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Since agricultural crops contribute > 70% of human cadmium (Cd) intake, modification of crops to reduce accumulation of this pollutant metal during plant growth is desirable. Here we describe Cd accumulation characteristics of seedlings and field grown tobacco plants expressing the Cd-chelating protein, mouse metallothionein I. The objective of the transformation is to entrap Cd in roots as Cd-metallothionein and thereby reduce its accumulation in the shoot. Transformed and control seedlings were exposed for 15 days in liquid culture at a field soil-solution-like Cd concentration of 0.02 microM. Transformed seedlings of Nicotiana tabacum cultivar KY 14 contained about 24% lower Cd concentration in shoots and about 5% higher Cd concentration in roots than control seedlings. Dry weights of transformed and control tissues did not differ significantly. In the field in 1990, mature transformed N. tabacum cv. KY 14 plants exposed only to endogenous soil Cd contained about 14% lower leaf lamina Cd concentration than did controls. Differences were significant at the p < or = 0.1 level in 13 of 16 leaf positions. Leaf dry weight did not differ significantly but transformed field plants had 12% fewer leaves and were 9% shorter than the controls. Copper (Cu) concentration was significantly higher (ca10%) in the bottom nine leaf positions of transformed plants suggesting that reduced leaf number and plant height may be due to Cu deficiency or toxicity. Alternatively, somaclonal variation or gene position effects may be involved. No differences were found in zinc levels. With N. tabacum cv. Petit Havana, transformed seedlings contained no less Cd in shoots but 48% higher Cd concentration in roots.(ABSTRACT TRUNCATED AT 250 WORDS)

Using Avidn-biotin-peroxidase complex (ABC) immunohistochemical method, 68 gastric cancer specimens and 20 non-cancerous stomach specimens were examined for ER. In 38 of 68 gastric cancer specimens, ER concentrations were also biochemically determined by Dextran-coated charcoal (DCC) assay. The results showed that ER was absent in noncancerous stomach tissues. ER was present in 21 (30.9%) of gastric cancers. The ER positive cases were chiefly poorly differentiated gastric cancers. There were no significant statistical differences in the patient's age or sex, size of primary tumor and the ER positive rates. The results indicate that endocrine therapy might be useful for the ER positive gastric cancer patients.

Norwalk virus capsid protein was produced by expression of the second and third open reading frames of the Norwalk virus genome, using a cell-free translation system and baculovirus recombinants. Analysis of the expressed products showed that the second open reading frame encodes a protein with an apparent molecular weight of 58,000 (58K protein) and that this protein self-assembles to form empty viruslike particles similar to native capsids in size and appearance. The antigenicity of these particles was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assays of paired serum samples from volunteers who developed illness following Norwalk virus challenge. These particles also induced high levels of Norwalk virus-specific serum antibody in laboratory animals following parenteral inoculation. A minor 34K protein was also found in infected insect cells. Amino acid sequence analysis of the N terminus of the 34K protein indicated that the 34K protein was a cleavage product of the 58K protein. The availability of large amounts of recombinant Norwalk virus particles will allow the development of rapid, sensitive, and reliable tests for the diagnosis of Norwalk virus infection as well as the implementation of structural studies.

Wilms tumor, an embryonic kidney malignancy, accounts for approximately 6% of all pediatric neoplasms. A gene implicated in the genesis of this tumor, the Wilms tumor suppressor gene (WT1), encodes a zinc-finger DNA-binding protein (WT1) that functions as a transcriptional repressor. In certain Wilms tumors, the platelet-derived growth factor A chain (PDGF-A) is overexpressed; it has therefore been suggested that it may play an autocrine role in development of these neoplasms. Since the PDGF-A promoter contains putative binding sites for WT1, we explored the role of WT1 in regulating A-chain expression. The major PDGF-A promoter activity was localized in transient transfection assays to a region spanning from -643 to + 8 relative to the transcription start site. WT1 bound to several sites in this region of the promoter, as demonstrated by gel-shift analysis and DNase I footprinting, and functioned as a powerful repressor of PDGF-A transcription in vivo. Maximal repression (> 50-fold) of the PDGF-A promoter was dependent on the presence of multiple WT1 binding sites in transient transfection assays. Our observations suggest a mechanism for normal downregulation of a growth factor gene and of an autocrine growth process of import in kidney development and other biological systems.

