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Expression of the epithelial-specific adhesion molecule E-cadherin has been assessed in paraffin-embedded tissue from a series of 72 colorectal carcinomas. Using immunocytochemistry and in situ hybridization it was found that E-cadherin expression was related inversely to tumor differentiation. Out of 44 well- and moderately differentiated tumors, 36 expressed good positivity, whereas 24 of 28 poorly differentiated tumors were E-cadherin-negative. Classification by Dukes stage revealed a highly significant difference (P << 0.001) between A and B (32 positive, four negative) and C1 and C2 (seven positive, 29 negative) stages in terms of immunoreactivity. Of the 32 lymph node metastases studied, 20 were negative for E-cadherin expression, as were seven of eight liver metastases. These results indicate that the down-regulation of E-cadherin levels in vivo is associated with the dedifferentiation, progression, and metastasis of colorectal cancer.

Genes on chromosomes 17q and 18q have been shown to code for putative tumor suppressors. By a combination of allele-loss studies on sporadic ovarian carcinomas and linkage analysis on a breast/ovarian cancer family, we have investigated the involvement of such genes in these diseases. Allele loss occurred in sporadic tumors from both chromosome 17p, in 18/26 (69%) cases, and chromosome 17q, in 15/22 (68%) cases. In the three familial tumors studied, allele loss also occurred on chromosome 17 (in 2/3 cases for 17p markers and in 2/2 cases for a 17q allele). Allele loss on chromosome 18q, at the DCC (deleted in colorectal carcinomas) locus, was not as common (6/16 cases [38%]) in sporadic ovarian tumors but had occurred in all three familial tumors. The results of linkage analysis on the breast/ovarian cancer family suggested linkage between the disease locus and 17q markers, with a maximum lod score of 1.507 obtained with Mfd188 (D17S579) polymorphism at 5% recombination. The maximum lod score for DCC was 0.323 at 0.1% recombination. In this family our results are consistent with a predisposing gene for breast/ovarian cancer being located at chromosome 17q21.

Many different types of chemical exposures can increase the incidence of tumors in animals and humans, but usually a long period of time is required before the carcinogenic risk of an exposure is manifested. Both of these observations can be explained by a multistep/multigene model of carcinogenesis. In this model, a normal cell evolves into a cancer cell as the result of heritable changes in multiple, independent genes. The two-stage model of initiation and promotion for chemical carcinogenesis has provided a paradigm by which chemicals can act by qualitatively different mechanisms, but the process of carcinogenesis is now recognized as more complex than simply initiation and promotion. Even a three-stage model of initiation, promotion, and progression, which can be operationally defined, is not adequate to describe the carcinogenic process. The number of genes altered in a cancer cell compared to a normal cell is not known; recent evidence suggests that 3-10 genetic events are involved in common adult malignancies in humans. Two distinct classes of genes, protooncogenes and tumor-suppressor genes, are involved in the cancer process. Multiple oncogenes may be activated in a tumor, while multiple tumor-suppressor genes may be inactivated. Identification of the genes involved in carcinogenesis and elucidation of the mechanisms of their activation or inactivation allows a better understanding of how chemical carcinogens influence the process of neoplastic evolution. The findings of multiple genetic changes (including point mutations, chromosomal translocations, deletions, gene amplification, and numerical chromosome changes) in activated protooncogenes and inactivated tumor-suppressor genes provide experimental support for Boveri's somatic mutation theory of carcinogenesis. In addition to mutagenic mechanisms, chemicals may heritably alter cells by epigenetic mechanisms and enhance the clonal expansion of altered cells. Most chemical carcinogens operate via a combination of mechanisms, and even their primary mechanism of action may vary depending on the target tissues. The classification of chemicals by mechanism of action or by nongenotoxic or genotoxic activity has certain inherent difficulties because no classification of chemicals is exhaustive or definitive.

