics of Zn accumulation.

Tumor etiology and chromosome pattern.

Fibrosarcomas induced in Chinese hamsters and rats by Rous sarcomla virus and 7,12-dimethylbenz(a)anthracene are associated with nonrandom chromosome variation. Although histologically indistinguishable, the tumors induced by the virus or chemical in each host species are characterized by completely different karyotypic patterns.

Amplicon structure in multidrug-resistant murine cells: a nonrearranged region of genomic DNA corresponding to large circular DNA.

Multidrug resistance (MDR) in tumor cell lines is frequently correlated with amplification of one or more mdr genes. Usually the amplified domain also includes several neighboring genes. Using pulsed-field gel electrophoresis, we have established a restriction map covering approximately 2,200 kb in the drug-sensitive mouse tumor cell line TC13K. The mapped region is located on mouse chromosome 5 and includes the three mdr genes, the gene for the calcium-binding sorcin protein, and a gene with unknown function designated class 5. Long-range maps of the amplified DNA sequences in five of six MDR sublines that had been independently derived from TC13K generally displayed the same pattern as did the parental cell line. All six MDR sublines exhibited numerous double minutes, and one of them displayed a homogeneously staining region in a subpopulation. Large circular molecules, most likely identical to one chromatid of the double minutes, were detected in four of the sublines by linearization with gamma irradiation. The size of the circles was about 2,500 kb, which correlated to a single unit of the amplified domain. We therefore propose that in four independent instances of MDR development, a single unit of about 2,500 kb has been amplified in the form of circular DNA molecules. The restriction enzyme map of the amplified unit is unchanged compared with that of the parental cell line, whereas the joining sites of the circular DNA molecules are not identical but are in the same region.

Chromosome ideograms of the laboratory rat (Rattus norvegicus) based on high-resolution banding, and anchoring of the cytogenetic map to the DNA sequence by FISH in sample chromosomes.

A detailed banded ideogram representation of the rat chromosomes was constructed based on actual G-banded prometaphase chromosomes. The approach yielded 535 individual bands, a significant increase compared to previously presented ideograms. The new ideogram was adapted to the existing band nomenclature. The gene locus positions in the rat draft DNA sequence were compared to the chromosomal positions as determined by dual-color FISH, using rat (RNO) chromosomes 6 and 15 and a segment of RNO4 as sample regions. It was found that there was generally an excellent correlation in the chromosome regions tested between the relative gene position in the DNA molecules and the sub-chromosomal localization by FISH and subsequent information transfer on ideograms from measurements of chromosomal images. However, in the metacentric chromosome (RNO15), the correlation was much better in the short arm than in the long arm, suggesting that the centromeric region may distort the linear relationship between the chromosomal image and the corresponding DNA molecule.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces genetic changes in murine intestinal tumours and cells with ApcMin mutation.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the mutagenic heterocyclic amines derived from cooked meat. In previous animal studies, spontaneous tumour formation in B6(Min/+) mice was associated with somatic loss of the wild-type Apc+ allele by loss of the entire chromosome 18 or by recombination. The objective of this study was to examine genetic changes caused by PhIP-exposure in a mouse intestinal cell line and in tumours from hybrid mice by keeping track of the chromosomes carrying the two Apc alleles. We transformed the SV40 T-immortalised intestinal epithelial cell line IMCE, derived from the B6(Min/+) mice by exposure to N-OH-PhIP, and studied the effect on Apc status and chromosome 18. Eighteen transformed cultures were obtained and all of them had retained the Apc+ allele. Five of seven transformed cultures were tumorigenic after implantation in nude mice. Chromosomal analysis of these five cultures and the parent IMCE cell line showed that the IMCE cells were near-tetraploid with an average of 77 chromosomes/cell, while the tumorigenic cell cultures were all triploid to hyper-triploid with a range of 61-69 chromosomes/cell. The number of copies of chromosome 18 was about four in the IMCE line and this copy number was retained in the transformed lines derived from IMCE. Changes in chromosome 18 and Apc during tumour development in vivo were examined in spontaneously formed and PhIP-induced intestinal tumours from two hybrid mice strains, i.e. B6(Min/+) - a murine FAP model - crossed with either AKR/J or A/J. We evaluated the allelic status of Apc, and the heterogenic microsatellite markers D18Mit19 and D18Mit4, located at the upper and lower ends of chromosome 18, respectively. In tumours from untreated animals, instability in the D18Mit19 and Apc was observed. Upon PhIP exposure, the B6(Min/A+) hybrid mouse tumours differed distinctly in genetic profile from those obtained from untreated animals and we detected three genetically different tumour groups, all of which had apparently retained Apc+. One group had allelic balance between the Apc(Min) and Apc+, the second had allelic imbalance between the Apc and D18Mit4 alleles, indicative of chromosomal stability in the first group and instability in the lower end of chromosome 18 in the second group, respectively. The third group showed variable allelic status of the three markers. A similar change in genetic profile was also seen in intestinal tumours of PhIP-exposed B6(Min/AKR+) hybrid mice, but it was less pronounced. Chromosomal breaks and/or recombinational events could be alternative explanations for the observed allelic imbalances in chromosome 18 markers in intestinal tumours from PhIP-exposed mice.

