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We have previously observed that the frequency of loss of heterozygosity (LOH) on chromosome 18q was low in adenomas and intramucosal carcinomas, whereas invasive carcinomas exhibited a high frequency in familial adenomatous polyposis patients (M. Miyaki et al., Cancer Res., 50: 7166-7173, 1990). In the present study, LOH at the DCC locus on chromosome 18q and the expression of DCC gene into mRNA were analyzed in colorectal tumors with distinct histopathological types. The carcinomas that showed 18q LOH also lost the DCC locus. The expression of DCC gene into mRNA was examined at the level of 233-base pair fragments of nucleotide 986-1218 in DCC complementary DNA. In a moderate-to-severe adenoma, 5 carcinoma-in-adenomas, and 4 intramucosal carcinomas, the level of expression was as high as in normal colorectal mucosa, whereas it was greatly reduced or not detectable in most (13 of 16) invasive carcinomas. Among these invasive carcinomas, 7 of 11 showed 18q LOH, but 4 showed no LOH. These results suggest that the DCC gene is included in the allelic deletion on chromosome 18q, and that the progression of colorectal carcinoma from early stage to advanced stage accompanies the inactivation of the DCC gene through LOH and other mechanisms.

We reviewed the records of all patients with a diagnosis of malignancy who were treated at our center and who had not had chemotherapy for at least 18 months, to assess the prevalence of chronic hepatitis B surface antigen (HBsAg)-negative hepatitis, to assess the prevalence of a marker of hepatitis C virus infection, and to determine the severity of chronic liver disease. Of 557 eligible patients, 38 (6.8%) had chronic HBsAg-negative hepatitis. Of these 38 patients, 20 (52.6%) had a marker of hepatitis C virus infection. The prevalence of chronic HBsAg-negative hepatitis was higher in patients previously treated for leukemia than in patients treated for another malignancy (11.8% vs 4.6%; p = 0.004). The liver biopsy revealed chronic active hepatitis or cirrhosis or both in 8 (28%) of 28 patients with clinical chronic HBsAg-negative hepatitis. Four patients without hepatitis C virus infection who underwent liver biopsy had hepatitis B virus antigen in the liver, confirmed by immunohistochemistry studies. One patient uninfected with hepatitis C virus had hemochromatosis. We conclude that infection with hepatitis C virus was the major cause of chronic HBsAg-negative hepatitis in pediatric patients previously treated for malignancy; the cause remained unidentified in 30% of the patients.

Estrogen receptors (ER) and progesterone receptors (PR) were determined on curettages from women with endometrial adenocarcinoma. The results obtained with the enzyme immunoassay (EIA) and the dextran-coated charcoal (DCC) assay were compared. A highly significant correlation was obtained between these methods for the ER measurement (Rs = 0.91). For PR determination, the Rs value between EIA and DCC assay was 0.57 and the mean value of PR-DCC is significantly higher than the mean value of PR-EIA. These results suggest that EIA is a suitable method for ER measurement. For PR determination on curettage material the DCC assay seems more accurate than EIA.

The immobilization of heparin onto polymeric surfaces using a hydrophilic spacer was effective in curtailing surface induced thrombus formation. In this study, the binding kinetics of immobilized heparin with antithrombin III (ATIII) and thrombin were investigated. Low molecular weight heparin (molecular weight, 6,000 daltons) was fractionated on an ATIII affinity column, and it was immobilized onto a styrene/p-amino styrene random co-polymer surface via hydrophilic spacer groups. This polymer substrate was coated onto glass beads (diameter range, 0.088-0.105 mm). PEO (molecular weight 3,400), modified by tolylene diisocyanate, was covalently coupled as a spacer group, followed by heparin. The bioactivity of immobilized heparin was approximately 16.2%, relative to free heparin, and nearly 1:1 binding between heparin and PEO was calculated. The binding constants of immobilized heparin and ATIII, and immobilized heparin and thrombin, were 0.958 x 10(7) M-1 and 1.76 x 10(8) M-1, respectively. The immobilized heparin bound with both ATIII and thrombin, and the binding mechanism was similar to that of free heparin.

