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This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.

In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.

Prekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAL-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G11. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with C1 inhibitor (C1 INH) and alpha 2-macroglobulin (alpha 2 M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-alpha 2 M complexes. Complexes of KAL-antithrombin III (ATIII) and the ratio of KAL-alpha 2 M/KAL-C1 INH were higher in activated C1 INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KAL-alpha 2 M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and alpha 2 M. Immunoblots of activated plasma also showed bands at the position of KAL-C1 INH and KAL-ATIII complexes. When alpha 1-antitrypsin Pittsburgh (alpha 1-AT. Pitts) was added to plasma before activation, KAL-alpha 1-AT. Pitts was the main complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Compounds representing chemically modified derivatives of natural polysaccharides and possessing heparin-like properties have been obtained basing on new methods of sulphatization++ of hydroxyl-containing polymers. The study of the anticoagulation activity of mannan, starch, dextran sulfates permits a suggestion that these compounds realize their anticoagulant potential not through antithrombin-III. A relationship has been established between the properties of semi-synthetic heparinoids and sulfur percentage, molecular mass and the type of polymeric matrix. The investigations conducted have made possible the development of effective thrombosis-preventing drugs and further thorough study of their effects.

A comparative study of estrogen and progesterone receptor bindings of breast carcinoma tissue was done by immunoperoxidase and dextran-coated charcoal (DCC) methods. Fifteen cases of paraffin embedded formalin fixed tissue of mammary carcinomas which had previously been evaluated by the DCC method were selected. Twelve cases were ductal carcinoma and 3 were of lobular origin. Lymph node tissue showing metastasis was available in 3 cases. By immunoperoxidase technique, 12 and 10 (80 and 66.7%) cases were positive for estrogen and progesterone receptor respectively compared with 8 and 3 (53.3 and 20%) cases by the DCC technique. Corresponding results of both methods to detect estrogen and progesterone bindings were 9 and 8 (60 and 53.3%) of all cases, respectively. Five cases for estrogen and 6 cases for progesterone positive by immunoperoxidase could not be detected by the DCC technique. Only one case of estrogen negative by the immunoperoxidase gave a positive result with the DCC technique. Variability of staining occurred between primary and metastatic lesions, 2 out of 3 cases displayed positive staining in both sites; one remaining case was positive only in the lymph node metastasis. Immunoperoxidase is a relatively simple, swift and inexpensive technique in comparison to the DCC technique. Using fixed embedded tissue makes it possible for retrospective studies and providing a permanent record for reevaluation. Moreover, morphology of the tumor can be determined at the same time as detection of hormonal receptor bindings.

The association of thrombosis with antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE) could be due to their interference with natural phospholipid dependent anticoagulant mechanisms. We studied antigenic protein C (APC), functional protein C (FPC), free protein S (FPS), protein S bound to C4 binding protein (C4bp-S), antithrombin III (ATIII), as well as IgG and IgM anticardiolipin antibodies (aCL) in 38 patients with SLE with a history of thromboses and 70 patients with SLE without such history. We found a high frequency of deficiencies of natural anticoagulants in both groups of patients with SLE but, because of patient selection, we could not determine the actual prevalence of these defects. Patients having had a venous thrombosis in the previous year had low C4bp-S more frequently than patients with older or no thromboses. When we divided our patients with SLE into those who had a definite, probable, questionable or no antiphospholipid syndrome (aPS) we found the frequency of C4bp-S deficiency to be significantly higher in those with definite aPS than in those without aPS. Intermediate proportions were found in patients with probable and questionable aPS. The levels of C4bp-S decreased as the levels of aCL, particularly IgG, increased. Stepwise discriminant analysis of natural anticoagulants selected deficiencies of C4bp-S and FPC with increased ATIII as a set of variables with highest predictive power for classification of patients with and without aPS. Thus, deficiencies of natural anticoagulants may occur frequently in patients with SLE.(ABSTRACT TRUNCATED AT 250 WORDS)