The Republic of Georgia High Blood Pressure Control Program.

INTRODUCTION: 52% of adults have uncontrolled hypertension in the Republic of Georgia. We incorporated a blood pressure control program into an existing primary healthcare system in an attempt to improve the rate of blood pressure control. METHODS: We conducted standardized trainings of rural primary care providers--doctors and nurses--in accurate measurement of blood pressure according to the Shared Care Method of Training and Certification. Our attention was focused especially on patient management based on Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC) guidelines. Antihypertensive treatment was implemented by a stepped-care approach; hydrochlorothiazide and atenolol were given to patients at follow-up visits at no cost. The treatment goal was < 140/ 90 mm Hg based on the office blood pressure. RESULTS: A total of 251 patients with uncontrolled hypertension were enrolled in the program; 32% had stage I hypertension, 41% had stage II hypertension, and 27% had stage III, as defined by JNC VI. During the first 30 months of followup, blood pressure decreased gradually from 170/95 to 140/ 82 mm Hg. The rate of high blood pressure control increased progressively up to 59%. CONCLUSIONS: We conclude that hypertension control can be improved in all groups of patients, even in a healthcare system with limited resources. We emphasize that Georgia or any other healthcare system should not wait for universal health care to improve high blood pressure control. It can be incorporated into whatever system exists today.

Absence of late-replicating X-chromosome in a female patient with acute myeloid leukemia and the 8;21 translocation.

Autoradiography was used to demonstrate that the x-chromosome of the 45,X,-X,t(8;21) stemline of a female patient with acute myeloid leukemia (AML) was the active X-chromosome. This suggested that in patients housing AML with the 8;21 translocation, the loss of the inactive X-chromosome in females and of the Y in males (which is known to occur in nearly half of the patients) entails selective advantage to the stemline.

Independent amplification of two gene clusters on chromosome 4 in rat endometrial cancer: identification and molecular characterization.

The BDII rat is genetically predisposed to hormone-dependent endometrial adenocarcinoma and was used to model human cancer. Tumors arising spontaneously in strain crosses involving BDII rats were analyzed by means of comparative genome hybridization. The most common aberration was amplification of the proximal region of rat chromosome 4, centered around bands q12-q22. The copy numbers of 15 cancer-related genes from the region were examined in tissue cultures of 11 endometrial carcinomas (10 endometrial adenocarcinomas and 1 endometrial squamous cell carcinoma) and one peritoneal mesothelioma. Amplification in rat chromosome 4 was detected in six tumors (50%), five of which carried two separate amplified regions, situated at 4q12-q13 and 4q21-q22, interrupted by a nonamplified segment at 4q13-q21.1. The genes Cdk6 (cyclin-dependent kinase 6) and Met (hepatocyte growth factor receptor) were located in the core of each amplified region and were amplified most recurrently and at the highest levels among the genes tested. Using fluorescence in situ hybridization on tumor metaphases, it was observed that the amplified Cdk6 and Met sequences were situated on typical homogeneously staining regions (HSRs). In three tumors, both genes were amplified in the same HSRs, whereas in two tumors, the amplified sequences of each gene were situated in separate HSRs. In addition, Cdk6 and Met amplification was consistently associated with a corresponding increase in gene expression, suggesting that the two genes might represent the targets for the amplifications. In the sixth tumor, which carried amplified sequences of Met but not of Cdk6, coexpression of Met and the normally silent hepatocyte growth factor gene (Hgf; the ligand of Met) was observed. This finding suggests that an autocrine signaling circuit might be operating in this particular tumor. Taken together, our findings suggest that up-regulation of Cdk6 and/or Met may contribute to the development of endometrial cancers in the BDII rat.

Tumorigenicity of SEWA murine cells correlates with degree of c-myc amplification.