The maize Suppressor-mutator (Spm) transposable element is subject to epigenetic inactivation in transgenic tobacco, as it is in maize. Spm inactivation in tobacco is correlated with increased methylation of sequences near the element's transcription start site. To determine whether element-encoded gene products can promote the reactivation of an inactive element, we investigated the effects of introducing individual CaMV 35S promoter-driven cDNAs for tnpA, tnpB, tnpC and tnpD, the element's four known protein-coding sequences. Introduction of the tnpA cDNA promoted the reactivation of the inactive resident Spm element, as judged by the appearance of regenerants with very early excision events and transposed elements. By contrast, the tnpB, tnpC and tnpD cDNAs had no affect on the activity of the resident Spm element. Similar results were obtained when the element-encoded cDNAs were introduced either by Agrobacterium-mediated retransformation or by a genetic cross. Reactivation of an inactive Spm by the tnpA cDNA is accompanied by reduced methylation of several methylation-sensitive restriction sites near the element's transcription start site. Maintenance of the reactivated Spm element in an active state requires the continued presence of the tnpA cDNA. Elimination of the tnpA cDNA locus by genetic segregation generally results in decreased element activity, as judged by a low frequency of excision events, and is accompanied by increased methylation of the element's 5'-end. Exceptions resembling the phenomenon of "presetting" are also observed in which progeny plants that did not receive the tnpA cDNA locus after meiotic segregation maintain high excision activity and exhibit low methylation levels.

The APC gene has been found to be mutated during the development of sporadic colorectal tumors as well as in the germ line of familial adenomatous polyposis patients. To facilitate the characterization of both normal and mutant APC protein, a series of monoclonal and polyclonal antibodies specific for the APC protein was produced. When lymphoblastoid cell lines derived from seven familial adenomatous polyposis patients with known mutations were analyzed by Western blot, an approximately 300-kDa protein corresponding to the predicted size of full-length APC was detected in all 7 cell lines. In addition, truncated APC proteins corresponding to the product of the known mutated alleles could be detected in 4 of the 7 lines. Similar analysis of 23 colon carcinoma and 9 adenoma cell lines revealed truncated proteins in 24 (75%) of the cell lines. Moreover, 26 (81%) of the colon tumor lines were totally devoid of the normal, full-length protein. In contrast, Western blot analysis of 40 cell lines derived from sporadic tumors of other organs detected only full-length APC. Immunohistochemical analysis of APC in normal colonic mucosa revealed cytoplasmic staining with more intense staining in the basolateral margins of the epithelial cell. This staining was markedly increased in the upper portions of the crypts, suggesting an increased level of expression with maturation. These studies provide some initial clues to the function of the cytoplasmic protein APC and demonstrate the feasibility of identifying APC mutations by direct analysis of the APC protein.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth. Assimilatory nitrate and nitrite reductases convert nitrate through nitrite to ammonium. We report here the molecular cloning of the nasA and nasB genes, which encode assimilatory nitrate and nitrite reductase, respectively. These genes are tightly linked and probably form a nasBA operon. In vivo protein expression and DNA sequence analysis revealed that the nasA and nasB genes encode 92- and 104-kDa proteins, respectively. The NASA polypeptide is homologous to other prokaryotic molybdoenzymes, and the NASB polypeptide is homologous to eukaryotic and prokaryotic NADH-nitrite reductases. The narL gene product positively regulates expression of the structural genes for respiratory nitrate reductase, narGHJI. Surprisingly, we found that the nasBA operon is tightly linked to the narL-narGHJI region in K. pneumoniae, even though the nitrate assimilatory and respiratory enzymes serve different physiological functions.

Antithrombin-III (AT-III) is a heparin-dependent inhibitor of thrombin and Factor Xa, two serine proteases that are crucial for blood coagulation. In order to assess whether it would be possible to target AT-III only towards Factor Xa, we replaced parts of the reactive site, or P region, of AT-III by sequences present in prothrombin, a substrate of Factor Xa in the coagulation cascade. We show that replacement of the P3 to P3' region generates the hypothesized phenotype. In fact, point mutation of the P1' site from Ser (present in AT-III) to Ile (present in prothrombin) is sufficient to dissociate heparin-dependent thrombin and Factor Xa inhibitory activities. Interestingly, a combined mutation at P3 and P3' brings about the same dissociation. We show that besides Ile, other amino acids at P1' can lead to the dissociation in inhibitory activity. Amino acids with small side chains (Gly, Ser, Ala, and Thr) have only a marginal effect on the inhibitory activity against either protease. However, larger residues at the P1' position abolish the heparin-dependent anti-thrombin activity, whereas the heparin-dependent anti-Factor Xa activity is not at all or only moderately affected. These results can be rationalized by a comparison of the x-ray structure and a three-dimensional model of the S1' binding pockets of thrombin and Factor Xa, respectively. It appears that the S1' pocket of Factor Xa leaves much more space for the P1' residue of AT-III than the S1' pocket of thrombin.

