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Adenomatous polyposis coli (APC) is a genetic disorder transmitted as an autosomal dominant trait. This syndrome is characterized by the development of numerous polyps during the first 20-30 years of life and classified into two phenotypes according to the number of polyps: the profuse and sparse types. If left untreated, most or all affected individuals are at a high risk of developing adenocarcinoma by as early as 40 years of age. Therefore, comparison of APC adenocarcinomas with non-polyposis colorectal carcinomas (NPCC) was thought to be useful for understanding genetically determined carcinogenesis. I investigated gene alterations in specimens obtained from 53 APC patients, of which 16 represented the profuse type and the others the sparse type, and from 15 NPCC patients. The results are summarized as follows: 1) K-ras gene mutations were detected more frequently in the profuse-type adenomas (43%) than in the sparse ones (14%) (p less than 0.05). 2) Loss of heterozygosity on the long arm of chromosome 5(5q), 18(18q) and the short arm of chromosome 17(17p) in the profuse-type adenomas was observed more frequently (22%) than in the sparse ones (7.3%) (p less than 0.05). 3) No significant differences were observed between APC adenocarcinomas and NPCCs regarding the allelic deletions on 5q, 17p and 18q in these tumors. 4) The alteration of the DCC gene, which is known to be involved in the formation of NPCC, was frequently detected in the APC adenocarcinomas, suggesting that similar genetic events are involved in the oncogenesis of adenocarcinomas from APC and NPCC.

To examine signal transduction events activated by oncogenic p21ras, we have studied kinases that are activated following the scrape loading of p21ras into quiescent cells. We observe rapid activation of 42 kDa and 46 kDa protein kinases. The 42 kDa kinase is the mitogen and extracellular-signal regulated kinase ERK2, (MAP2 kinase), which is activated by phosphorylation on tyrosine and threonine in response to oncogenic p21ras, while the 46 kDa kinase is likely to be another member of the ERK family. Stimulation of these kinases by oncogenic p21ras does not require the presence of growth factors, showing that oncogenic p21ras uncouples kinase activation from external signals. In ras transformed cell lines, these kinases are constitutively activated. We propose that the kinases are important components of the signal transduction pathway activated by p21ras oncoprotein.

One hundred patients with multiple injuries (mean ISS 37 patients) were prospectively evaluated over a period of 14 days following trauma. Significant differences in the blood levels of PMN elastase, cathepsin B, lactate, neopterin, C-reactive protein (CRP) and antithrombin III (ATIII) were found in non-survivors and in survivors with and without organ failure. On admission, a prediction of organ failure was possible with an accuracy of 63% to 69% (PMN elastase, cathepsin B, ATIII). Death was predictable with an 80% to 90% accuracy within the first 4 days (PMN elastase, lactate, CRP, neopterin). The prognostic value of these factors was comparable to trauma scores regarding organ failure and better with respect to death. Biochemical parameters may be helpful in estimating the severity of the injury and prognosis and in monitoring the ICU course of such patients.

Blood and urine concentrations of antithrombin-III, plasminogen, alpha 2-macroglobulin, alpha 1-antitrypsin, degradation products of fibrinogen-fibrin were studied in patients with abdominal suppuration. Noticeable deviations from the normal values especially marked in the severe process indicated the development of DIC syndrome and renal failure. Heavy combined treatment promoted normalization of the hemostasis shifts and eliminated pyo-inflammatory processes in the abdominal cavity.

Coagulation changes due to polytrauma are considered to be an important determinant for the outcome. In this context, physiological inhibitors of activated coagulation are highlighted with special reference to antithrombin-III (AT-III). Blood samples of 20 randomly selected adults with polytrauma (Injury Severity Score mean = 36.7 +/- 8.6) were investigated. To investigate the very early onset of coagulation changes, samples were taken as early as possible at the site of emergency (mean = 18.3 +/- 5.5 min. after trauma) as well as at hospital admission (mean = 78.0 +/- 10.4 min.). By means of a specially designed "mini-lab", basic processing of the samples harvested (centrifugation, pipetting, freezing) was done on the spot to obtain haemostaseological results that agree as closely as possible with the subsequent analyses. Due to the activation of intravascular coagulation as well as the consumption of physiological inhibitors, comprehensive coagulation disturbances become obvious at the time of hospital admission. These are intensified by haemodilution as a consequence of high-dose fluid replacement. However, significantly elevated levels of specific coagulation reaction products (thrombin-antithrombin-III-complex) give evidence of the consumption of inhibitory potential exceeding haemodilution.

Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD+ abolished the burst. The Km for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD+ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested the some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.

Using the fluorescent indicators 2',7'-bis(2-carboxyethyl)-5'-(6')-carboxyfluorescein and Oxonol V to monitor intracellular pH (pHi) and cell membrane potential respectively, we have investigated the involvement of H(+)-dependent ATPase and H(+)-dependent K+ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24 mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pHi of 6.42. The subsequent recovery of pHi was unimpaired by the absence of extracellular K+, but was reduced in the presence of the Na+ antagonist amiloride (1 mmol/l), recovering by 0.11 +/- 0.003 units, compared with 0.27 +/- 0.02 units under amiloride-free conditions. In the presence of the H(+)-dependent ATPase antagonist N,N'-dicyclohexylcarbodiimide (DCC), the pHi recovery observed in amiloride-containing, K(+)-free buffer was abolished. The recovery of pHi in Na(+)- and K(+)-containing buffer was accompanied by hyperpolarization of the cell membrane, the later stage of which was reduced after blockade of K+ channels with BaCl2, implying a major contribution of transmembrane K+ movement to such events. In contrast to its attenuating effect on pHi recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H(+)-dependent ATPase may not be a major contributory factor to this event. However, when K+ channels were blocked by addition of BaCl2, addition of DCC abolished the butyrate-induced membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxisome proliferators are a diverse group of chemicals, including several hypolipidaemic drugs, that activate a nuclear hormone receptor termed the peroxisome proliferator activated receptor (PPAR). The peroxisomal enzyme acyl CoA oxidase (ACO) is the most widely used marker of peroxisome proliferator action. We have examined the 5' flanking region of the rat ACO gene for sequences that mediate the transcriptional effect of peroxisome proliferators and have identified an element located 570 bp upstream of the ACO gene that confers responsiveness to the hypolipidaemic peroxisome proliferator Wy-14,643. This peroxisome proliferator response element (PPRE) contains a direct repeat of the sequence motifs TGACCT and TGTCCT and binds PPAR. These data therefore indicate an important role of PPAR in mediating the action of peroxisome proliferators including the induction of ACO.

The envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) binds the cellular protein CD4 with high affinity. By deletion we show that 62 N- and 20 C-terminal residues along with the V1, V2 and V3 variable regions of gp 120 are unnecessary for CD4 binding. A 287 residue variant (ENV59), missing those 197 amino acids, binds to CD4 with high affinity. A polyclonal antibody failed to efficiently precipitate ENV59 which is consistent with the loss of immunodominant antigenic structures in the regions deleted. This suggests that ENV59 may have potential as an immunogen, able to elicit antibodies against more conserved regions of gp120. Additionally, complementing co-expressed gp120 fragments as well as a circularly permuted molecule bind CD4, and suggest either that the molecular termini are adjacent in the folded structure, or that an N-terminal region folds into the structure unconstrained by its method of attachment to the rest of the molecule.

<h4>Aims</h4>To evaluate the feasibility of an interlaboratory quality control programme in immunohistochemistry.<h4>Methods</h4>Several pathology laboratories were asked to carry out immunohistochemical oestrogen receptor staining on a set of freeze dried cryostat sections of breast cancer tissue. The sections and protocols for staining and semi-quantitative scoring were mailed to the participating laboratories in two trials. The oestrogen receptor content of the breast cancer samples was determined by radioligand binding assay on the tumour cytosol.<h4>Results</h4>In the first trial 11 laboratories (response rate 60%) participated. Eight (73%) of the participants scored within a 95% confidence interval and all but one correctly classified the tumour as receptor positive. In the second trial all 20 participating laboratories (response rate 55%) correctly scored one tumour sample as negative and 18 of them (90% of respondents) correctly classified the two other tumour samples as receptor positive. In a quantitative evaluation a histochemical score within 95% confidence interval limits was provided by eight (40%) and 12 (60%) of the participants.<h4>Conclusions</h4>Semiquantitative scoring of immunocytochemical staining is valuable for performing correlative inter-laboratory studies, although this scoring protocol may not be required for diagnosis or prognosis. Significant inter-laboratory variability exists, leading to qualitatively correct receptor classification in 100% of receptor negative and 80% of receptor positive cases, and quantitative agreement in only about half of the cases. The perceived variability is not caused by systematic differences in the choice of the immunocytochemical technique, or the mailing of freeze dried sections. Quality control programmes should be included in the standard procedures of each diagnostic immunohistochemistry laboratory.