Previous studies have shown that cells of the SEWA mouse tumor contain amplified copies of the proto-oncogene c-myc in the aberrant chromosomal structures of double minutes (DMs), homogeneously staining regions (HSRs) and C-bandless chromosomes (CMs). DMs, and to a lesser degree CMs, tend to disappear from the cells grown in vitro and again reappear after transfer back in vivo, as if DNA amplification confers a growth advantage upon the tumor cells. We have now isolated five in vitro clones that exhibit different degrees of c-myc amplification. When we inoculated cells of the different clones into compatible hosts, we found that there was a positive correlation between degree of c-myc amplification, level of c-myc RNA, and tumorigenicity. Our results lend further support to the idea that gene amplification contributes to the higher malignant phenotype, and to progression of tumors.

Chromosomal assignment of five cancer-associated rat genes: two thyroid hormone receptor (ERBA) genes, two ERBB genes and the retinoblastoma gene.

Using a panel of somatic cell hybrids that segregate rat chromosomes, the localization of five cancer-related rat genes was determined: (i) two thyroid receptor genes, THRA1/ERBA1 and THRB/ERBA2 on chromosomes 10 and 15 respectively, (ii) two ERBB genes, namely the epidermal growth factor gene (EGFR, also called ERBB1) and the ERBB2 gene (also designated neu) on chromosomes 14 and 10 respectively, and (iii) the retinoblastoma gene, RB1, on chromosome 15. The THRA1/ERBA1 and ERBB2/neu genes are thus included in a synteny group, conserved on rat chromosomes 10 and human chromosome arm 17q.

Amplification and overexpression of the hepatocyte growth factor receptor (HGFR/MET) in rat DMBA sarcomas.

In the present study subcutaneous fibrosarcomas were induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in rats from F1 generation cross breedings of two different inbred strains. Comparative genomic hybridization (CGH) analysis, which allows detection of DNA sequence copy changes, was applied to one of the tumors and it was found that there were increased copy numbers of sequences at chromosome 4q12-q21 in this tumor. We have previously determined that the loci for the hepatocyte growth factor (Hgf) and hepatocyte growth factor receptor (Hgfr/Met), a protooncogene, are situated in this particular chromosome region. Using probes for the two genes in FISH (fluorescence in situ hybridization) and in Southern blots we found that the Hgfr/Met gene was amplified in five of the 19 sarcomas studied, and that the Hgf gene was coamplified in two of them. Northern and Western blots and tyrosine phosphorylation analysis showed that the HGF receptor was overexpressed and functional in all five tumors, as well as in two additional tumors. In summary, both amplification and overexpression of the Hgfr/Met gene was found in about 25% of DMBA-induced experimental rat sarcomas, and HGF receptor overexpression alone was seen in two additional tumors. Possibly this reflects an involvement in paracrine or autocrine stimulation of growth and invasiveness by HGF. Our finding could provide a rodent model system to increased knowledge about causality and therapy, which may be applicable to the sizeable fraction of human musculoskeletal tumors displaying MET overexpression.

Establishment and characterization of a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia.

In this study, a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia with an early onset and high incidence was established and characterized. All tumors analyzed were found to express the alpha,beta T-cell receptor, and the majority of them had a mature, CD3+CD4+CD8- immunophenotype. In a few cases, tumors with a more immature CD3+CD4+CD8+ phenotype were isolated. Expanded phenotyping using a broad panel of lymphocyte differentiation markers confirmed the mature T-cell phenotype of the tumors. Histologic and cell cycle analysis of the tumors revealed an aggressive lymphoblastic malignancy with a very high proliferation rate and widespread engagement of bone marrow and lymphoid as well as nonlymphoid organs. Thus, the TLL mouse strain represents a unique model for the analysis of the oncogenesis and progression of mature T-cell tumors and for the development of therapeutic measures to combat such tumors.

The related genes encoding growth hormone and prolactin have been dispersed to chromosomes 10 and 17 in the rat.

We have assigned the rat GH gene to chromosome 10 and the rat PRL gene to chromosome 17. DNA from a series of mouse BWTG3 x rat hepatocyte somatic cell hybrids, each of which has retained a unique complement of rat chromosomes, was analyzed for the presence of rat GH and PRL genomic fragments by Southern blotting. Radiolabeled complementary DNAs (cDNAs) encoding rat GH and rat PRL were used as molecular probes. Based upon these assignments, we conclude that the evolutionarily related GH and PRL genes have been dispersed to different chromosomes in rat as in man.

