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Antithrombin III (ATIII) deficiency has been implicated in adults as a predisposing factor to thrombosis; however, thromboembolic complications are rare in children with the same deficiency. We hypothesized that because of the elevated levels of plasma alpha-2-macroglobulin (alpha 2M) throughout childhood, plasmas of ATIII-deficient children inhibit thrombin more efficiently than those of ATIII-deficient adults. In total, 14 ATIII-deficient adults (ages 25 to 46 years), 13 ATIII-deficient children (ages 2 to 13 years), 9 normal children (ages 3 to 15 years), and 16 normal adults were studied. We measured thrombin inhibition in these plasmas, as well as the contributions of ATIII, alpha 2M, and heparin cofactor II (HCII) as thrombin inhibitors in each plasma. 125I-alpha-thrombin, 25 nmol/L, was added to each plasma (defibrinated with Arvin at 37 degrees C), and 90 seconds later the free thrombin and thrombin-inhibitor complexes were quantitated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric scanning. Plasma from ATIII-deficient adults inhibited significantly less thrombin (12.8 +/- 0.6 nmol/L) than both normal adults (16.1 +/- 0.3 nmol/L, P less than .01), normal children (15.7 +/- 0.4 nmol/L, P less than .01), or ATIII-deficient children (15.5 +/- 0.3 nmol/L, P less than .01). There was no significant difference between the total concentration of thrombin inhibited by ATIII-deficient children and either normal adult or normal children groups. In addition, plasmas of ATIII-deficient children inhibited thrombin significantly more efficiently than plasma of ATIII-deficient adults (P less than .01). In the ATIII-deficient patients there was a significant correlation between the alpha 2M level and ability to inhibit thrombin (P less than .01), but no correlation between either ATIII or HCII levels and thrombin inhibition. On the addition of heparin (0.4 U/mL) to plasma, all four types of plasma inhibited thrombin to the same extent. Although ATIII was the predominant inhibitor in all heparinized plasmas, HCII inhibited more thrombin in the ATIII-deficient patients than in normal patients (2.8 +/- 0.3 v 1.2 +/- 0.2 nmol/L, P less than .01). We hypothesize that the lower risk of thromboembolic complications in ATIII-deficient children may be due in part to the protective effect of elevated alpha 2M levels during childhood.

An efficient solid-phase synthesis of the TRH analogue Glp-His(Nim-Trt)-Hyp-OH is described. Na-Fmoc protected amino acids and DCC/HOBt activation were employed. The bulky and mild-acid-sensitive 2-chlorotrityl resin, utilised as the solid support, completely suppressed dioxopiperazine formation. The tripeptide is a key intermediate in the synthesis of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline. The tripeptide was converted, with inversion of configuration at C-4 of the Hyp residue, to Glp-His(Nim-Trt)-cHyp lactone in the presence of triphenylphosphine-diethyl azodicarboxylate (TPP-DEAD). One-pot MeOH-TPP-DEAD transesterification of the lactone, followed by Nim-detritylation, provided Glp-His-cHyp-OMe. This ester gave the corresponding amide and acid on ammonolysis and saponification, respectively. A high-field 1H NMR investigation of Glp-His-cHyp-OH and its diastereomer Glp-His-Hyp-OH, obtained by Nim-detritylation of the key tripeptide, showed that the configuration at C-4 of the prolyl residues is critical for the determination of the preferred three-dimensional structure of the molecules.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.

The first part of this study deals with well known as well as new data on normal iron metabolism. The second part will concern Genetic Haemochromatosis, a recessively transmitted disease principally determined by a gene located on the sixth chromosome near the A locus of HLA system: phenotypic expression of the gene, clinical features, iron overload assessment, mechanism of iron toxicity, pathogenesis of iron overload. The third part considers iron overload secondary to anaemias, to chronic alcoholic liver diseases, to porphyria cutanea tarda, to chronic haemodialysis... and their relation to the Genetic Haemochromatosis. Beyond what is already well established still lies a large number of questions with answers, at this stage, uncertain or incomplete.