Several reports demonstrate estrogen receptor involvement in specific brain functions. In addition, estrogen receptors are expressed at early stages of brain development, suggesting that estrogens or related molecules may play an instructive role in the differentiation of specific brain areas. The lack of model systems in which these phenomena could be studied prompted us to develop a neuroblastoma cell line expressing the estrogen receptor. The cell line expresses the hormone receptor at levels compatible with a physiological activity. The activated estrogen receptor is capable of blocking proliferation of the cells without exerting toxic effects. Following growth arrest, the cells display a neuron-like morphology and express tau and synaptophysin, two proteins synthesized in differentiating neurons. The cell line generated will provide a valuable model system for molecular and biochemical studies of the activity of estrogens in neural-derived cells.

The natural anticoagulants (antithrombin III, protein C, protein S), plasminogen and tissue plasminogen activator antigen (t-PA ag), were measured in 27 consecutive patients following allogeneic BMT. Thrombosis and veno-occlusive disease were not seen in this study. Changes in the levels of these proteins occurred mainly during acute GVHD. There were 14 patients who had no acute GVHD (group I) and 13 patients who had acute GVHD (group II). No changes in antithrombin III (ATIII), protein C, protein S and t-PA levels were found in group II before the appearance of acute GVHD when compared with group I. However, we noted a significant rise in protein S (p = 0.01), antithrombin III (p = 0.001) and t-PA ag (p = 0.0004) levels during acute GVHD. In contrast, protein C levels decreased early in GVHD (p = 0.005), and then increased progressively over the course of a month post-GVHD. No changes in plasminogen levels were observed. These results might reflect activation of and/or damage to endothelial cells during GVHD.

We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.

The relationship between nuclear DNA ploidy pattern and cytological atypia in aspiration cytology of breast cancer was studied in 40 cases of primary breast cancers (T1: 14 cases; T2: 26 cases). They were divided into two groups, according to cytological specimens, high degree (H group) and low degree (L group). Twelve of 40 cases were classified into H group and 28 cases into L group. Fresh frozen materials were obtained from mastectomy specimens and nuclear DNA analysis was carried out by flow cytometry (FCM). Estrogen receptor (ER) and progesterone receptor were measured with the DCC method. Lymph node metastases were histologically seen in 17 cases; the ER positive rate was 55.0%, and the PgR positive rate was 42.5%. FCM revealed 21 cases (52.5%) with diploidy pattern and 19 cases (47.5%) with aneuploidy pattern. S-phase fraction in 17 cases (48.6%) exceeded 20%. L group had more diploid cases and ER positive cases than H group (p < 0.05, p < 0.01). No differences in other factors were seen between the two groups. In diploid cases, L group had more ER positive cases than H group (p < 0.02).

We investigated the potential pituitary origin of gonadal insufficiency in hemochromatosis. Gonadotropin secretion was studied in seven patients with hemochromatosis and hypogonadism, before and after chronic pulsatile GnRH therapy. Pulsatile LH secretion was studied before (sampling every 10 min for 6 h) and after 15-30 days of chronic pulsatile GnRH therapy (10-12 micrograms per pulse). Prior to GnRH therapy, all the patients had low serum testosterone, FSH and LH levels. LH secretion was non-pulsatile in four patients, while a single pulse was detected in the remaining three. Chronic pulsatile GnRH administration did not increase serum testosterone levels; similarly, serum LH levels remained low: neither pulse frequency nor pulse amplitude was modified. We conclude that hypogonadism in hemochromatosis is due to pituitary lesions.