A positive selection system designed to identify and recover candidate tumor-suppressor genes is described. The system compares mRNA expression of genes from normal and tumor-derived human mammary epithelial cells grown in a special medium that supports similar growth rates of the two cell types. mRNAs uniquely expressed in normal cells are recovered as cDNAs after subtraction with mRNA from tumor cells. Seven different clones, from 0.6 to 4.8 kilobases in transcript size and including both rare and abundunt transcripts, were recovered in the first 23 clones analyzed. Among the isolated clones were genes encoding the gap-junction protein connexin 26, two different keratins, and glutathione-S-transferase pi, as well as an unknown gene in the S100 family of small calcium-binding proteins. In principle, tumor-suppressor genes include two classes: class I, in which loss of function results from mutation or deletion of DNA and class II, in which loss of function is from a regulatory block to expression. A class II suppressor gene is assumed to be regulated by a different suppressor gene that lost its function by mutation or deletion. Both classes of tumor-suppressor genes may provide valuable proteins with clinical applications in cancer diagnosis or therapy. Class II suppressors may be especially useful because the normal genes are present and their reexpression may be inducible by drugs or other treatments.

This report reviewed recent remarkable progresses on the cytomolecular mechanisms in colorectal carcinogenesis. Colorectal carcinoma is a good model for the study of multi-step progression, because we can obtain adenomatous polyps which are considered as a precancerous form. Furthermore, a familial syndrome, which is characterized by numerous adenomas of the colon, is available for linkage analysis. Recently, the p53 and DCC genes have been identified as candidate tumor suppressor genes on chromosome 17p and 18q respectively. In this paper, we present the multiple genetic alterations in colorectal carcinoma, including activation of K-ras gene and inactivation of tumor suppressor gene such as p53 and DCC genes as well as loss of heterozygosity and approach to the gene responsible for adenomatous polyposis coli by reverse genetics.

With a view to investigating whether in circadian variations of platelet aggregation (PA) and fibrinolytic activity there is an elevated risk period for incidence and development of ischemic stroke, 25 healthy subjects (5 females and 20 males), their age ranging from 29 to 51, were exposed to the analysis of PA, euglobulin lysis time (ELT), fibrinogen degradation products (FDP), antithrombin III (AT III) and heparin tolerance test (HTT) in blood samples drawn by venepuncture at 08.00, 11.00, 13.00, 15.00, 17.00 and 18.00 h; beside these intervals in the case of 10 healthy males, whose age ranged from 32 to 45, blood samples were taken at midnight as well. The group of 25 subjects comprised those who usually worked daily and nightly shifts, as well as those who were either at bed rest or doing their duties during daytime. The findings of this investigation have demonstrated that all the parameters studied exhibited circadian variations irrespective of sex, age or daily/nightly activities of the subjects. The most pronounced PA interval, which was not accompanied by corresponding increase of fibrinolytic activity, was that around 11.00 h and it is marked as the highest risk period for onset of ischemic stroke.

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. Abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms-1 at a 0.63 nM concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.

In 21 women who had an abortion by hysterotomy between the 15th and 23rd week of pregnancy, the possibility that unfragmented heparin or low molecular weight heparin (LMWH) passed the placental barrier to the foetus was studied. Laboratory analyses included amidolytic assays of factor Xa inhibitory activity (XaI), antithrombin III (ATIII) and a direct measurement of heparin-like substances in plasma with a competitive binding assay. The ATIII concentration in foetal plasma was about 20% of that in normal human plasma and varied considerably between individuals (2-27%). The XaI activity did not differ between the two treated groups, but the mean XaI activity of the combined groups differed from zero (P less than 0.05). If the XaI activity was corrected for the ATIII concentration, the heparin activities no longer differed significantly from zero. As the concentration of heparin-like substances were above the detection limit (0.35 microgram/ml) in 6/16 analysable samples of foetal plasma, a further 15 women who had not received any heparin were included as controls. In 12/14 analysable foetal plasmas heparin-like substances in concentrations above 0.35 micrograms/ml could be detected. Determination of heparin activity in foetal plasma is thus difficult due to the influence of endogenous ATIII on heparin assays. In conclusion, this study did not demonstrate any evidence for the passage of heparin or LMWH across the placental barrier. No differences were detected whether unfragmented heparin or LMWH had been given to the mothers. Our results also indicate the presence of an endogenous glycosaminoglycan in foetal plasma.