Prolactin-like protein-C variant: complementary deoxyribonucleic acid, unique six exon gene structure, and trophoblast cell-specific expression.

The rat placental PRL family consists of proteins structurally related to pituitary PRL. As a consequence of attempting to characterize the gene for one of the members of the family, PRL-like protein-C (PLP-C), we identified a related gene that we have termed PLP-C variant (PLP-Cv). In this study, we present information on the PLP-Cv gene and its pattern of expression. Screening of a rat genomic library with a PLP-C cDNA resulted in the isolation of four phage clones. Nucleotide sequence analysis of the clones revealed a gene, PLP-Cv, closely related but distinct from PLP-C. The PLP-Cv gene possessed a six exon/five intron organization, unique among members of the PRL family, and was localized to chromosome 17 of the rat genome, similar to other PRL family members. A PCR strategy involving primers based on the PLP-Cv gene was used to isolate a placental PLP-Cv cDNA. PLP-Cv showed 90 and 78% sequence identity with PLP-C at nucleotide and amino acid levels, respectively. Expression of PLP-Cv was restricted to the trophoblast lineage and was coordinately activated with PLP-C beginning at day 11 of gestation and continuing until term. Primer extension analysis revealed multiple putative transcription start sites. A 2.1-kilobase pair PLP-Cv promoter-luciferase reporter construct was specifically activated in differentiating rat trophoblast cells but not in other cell types. In conclusion, we have identified a new member of the PRL family possessing considerable homology to PLP-C, a unique gene structure, and displaying a trophoblast-specific pattern of transcriptional activation.

Rcho-1 trophoblast cell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities.

In this report, we have investigated placental lactogens (placental lactogen-I, PL-I; PL-I variant, PL-Iv; PL-II) expressed by differentiated Rcho-1 trophoblast cells. A complementary DNA (cDNA) library to differentiated Rcho-1 trophoblast cells was constructed and screened with probes to detect PL-I and PL-II. Sequence analysis of three independent Rcho-1 PL-I cDNAs indicated that they significantly differed from the previously reported PL-I sequence but more closely resembled a related cDNA referred to as PL-I mosaic (PL-Im). Upon further analysis, Rcho-1 PL-I/PL-Im transcripts could be detected in Rcho-1 trophoblast cells and normal developing placental tissue; however, the previously reported PL-I transcript could not be identified from the same sources. Given these results, we examined the original PL-I cDNA by PCR and nucleotide sequence analyses. The sequence differed from the original report and was found to be identical to the Rcho-1 PL-I and PL-Im cDNA clones. Thus, PL-I, Rcho-1 PL-I, and PL-Im are equivalent and should be referred to as PL-I. The PL-I gene was localized to chromosome 17 of the rat genome, similar to other PRL family members. Rcho-1 PL-II cDNAs were identical to the published PL-II sequence. PL-Iv cDNAs were isolated from differentiated Rcho-1 cells via an RT-PCR strategy and found to be identical to previously isolated PL-Iv cDNAs. Rcho-1 PL-I and PL-II cDNAs were subcloned into the pcDNA3 expression vector and recombinant protein produced in HRP-1 cells. Both recombinant Rcho-1 PL-I and PL-II proteins significantly stimulated the proliferation of lactogen-dependent rat Nb2 lymphoma cells and mouse mammary epithelial cells. In summary, we show that the Rcho-1 PL-I corresponds to PL-Im and Rcho-1 PL-Iv and PL-II are identical to their previously described placental counterparts. Additionally, both recombinant Rcho-1 PL-I and PL-II proteins are biologically active.

Characterization and chromosomal localization of rat scavenger receptor class B type I, a high density lipoprotein receptor with a putative leucine zipper domain and peroxisomal targeting sequence.

High density lipoprotein (HDL) participates in reverse cholesterol transport and in the delivery of cholesterol to steroid-producing tissues. Scavenger receptor class B type I (SR-BI) was recently shown to bind HDL and mediate internalization of its cholesterol content. We have cloned the rat homolog of this receptor, determined its chromosomal location, and examined its expression in rat tissues and in a model of follicular development, ovulation, and luteinization. The predicted protein contained two transmembrane domains, a leucine zipper motif, and a peroxisomal targeting sequence. The rat and human SR-BI genes were mapped to a region previously linked between rat and human chromosomes 12. SR-BI gene expression was detected in several rat tissues, with high levels in ovarian tissue, liver, and adrenal cortex, as determined by ribonuclease protection assay and in situ hybridization. A significant increase in SR-BI gene expression was detected in the late phase of corpus luteum formation, and transcripts were abundant in corpus luteum and in thecal cells at all stages of follicular development. In conclusion, the rat SR-BI complementary DNA predicted a protein with several conserved motifs, including a putative leucine zipper and a peroxisomal targeting sequence. The chromosomal locations of the rat and human SR-BI homologs suggest that this gene is a new member of a previously reported, conserved synteny group. SR-BI gene expression was high in steroid-producing tissues and in the liver, consistent with a role of this receptor in the uptake of HDL cholesterol.