So far, the few studies performed on endometrial steroid receptors in in vitro fertilization (IVF) cycles have been carried out by the dextran-coated charcoal (DCC) method. Despite widespread efforts to standardize the DCC assay method, assay results still vary and thus, data obtained in IVF cycles are controversial. In the present article, late luteal estradiol (E2) and progesterone (P) receptor (R) levels were measured by a new monoclonal enzyme immunoassay (EIA) method in endometrial samples obtained from 21 patients in an IVF program who received no embryo transfer (ET) after ovarian stimulation with follicle-stimulating hormone/human menopausal gonadotrophin/human chorionic gonadotrophin under pituitary suppression with buserelin. Plasma levels of E2, P and prolactin (PRL) were measured in three blood samples collected in the midluteal phase. Results were compared with those obtained in a control group of 21 spontaneous cycles. E2, P and PRL were significantly higher in stimulated than in spontaneous cycles. The level of E2R was decreased in endometrium in IVF stimulated cycles, but PR remained unchanged.

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.

Primary hemochromatosis is a genetically determined autosomal recessive disorder characterized by the excessive accumulation of body iron, most of which is deposited in the parenchymal cells of various organs. alpha 1-Antitrypsin deficiency is characterized among others by defective secretion of alpha 1-antitrypsin from liver cells. Whereas the risk of cirrhosis is increased in homozygous patients (PI ZZ) and possible in heterozygous patients (non-PI MM) as well, a greater risk for hepatocellular carcinoma has been suggested only in homozygous patients. Because these two metabolic disorders are relatively common, it has been difficult to determine whether they are associated with each other. In this study, we tried to determine the relationship between these two disorders using the case material seen at the University of Pittsburgh during a 7-yr period. We studied 15 patients with genetic hemochromatosis. alpha 1-Antitrypsin quantitation and phenotyping were performed in each case using standard methods. The distribution of the various Pi phenotypes was compared with that found in a normal population and reported elsewhere. Odds ratio and chi 2 tests were used to measure the relative risk and significance of association, respectively. Eleven patients (73%) were found to be PI M and four (27%) were identified as being heterozygotes: three (20%) were PI MZ, and one (7%) was PI MS. The prevalence of the PI MS phenotype was similar to that in the general population (7% vs. 6.4%; NS). The PI MZ phenotype, however, was statistically more common in patients with hemochromatosis than in the general population (20% vs. 2.2%; p less than 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

<h4>Objective</h4>To assess whether growth factors are produced by early human embryos in culture.<h4>Design</h4>We studied various growth factors in the culture media of human embryos (n = 6) cultured from days 3 to 8 after fertilization.<h4>Main outcome measures</h4>Four growth factors were measured: Insulin growth factors I and II (IGF-I and IGF-II), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) activity.<h4>Results</h4>Nonconditioned INRA Menezo B2 (Biomerieux, S.A., Paris, France) culture medium contained significant levels of TGF alpha activity (5.2 ng/mL) and low levels of IGF-I (1.02 ng/mL) and IGF-II (2.8 ng/mL), whereas EGF was below detection of our assay. With human embryo, the culture media contained lower TGF alpha activity on days 3 and 4 after fertilization (2.5 ng/mL and 2.8 ng/mL, P less than 0.05). From days 5 to 8 after fertilization, a significant increase in TGF alpha activity and IGF-II was detected (TGF alpha activity: day 5: 3.7 ng/mL; day 6: 4.4 ng/mL; day 7: 6.4 ng/mL; day 8: 8.4 ng/mL) (IGF-II: day 5: 3.4 ng/mL; day 6: 3.1 ng/mL; day 7: 4.1 ng/mL; day 8: 4.2 ng/mL). Epidermal growth factor was undetectable, and IGF-I did not vary significantly.<h4>Conclusion</h4>Transforming growth factor alpha activity and IGF-II are produced by human embryos in culture at a time when they could play a role in morula to blastocyst transformation.