A predisposition to the development of certain specific and familial cancers is associated with the inheritance of a single mutated gene. In the best-characterized cases, this primary mutation is a loss of function mutation consistent with viability but resulting in neoplastic change consequent to the acquisition of a second somatic mutation at the same locus. Such genes are referred to as tumor-suppressor genes. Classical examples are the Rb-1 gene associated with the development of retinoblastoma and the p53 gene, which is associated with a wider range of neoplasms, including breast cancer. Other tumor-suppressor genes have been isolated which are associated with Wilms' tumor, neurofibromatosis, and inherited and sporadic forms of colorectal cancer. Some of these genes appear to act as negative regulators of mitotic cycle genes, and others may have different properties. The nature of these genes is discussed, as is the evidence for the involvement of tumor-suppressor genes in other inherited, and sporadic, forms of cancer. Some recent data on the Wilms' tumor gene, WT1, and on the involvement of the p53 gene in breast cancer are presented, and the importance of genomic imprinting in contributing to the excess of suppressor gene mutations in chromosomes of paternal origin is considered.

Heparin is indispensable anticoagulant for cardiopulmonary bypass, but the dose of heparin is even now under discussion. In this study, hemostatic fluctuation was analyzed during and after the bypass using hemostatic molecular markers. The subjects were 16 adult cases of open heart surgery, 12 males, 4 females. The average age was 55.0 year. Operations were aortocoronary bypass in 12, valvular surgery in 3 and ASD patch closure in one with moderate hypothermic cardiopulmonary bypass. At the beginning of cardiopulmonary bypass, 3 mg/kg heparin was administered and the equivalent amount of protamine sulfate was used for neutralization at the end of the bypass. Platelet count, hematocrit, antithrombin III (ATIII), beta-thromboglobulin, platelet factor 4, fibrinopeptide A, thrombin antithrombin III complex, FDP, D dimer FDP, plasmin alpha 2 plasmin inhibitor complex, tissue plasminogen activator (t-PA), and thrombomodulin (TM) were measured through the operation up to two weeks after surgery. ATIII decreased to 50% of control value all through the bypass. Platelet markers increased immediately, and the activated state continued 3 hours after the bypass. Coagulation markers increased markedly after the aortic declamping, and reached at its peak by three times as control value, immediately after the protamine neutralization and continued for 3 hours. During the bypass, fibrinogenolysis caused by t-PA which was stimulated by non-physiological circulation and stimulating substances, was observed. Fibrinolysis occurred following the hypercoagulability after the neutralization. TM was within normal range before the aortic declamping. But increased gradually after the declamp, and reached twice as much as the base line. It could be concluded that hypercoagulability and high platelet activation might play a role of perioperative thrombosis. Hypercoagulability and increase of serum TM would be related to reperfusion of the lung. The increasing of TM would reflect broad injury of vessel walls after the bypass, because plasma TM increased following the generalized injury of endothelial cells.

Inbred, congenic, and intra-H-2-recombinant mouse strains were given subcutaneous injections of either 1.6 mg HgCl2/kg body wt or 0.1 ml NaCl thrice weekly for 5-6 weeks. Mercury-treated mice from strains carrying the H-2s haplotype developed antinucleolar antibodies (ANoA), which targeted the 34-kDa nucleolar protein fibrillarin, and in some instances also nucleolar proteins of 60-70 and 10-15 kDa, the latter corresponding to histones. Strains with H-2b and H-2d haplotypes were resistant to induction of ANoA. The susceptibility to development of AnoA/antifibrillarin antibodies (AFA) was mapped to the H-2A-region using intra-H-2-recombinant strains. We were not able to confirm earlier reports that expression of H-2E genes dampens the development of ANoA. Mercury treatment caused a substantial increase in the titer of antichromatin (ACA) and/or antihistone (AHA) antibodies in a fraction of SJL/J, A.SW, A.TH, B10.S, and B10.HTT mice (H-2s), and in A/J (H-2k) mice, whereas mice from the C57BL/6J and C57BL/10J (H-2b), and the DBA and BALB/c (H-2d) strains were low or nonresponders. The development of AHA and ACA could not be linked to the H-2 complex. A significant, substantial increase of granular mesangial and systemic vessel wall IgG deposits occurred in mice with serum ANoA/AFA. However, the B10.S(9R) and B10.HTT strains, which express the H-2E genes, developed only an intermediately increased titer of mesangial IgG deposits. Systemic vessel wall IgG deposits occurred in only 60-80% of the B10.S(9R) mice and in none of the B10.HTT mice. This contrasted with the high titer of mesangial IgG deposits and uniform development of systemic vessel wall IgG deposits observed in B10.S mice not expressing H-2E. Mice with mesangial IgG deposits showed a mild glomerulonephritis. There was no systemic vasculitis. The susceptibility to development of ANoA, AHA, ACA, and systemic, granular IgG deposits in the B10.S strain was influenced by the sex, since males showed less uniform development of these immunopathologic features than females.