A single gene (xylB) encoding both beta-D-xylosidase (EC 3.2.1.37) and alpha-L-arabinofuranosidase (EC 3.2.1.55) activities was identified and sequenced from the ruminal bacterium Butyrivibrio fibrisolvens. The xylB gene consists of a 1.551-bp open reading frame (ORF) encoding 517 amino acids. A subclone containing a 1.843-bp DNA fragment retained both enzymatic activities. Insertion of a 10-bp NotI linker into the EcoRV site within the central region of this ORF abolished both activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytoplasmic proteins from recombinant Escherichia coli confirmed the presence of a 60,000-molecular-weight protein in active subclones and the absence of this protein in subclones lacking activity. With p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside as substrates, the specific activity of arabinosidase was found to be approximately 1.6-fold higher than that of xylosidase. The deduced amino acid sequence of the xylB gene product did not exhibit a high degree of identity with other xylan-degrading enzymes or glycosidases. The xylB gene was located between two incomplete ORFs within the 4,200-bp region which was sequenced. No sequences resembling terminators were found within this region, and these three genes are proposed to be part of a single operon. Based on comparison with other glycosidases, a conserved region was identified in the carboxyl end of the translated xylB gene which is similar to that of glucoamylase from Aspergillus niger.

The levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI) and other substances of coagulation-fibrinolysis, such as fibronectin (Fn) and von willebrand factor (vWF) as well as the activity content of antithrombin-III(AT-III) in plasma were determined in 20 patients with acute myocardial infarction (AMI). In 11 of them these measurements were carried out before and after the treatment with urokinase (UK1000 000 IU). The results suggested that the function of coagulation-fibrinolytic system was disturbed in AMI. Thrombolytic treatment with UK could interfere and improve the stabilization of fibrinolytic activity in the body, but these actions last only short time. Some substances of coagulation showed change with UK treatment.

Correlation between survival and hormonal receptor status in patients with breast cancer is still controversial. The results of a study carried out, from 1973 to 1988, on 55 patients (average age 56.7) affected with breast cancer and submitted to radical (Halsted) mastectomy, are reported. Estradiol receptors (ER) and progesterone receptors (PR) were determined using the dextran-coated charcoal (DCC) method and the enzymatic immuno assay (EIA). The threshold value for defining the ER positivity was 10 f/mole/mg of cytosol protein and 50 f/mole/mg for PR positivity. Forty-one patients resulted as ER+, 31 were also PR+. Moreover, 41 patients were treated with tamoxifen (10 mg x 2 die per os). In a 15-year follow-up, no significant difference in survival rate between patients with receptor-positive tumors or treated with tamoxifen and patients with receptor-negative tumors was recorded.

Bovine antithrombin (ATIII) is a glycoprotein of Mr 56,600. Its primary structure was established using peptide sequences from five different digests. Bovine ATIII exhibits four glcosylation sites as well as human ATIII. The primary structures of bovine and human ATIII were compared: all the residues required for the integrity of the heparin-binding domain are strictly conserved. However, there are differences in the secondary structures of both proteins, bovine and human ATIII.

The ability of carcinomas to invade and to metastasize largely depends on the degree of epithelial differentiation within the tumors, i.e., poorly differentiated being more invasive than well-differentiated carcinomas. Here we confirmed this correlation by examining various human cell lines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioid phenotype were noninvasive and expressed the epithelium-specific cell-cell adhesion molecule E-cadherin (also known as Arc-1, uvomorulin, and cell-CAM 120/80), as visualized by immunofluorescence microscopy and by Western and Northern blotting, whereas carcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin mAbs. These findings indicate that the selective loss of E-cadherin expression can generate dedifferentiation and invasiveness of human carcinoma cells, and they suggest further that E-cadherin acts as an invasion suppressor.

Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.