A 1.08 kbp cDNA encoding rat liver mitochondrial delta 3, delta 2-enoyl-CoA isomerase (ECI) of 298 amino acid residues (Mr 32,895) was isolated from rat liver lambda gt11, lambda gt10 cDNA libraries by the combination of an immunochemical method with a rabbit-antibody against rat liver ECI and a plaque hybridization method. The deduced amino acid sequence from the cDNA indicates that ECI is synthesized with an amino-terminal extrasequence of 35 amino acid residues and processed to the mature enzyme (Mr 29,256).

Anticoagulant properties were first described in extracts from marine algae over 50 years ago. Currently over 60 species, representing the three major divisions of marine algae, have been reported to have such properties. The major active components are a variety of sulphated polysaccharides, some of which characterize as proteoglycans. These sulphated polysaccharides/proteoglycans exert antithrombin properties, primarily mediated by heparin cofactor II (HCII). However antithrombin III (ATIII) mediated anticoagulant activities, as well as an element of direct antithrombin activity, have also been observed.

No abstract available.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.

To assess the clinical value of human leukocyte antigen typing in the diagnosis and management of hereditary hemochromatosis, 105 siblings of 35 proband cases of hemochromatosis were retrospectively analyzed to study whether the exclusion of human leukocyte antigen typing would have adversely affected management. All siblings and probands had already been tested for human leukocyte antigen-A and human leukocyte antigen-B typing, serum ferritin and transferrin saturation. The median age of siblings was 55 yr (range = 11 to 82). Siblings were categorized according to putative genotype (homozygote, heterozygote and normal) using human leukocyte antigen typing. Phenotypic expression of hemochromatosis was considered to be iron overload as indicated by an elevated ferritin (male = greater than 350 micrograms/L, female = greater than 200 micrograms/L) and/or transferrin saturation (greater than 55%). Six of 37 homozygotes had a normal ferritin and transferrin saturation, with five of these patients under 32 yr old. No putative heterozygotes with both an abnormal ferritin and transferrin saturation were seen, although 12 of 48 (25%) heterozygotes had either an elevated ferritin or transferrin saturation. Twenty of 20 normal siblings had a normal ferritin and transferrin saturation. To assess the cost of screening with and without human leukocyte antigen typing, a cost model simulation was used that compared the costs of both methods in a hypothetical family (proband, homozygote, heterozygote and normal sibling).(ABSTRACT TRUNCATED AT 250 WORDS)

S115 mouse mammary epithelial cells lose their epithelial morphology and become tumorigenic when exposed to steroids. We have recently reported that testosterone exposure results in the suppression of syndecan expression, suggesting that this cell surface proteoglycan may influence S115 cell phenotype. We now report that a similar suppression and morphological response of S115 cells can be achieved by glucocorticoid exposure. We introduced into S115 cells an exogenous gene construct containing the full-length human syndecan cDNA under the control of a glucocorticoid-inducible retroviral promoter, in order to study the effect of syndecan expression on S115 cell behavior. Glucocorticoid-induced re-expression of syndecan in S115 cells restored an epithelial phenotype, while control transfectants and parental S115 cells exhibited an altered, nonepithelial phenotype. Moreover, the S115 cells expressing exogenous syndecan revealed a reduced ability to form colonies in soft agar. Therefore, the maintenance of epithelial morphology and normal growth of S115 cells are dependent on syndecan expression.