Structure and chromosomal localization of the rat salivary Psp and Smgb genes.

SMGB and PSP are among the most abundant products of the immature acinar cells in developing rat parotid and submandibular glands and are also products of the sublingual gland serous demilunes. Previous analysis of Smgb and Psp cDNA clones demonstrated a high degree of sequence similarity between the signal peptide-encoding and 3' untranslated regions of these transcripts, although the secreted proteins themselves are more divergent. The current study reports the upstream sequences, genomic organization and localization of the Psp and Smgb genes. Both structural genes contain nine exons and are present at 3q41-3q42, where they are arranged in tandem and separated by 21kb. In addition to the previously observed sequence similarity, Psp and Smgb are highly homologous throughout exon 1 and at 365 of 600bp immediately upstream of the transcription start site. These findings indicate that the Psp and Smgb genes arose by tandem duplication and divergence. The similar neonatal submandibular and parotid gland expression patterns observed for these genes are likely to be due to closely conserved or shared enhancer(s).

Characterization of the aldose reductase-encoding gene family in rat.

Although the enzyme aldose reductase (AR) is implicated in the development of tissue pathology in diabetes, the exact mechanism of this involvement remains unclear. To better understand the role that expression of the aldose reductase-encoding gene (ALR) may play in diabetic complications, we have begun to analyze the gene and its regulatory regions, and we present here the sequence of four ALR genes in the rat. The putative functional gene is 14.1 kb long, has ten exons which show perfect sequence identity to the rat lens AR RNA sequence, and nine introns with classical splice-site consensus sequences. Potential regulatory elements in the 5'-flanking region of this gene include a TATA box and two CCAAT boxes. Probing rat genomic Southern blots with a fragment from the first intron indicates that there is probably only one copy of this gene in the rat genome. The other three genes are processed pseudogenes which show approx. 90% identity to the rat lens AR RNA sequence, contain no introns, and have poly(A) regions at their 3' ends. Chromosomal localization studies show the presence of ALR genes on chromosomes 3, 4 and 6 in the rat with the putative functional gene mapped on chromosome 4.

Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase.

The gene (PP63) encoding the inhibitor (PP63) of the insulin receptor tyrosine kinase was isolated from a rat genomic library. The intron/exon organization was deduced from Southern-blot analysis and sequence data (i.e., the exons + the boundaries). The PP63 gene, which maps to chromosome 11, spans approx. 8 kb and contains seven exons separated by six introns of different sizes. All of the boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Primer extension and S1 mapping experiments were used to locate the transcription start point (tsp) 73 nt upstream from the translational initiator. Both in vitro transcription assays and transcription of a chimeric gene in intact hepatoma cells indicated that the sequence located immediately upstream from the tsp contained a promoter. Several putative cis-regulatory elements, including a TATA box and a C/EBP-binding site were found within the 250 bp preceding the tsp.

Primary structure and assignment to chromosome 6 of three related rat genes encoding liver serine protease inhibitors.

Three closely related SPI genes which encode highly homologous proteins of the serine protease inhibitor family secreted by rat liver (SPI-1, SPI-2 and SPI-3), were isolated from genomic libraries and sequenced, totally (SPI-2) or partially (SPI-1 and SPI-3). These genes all map on rat chromosome 6. Each of them spans about 10 kb and contains five exons separated by four introns, located at equivalent positions. S1 mapping analysis indicated that initiation of transcription occurs at the same position (tsp) in each of the three genes. In vitro transcription experiments demonstrated the presence of promoter elements upstream from the putative tsp. Detailed analysis of 5'-flanking sequences in the three SPI revealed major differences. A high degree of identity (70%) was found within a 350-bp region preceding the 'cap' site, with the exception of a 42-bp spacer, which was only found in SPI-3. Upstream from that point, SPI-1 and SPI-2 sequences remain largely homologous over at least 1 kb but completely diverge from the corresponding sequence in SPI-3. This may, at least partly, account for the differential regulation of the three SPI observed during acute inflammation and upon hypophysectomy.