Cytogenetic analysis of meningioma cells from one particular patient (MN32) displayed the stem-line karyo-type 45, XY, -1, 4p+, 22q-, 22q+, which thus had rearrangements of both chromosomes 22. The 22q+ marker appeared as a dicentric: 22 pter----q11::1p11----qter. The reciprocal product of this translocation has presumably been lost because it lacked a centromere. The 22q- chromosome also appeared to have lost sequences distal to band q11. We assumed that this marker could have been the result of a reciprocal translocation between chromosomes 4 and 22. To investigate the 4p+ and 22q- chromosomes in more detail, human-hamster somatic cell hybrids were constructed that segregated the 22q- and 4p+ chromosomes. Southern blot analysis with DNA from these hybrids showed that sequences from 22q were indeed translocated to 4p+ and that reciprocally sequences from 4p were translocated to 22q-, demonstrating a balanced t(4;22)(p16;q11). On the basis of these results we presume that in this tumor a tumor-suppressor gene is deleted in the case of the 22q+ marker and that the t(4;22) disrupts the second allele of this gene. The latter translocation was mapped between D22S1 and D22S15, a distance of 1 cM on the linkage map of this chromosome. The area in which we have located the translocation is within the region where the gene predisposing to neurofibromatosis 2 has been mapped.

The nucleic acid sequence of the androgen receptor (AR) gene predicts that the protein structure possesses DNA- and steroid-binding domains that show high degrees of sequence similarity with those of other steroid receptors. Since the steroid-binding domain of the AR corresponds to a 30 kDa portion of the protein, and the AR structure may be monomeric or hetero-oligomeric depending on its transformation state, we have herein determined the AR radiation-inactivation size (RIS) in relation to the molecular structure whose binding activity toward methyltrienolone (R1881) is abolished by a radiation 'hit'. Soluble fractions from whole canine prostatic tissue were used as a source of non-transformed AR. The AR transformation was induced by the addition of 0.6 M-KCl, and these preparations were used together with high-salt nuclear extracts as a source of transformed AR. To maximize the binding activity, molybdate and dithiothreitol were included during AR extraction. Receptor transformation was verified by modifications of both the sedimentation coefficients (from 7.5 S to 4.1 S on sucrose gradients) and molecular masses (from 260 kDa to 115 kDa by gel filtration). The RIS values of the non-transformed and transformed ARs were not statistically different: 92 +/- 19 kDa and 110 +/- 25 kDa respectively. In addition, the inactivation of AR binding activity by radiation was attributed to a loss of binding sites, with no significant change in the Kd. When benzoic acid, a free-electron scavenger, was added together with dithiothreitol before and after irradiation, no change in the RIS value was observed. Thus, in the canine prostate, the RIS value of the AR represents the monomeric protein, independently of its association with other proteins, and this value corresponds to that predicted by cloning studies and photoaffinity-labelling of AR.

No abstract available.

We compared blood loss and hemostasis in pigs which had undergone either orthotopic liver transplantation (OLT) (group A, n = 12) or auxiliary heterotopic partial liver transplantation (APLT) (group B, n = 11). Blood samples were taken at regular intervals during and after the operations. In both groups, nine animals survived longer than 24 h and data from these animals were used for analysis. Median (range) intraoperative blood loss was 825 ml (250-1500 ml) in OLT and 425 ml (300-750) in APLT (P less than 0.01). Routine clotting times, as the activated partial thromboplastin time, prothrombin time and thrombin time, showed no major intraoperative changes in either group. Fibrinogen levels decreased in both groups, but no significant difference was found between the two groups. The only significant difference between group A and B was a more sustained increase in fibrinolytic activity after graft recirculation in group A. Postoperatively, restoration of fibrinogen, antithrombin-III and alpha 2-antiplasmin levels was slightly faster in group B, resulting in significantly higher levels during the first day. We conclude that, in this animal model, APLT is associated with significantly lower blood loss and less severe fibrinolytic activity, than OLT. This difference might result from the lack of an anhepatic period and the reduced surgical trauma in auxiliary heterotopic liver transplantation.