Five genes, tmoABCDE, encoding toluene-4-monooxygenase (T4MO) were previously mapped to a 3.6-kb region of a 10.2-kb SacI DNA fragment isolated from Pseudomonas mendocina KR1 (K.-M. Yen, M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt, and K. K. Chen, J. Bacteriol. 173:5315-5327, 1991). In this report, we describe the identification and characterization of a DNA region in the SacI fragment whose expression enhances the T4MO activity determined by the tmoABCDE gene cluster. This region was mapped immediately downstream of the putative transcription termination sequence previously located at the end of the tmoABCDE gene cluster (Yen et al., J. Bacteriol., 1991) and was found to stimulate T4MO activity two- to threefold when expressed in Escherichia coli or Pseudomonas putida. Determination of the nucleotide sequence of this region revealed an open reading frame (ORF) of 978 bp. Expression of the ORF resulted in the synthesis of an approximately 37-kDa polypeptide whose N-terminal amino acid sequence completely matched that of the product predicted from the ORF. The ORF thus defines a gene, which has now been designated tmoF. The TmoF protein shares amino acid sequence homology with the reductases of several mono- and dioxygenase systems. In addition, the reductase component of the naphthalene dioxygenase system, encoded by the nahAa gene of plasmid NAH7 from P. putida G7, could largely replace the TmoF protein in stimulating T4MO activity, and TmoF could partially replace the NahAa protein in forming active naphthalene dioxygenase. The overall properties of tmoF suggest that it is a member of the T4mo gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system.

Genetic studies have implicated the early involvement of a gene on chromosome arm 9p in the development of cutaneous melanoma. We have performed loss-of-heterozygosity studies to confirm these original findings and identify the most frequently rearranged or deleted region of 9p. Eight markers were analyzed, including (from 9pter to proximal 9q) D9S33, the beta-interferon (IFNB1) locus, the alpha-interferon (IFNA) gene cluster, D9S126, D9S3, D9S19, the glycoprotein 4 beta-galactosyltransferase (GGTB2) gene, and the argininosuccinate synthetase pseudogene 3 (ASSP3). Two or more of these loci were found to be hemizygously reduced in 12 of 14 (86%) informative metastatic melanoma tumor and cell line DNAs, and homozygous deletions of the marker D9S126 were observed in 2 of 20 (10%) melanoma cell lines. These findings have resulted in the identification of a small critical region of 2-3 megabases on 9p21 in which a putative melanoma tumor-suppressor gene appears likely to reside. Several 9p candidate genes, including IFNB1, the IFNA gene cluster, GGTB2, and the tyrosinase-related protein (TYRP) locus, have all been eliminated as potential targets because they are located outside of the homozygously deleted regions.

Cancer development in man appeared to be a multistage process as suggested by epidemiological studies on commonly occurring gastric, colon, and breast cancers and also on human retrovirus-related leukemia, and by the finding by physicians and surgeons of precancerous lesions for many types of neoplasias. In the last 10 years it has become evident that human cancers have multiple genetic alterations caused by point mutations, recombinations, amplifications, and/or deletions. The genes affected include both oncogenes and tumor-suppressor genes and genes that accelerate cell proliferation and metastasis. Cancers with more malignant properties and poorer prognosis are generally associated with larger numbers of genetic alterations. These multiple genetic alterations are considered to be a direct reflection of the multiple steps involved in carcinogenesis. The multiple genetic alterations are caused by multiple environmental carcinogenic substances or factors, each of which usually exists only at minute concentrations and does not exert any major impact alone except under particular occupational, iatrogenic, and locally geographic conditions. The fact that carcinogenesis is a multistep process involving multiple genetic alterations clearly needs to be taken into consideration in assessing the risks of environmental carcinogenic substances or factors. The increasing incidence of multiple primary cancers is also most easily understood from the viewpoint of multiple steps in carcinogenesis. Possible multiple approaches to cancer prevention should therefore be considered in relation to multistep carcinogenesis and multiple carcinogenic factors.

Thirty elderly patients with T3 or T4 breast cancer underwent a wedge biopsy for radioligand-binding assay (RLA) of oestrogen receptor (ER) activity. A second, separate group of 21 elderly patients with T3 and T4 breast cancers underwent fine needle aspiration biopsy (FNA) for immunocytochemical assay of ER (ER-ICA). All the women received tamoxifen as primary treatment and response was assessed by UICC criteria. Tumour ER concentration by RLA was correlated with both response (Spearman's R = + 0.52) and time to progression (R = + 0.76). Nine patients with receptor-rich tumours (> 100 fmol/mg protein) failed to show a response. However, the percentage of cells staining for ER by ER-ICA assay was much more strongly related to the likelihood of response (R = + 0.89); no patient with < 20% cells staining responded. Wedge biopsy and the biochemical determination of ER activity is of limited value in predicting the likely response to tamoxifen; ER-ICA assays on such tumours may be more informative.