What then are the lessons to be learned about prevention and treatment of hemochromatosis? Early diagnosis is essential. The best indicator would be testing of serum iron and total saturation followed by a serum ferritin if elevated. Once these indices are abnormally high, MRI and or a liver biopsy should confirm the stage of the iron over-loaded state. If indeed the patient is not iron-overloaded (normal liver biopsy in the face of high saturation and ferritin level) phlebotomies should be performed until these indices are normal and then maintained at a normal level. This should entail four to six phlebotomies a year. Family members should also be screened and managed in a like manner. HLA typing may be a partially helpful screening device. The abnormal gene is closely linked on chromosome 6 with HLA histocompatibility loci. Now, by means of HLA typing, we can identify heterozygote carriers and homozygous (abnormal) among first degree relatives of patients with hemochromatosis. Unfortunately, HLA typing can only be used within a given family and cannot be used to screen the general population. It is estimated that 70% of hemochromatoics have the antigen HLA-A3; however, so does 28% of the (well) general population. Patients with unexplained cirrhosis, arthritis, liver disease, diabetes, impotency, cardiomyopathy and neurological symptoms should be screened in a like manner. Routine health practice profile chemistries must include a serum iron and iron saturation, and if high followed by a serum ferritin. Once diagnosed, therapy must be maintained with phlebotomy for the life time of the patient.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effect of heparin cofactor II (HCII) on thrombin-induced prostacyclin (PGI2) production by A10 cells, an established cell line of vascular smooth muscle cells from murine aorta. Confluent monolayers of A10 cells were incubated with 0.1 NIH U/ml of thrombin for 30 min in the presence of antithrombin III (ATIII) or HCII, and PGI2 production by the cells was measured by radioimmunoassay as 6-keto-prostaglandin F1a, the stable metabolite of PGI2. ATIII at 40 mInh.U/ml and more significantly inhibited thrombin-induced PGI2 production by A10 cells, but HCII at the same doses did only slightly. However, when A10 cells were preincubated with HCII for 30 min before exposure to thrombin, the PGI2 production was markedly inhibited. The medium conditioned by A10 cells for 30 min did not enhance the inhibitory effect of HCII on thrombin-induced PGI2 production by the cells. On the other hand, A10 cells synthesized both dermatan sulfate and heparan sulfate which are capable of activating HCII. From these results, it was suggested that HCII would be activated by glycosaminoglycans (GAGs) such as dermatan sulfate of the cell layers and could inhibit thrombin-induced PGI2 production. HCII may be a modulator of thrombin on the physiological functions of vascular smooth muscle cells, reacting to the cell surface GAGs.

Cancer is caused by specific DNA damage. Several common mechanisms that cause DNA damage result in specific malignant disorders: First, proto-oncogenes can be activated by translocations. For example, translocation of the c-myc proto-oncogene from chromosome 8 to one of the immunoglobulin loci on chromosomes 2, 14, or 22 results in Burkitt's lymphomas. Translocation of the c-abl proto-oncogene from chromosome 9 to the BCR gene located on chromosome 22 produces a hybrid BCR/ABL protein resulting in chronic myelogenous leukemia. Second, proto-oncogenes can be activated by point mutations. For example, point mutations of genes coding for guanosine triphosphate-binding proteins, such as H-, K-, or N-ras or G proteins, can be oncogenic as noted in a large variety of malignant neoplasms. Proteins from these mutated genes are constitutively active rather than being faithful second messengers of periodic extracellular signals. Third, mutations that inactivate a gene can result in tumors if the product of the gene normally constrains cellular proliferation. Functional loss of these "tumor suppressor genes" is found in many tumors such as colon and lung cancers. The diagnosis, classification, and treatment of cancers will be greatly enhanced by understanding their abnormalities at the molecular level.

The complete nucleotide sequence of the slt gene encoding the soluble lytic transglycosylase (Slt; EC 3.2.1.-) from Escherichia coli has been determined. The largest open reading frame identified on a 2.5-kb PvuII-SalI fragment indicates that the enzyme is translated as a preprotein of either 654 or 645 amino acids, depending on which of two potential start codons is used. The two possible translation products differ only in the lengths of their predicted signal peptides, 36 or 27 amino acids, respectively. In both cases, processing results in a soluble mature protein of 618 amino acids (Mr = 70,468). The deduced primary structure of the mature protein was confirmed by N-terminal sequencing and determination of the amino acid composition of the isolated transglycosylase. The slt gene contains a high percentage of rare codons, comparable to other low-expressed genes. A hairpin structure that could serve as a transcriptional terminator is located downstream of the slt coding region and precedes the trpR open reading frame at 99.7 min on the E. coli chromosomal map. A computer-assisted search did not reveal any significant sequence similarity to other known carbohydrate-degrading enzymes, including lysozymes. Interestingly, a stretch of 151 amino acids at the C terminus of the transglycosylase shows similarity to the N-terminal portion of the internal virion protein D from bacteriophage T7. Overexpression of the slt gene, under the control of the temperature-inducible phage lambda pR promoter, results in a 250-fold overproduction of the mature transglycosylase, whereas after deletion of the signal peptide a 100-fold overproduction of the enzyme is observed in the cytoplasm.

No abstract available.

The human XPBC/ERCC-3 was cloned by virtue of its ability to correct the excision repair defect of UV-sensitive rodent mutants of complementation group 3. The gene appeared to be in addition implicated in the human, cancer prone repair disorder xeroderma pigmentosum group B, which is also associated with Cockayne's syndrome. Here we present the genomic architecture of the gene and its expression. The XPBC/ERCC-3 gene consists of at least 14 exons spread over approximately 45 kb. Notably, the donor splice site of the third exon contains a GC instead of the canonical GT dinucleotide. The promoter region, first exon and intron comprise a CpG island with several putative GC boxes. The promoter was confined to a region of 260 bp upstream of the presumed cap site and acts bidirectionally. Like the promoter of another excision repair gene, ERCC-1, it lacks classical promoter elements such as CAAT and TATA boxes, but it shares with ERCC-1 a hitherto unknown 12 nucleotide sequence element, preceding a polypyrimidine track. Despite the presence of (AU)-rich elements in the 3'-untranslated region, which are thought to be associated with short mRNA half-life actinomycin-D experiments indicate that the mRNA is very stable (t 1/2 greater than 3h). Southern blot analysis revealed the presence of XPBC/ERCC-3 cross-hybridizing fragments elsewhere in the genome, which may belong to a related gene.