The POU domain gene family of transcription factors share a conserved bipartite DNA binding domain, and exhibit distinct temporal and spatial patterns of expression during development, particularly in the forebrain. A cDNA encoding a new member of the POU-III class of the POU domain gene family, referred to as Brn-4, was isolated from a rat hypothalamic cDNA library. Like other mammalian POU-III genes previously characterized (Brn-1, Brn-2, Tst-1), Brn-4 transcripts are initially widely expressed at all levels of the developing neural tube, but in contrast to other previously described POU-III genes, are subsequently restricted to only a few regions of the adult forebrain, including the supraoptic and paraventricular nuclei of the hypothalamus. Brn-4 was shown to bind to DNA sequences containing the octamer motif and to trans-activate promoters containing this DNA binding motif, based on the actions of a unique N-terminal information. This ontogenic pattern of Brn-4 expression in concert with that of Oct-2 and Pit-1, indicates that certain POU domain genes potentially exert their primary functions widely during early neural development, and in a very limited set of neurons in the mature brain.

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

The effect of burn wound size on the activation of fibrinolysis, coagulation, and contact factors was analyzed in 60 thermal injury patients. Blood samples from 47 male patients and 13 female patients, (average age 37 years; range 1.5-70 years) were collected within the first 36 hours and at 5-7 days following injury. The patient population was categorized by percentage of burn (second degree and/or third degree): less than 20%, n = 22; 20%-40%, n = 18; greater than 40%, n = 20. The average percentage of burn was 32% (range, 4%-95%). The mechanism of injury was by flame (25), explosion and flame (19), scald (12), electric (3), or chemicals (1). An associated inhalation injury was present in 12 patients. The overall mortality rate was 13% (8). Sepsis or serious infection occurred in 23% (14) of the patients. On admission, 83% of the patients had normal prothrombin times (PT) and activated partial thromboplastin times (APTT). However, specific hemostatic variables showed marked changes. Admission hemostatic markers that correlated with the severity of injury were: tissue-plasminogen activator (tPA), plasminogen activator inhibitor (PAI), D-dimer (D-di), plasminogen (Plg), proteins C and S (PrC and PrS), antithrombin III (ATIII), thrombin-antithrombin complex (TAT), kallikrein (Kal:c), kinin (Kin), C1 esterase inhibitor (C1Inh), and factor VII clotting and antigen (FVII:c, FVII:ag). These data suggest that during the early course following burn injury, thrombogenicity is increased (TAT increases) because of a decrease in ATIII, PrC, and PrS; and fibrinolysis activation (D-di increases) occurs via an increase in tPA with a p value increase in PAI.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood coagulation studies showed that patients with non-insulin-dependent diabetes mellitus (NIDDM) had significantly higher fibrinogen, FVIII:C, ristocetin co-factor, FV, FIX, lower ATIII, and PCV than those with insulin-dependent diabetes mellitus (IDDM). Diabetics with IDDM had a significantly higher ATIII, ristocetin co-factor, lower plasminogen and alpa-2-antiplasmin, and more enhanced platelet aggregation responses to ristocetin than age-matched controls. Patients with NIDDM as compared with controls, exhibited higher levels of fibrinogen, ristocetin co-factor, FVIII:C, FIX, and platelet count, but lower plasminogen, alpha-2-antiplasmin and PCV, reduced platelet aggragability to collage, ADP, and ristocetin. Diabetics with retinopathy and nephropathy had still higher levels of fibrinogen, FVIII:C, ATIII and ristocetin co-factor than those without complications. These results are in accord with many similar studies in Caucasians. It is concluded that the pattern of the changes in hemostatic variables noted in Saudi diabetic do not confirm the existence of racial and/or geographical variations in the hemostatic changes associated with diabetes mellitus.