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Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.

The effects of different buffer concentrations and compositions on the elution order and separation of H1 histone subtypes and their phosphorylated modifications isolated from several species was studied using high-performance capillary electrophoresis (CE). Various cations and anions were tested in an untreated silica capillary and low pH buffers, in the presence of the dynamic coating agent hydroxypropylmethyl cellulose. It was found that the cations and anions of buffers have a remarkable influence on both the efficiency and the selectivity of protein separations. A triethylammonium methanephosphonate system proved efficacious for the separation of rat histone subtype H1c from H1e and a perchlorate/triethylammonium phosphate system for the analysis of chicken and mouse linker histones. CE provides an attractive alternative to high-performance liquid chromatography and conventional gel electrophoresis.

Seventeen parameters of coagulation and fibrinolysis were measured in 33 patients with sickle cell disease; 30 were tested in steady state (SS) and 19 in crisis (Cr). There were 16 patients in both groups. The same parameters were measured in 16 controls of similar ethnic origin (Black controls; BC) and 20 Caucasian controls (CC), all with HbA only. Highly significant differences (P < 0.001) between Black and Caucasian control groups were noted for: fibrinogen, fibrinopeptide-A (FPA), beta-thromboglobulin (beta TG) and D-dimer. Significant differences (P < 0.03) in plasminogen activator inhibitor (PAI) and functional antithrombin III levels were also noted. Results of the sickle cell patients were therefore compared with those of the Black controls. Sickle cell patients in SS had raised v Wf compared with BC, which increased further during Cr (P = 0.001), but showed no significant increase in fibrinogen. Functional protein C was reduced in SS (P = 0.004) but with no further fall in Cr, while free protein S was normal in SS but reduced in Cr (P = 0.02). Total protein S and ATIII were normal in SS and Cr. FPA and beta TG were not significantly raised in SS or Cr compared with BC. There were, however, highly significant increases in D-dimer and thrombin-antithrombin complexes (TAT) in both SS and Cr compared with BC (P < 0.001 for SS and Cr vs BC). Thus significant activation of coagulation with consequent increase in fibrinolysis occurs during both the sickle cell crisis and in the steady state.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatibility antigen (HLA) class I region, on 6p21.3. Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available. The gene content of this region is high, and the HFE gene has not so far been identified. We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences. This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region. Five new polymorphisms have been detected among 20 specific fragments isolated. Four of them are tightly linked to the HFE locus. Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene. The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.

Hepatic fibrosis and cirrhosis are common findings in humans with hemochromatosis. In this study we investigated the molecular pathways of iron-induced hepatic fibrosis and evaluated the anti-fibrogenic effect of vitamin E. Male gerbils were treated with iron-dextran and fed a standard diet or a alpha-tocopherol enriched diet (250 mg/Kg diet). In gerbils on the standard diet at 6 wk after dosing with iron, in situ hybridization analysis documented a dramatic increase of signal for collagen mRNA around iron foci onto liver fat storing cells (FSC), as identified by immunocytochemistry with desmin antibody. After 4 mo, micronodular cirrhosis developed in these animals, with nonparenchymal cells surrounding hepatocyte nodules and expressing high level of TGF beta mRNA. In this group, in vivo labeling with [3H]-thymidine showed a marked proliferation of nonparenchymal cells, including FSC. In iron-dosed gerbils on the vitamin E-enriched diet for 4 mo, in spite of a severe liver iron burden, a normal lobular architecture was found, with a dramatic decrease of collagen mRNA accumulation and collagen deposition. At the molecular level, a total suppression of nonparenchymal cell proliferation was appreciable, although expression of collagen and TGF beta mRNAs was still present into microscopic iron-filled nonparenchymal cell aggregates scattered throughout the hepatic lobule. In conclusion, our study shows that anti-oxidant treatment during experimental hepatic fibrosis arrests fibrogenesis and completely prevents iron induced hepatic cirrhosis mainly through inhibition of nonparenchymal cell proliferation induced by iron.

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.

Loss of heterozygosity and loss of expression of the deleted in colon cancer (DCC) gene is frequently observed in a number of different cancer types. To determine if the DCC gene plays a direct role in tumor suppression, wild-type full-length or truncated DCC cDNA constructs were transfected into nitrosomethylurea (NMU) transformed tumorigenic HPV-immortalized human epithelial cells that had allelic loss and reduced expression of DCC. Full-length DCC suppressed tumorigenicity whereas truncated DCC did not. Tumorigenic reversion of initially suppressed transfectants was associated with loss of DCC expression and loss or rearrangement of transfected DCC sequences. These results provide the first direct evidence that DCC is a tumor suppressor gene.

The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.

The synthetic pentasaccharide Org 31540/SR 90107A represents the antithrombin III (ATIII) binding region of heparin and accelerates the ATIII-mediated inhibition of coagulation factor Xa. This compound and 15 structural analogues with ATIII binding constants (Kd) ranging from 2.7 to 2600 nmol/L were compared for their plasma elimination in rats as measured from their factor Xa inhibiting activity. After administration of a low dose (100 nmol/kg body wt IV), each pentasaccharide showed a characteristic plasma half-life varying from a minimum of 0.3 hour for pentasaccharides with low affinity for ATIII to 10.9 hours for pentasaccharides with high affinity for the protein. The latter value was close to the half-life measured for radioiodinated rat ATIII (11.8 hours). We hypothesized that the elimination half-life of pentasaccharides is markedly extended by ATIII binding, of which the extent is governed by the Kd of the complex. The following observations support this hypothesis. The low-dose, low-affinity pentasaccharides were almost fully recovered in the urine without having lost anti-factor Xa activity, whereas compounds with high ATIII binding affinity were only partly recovered in the urine. With a high dose (500 nmol/kg body wt), a rapid plasma clearance of pentasaccharide was observed until a concentration similar to that of endogenous ATIII was reached, in accordance with their expected 1:1 stoichiometric interaction. The elimination of half-life was similar to that of the low dose. The relation between Kd values and plasma half-lives could be explained by assuming rapid clearance of free and coclearance of ATIII-bound pentasaccharide with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The linear antigenic epitopes of the Epstein-Barr virus replication activator protein (ZEBRA), recognised by specific serum IgG in nasopharyngeal carcinoma (NPC), were determined. This was achieved by synthesizing the entire amino acid sequence of ZEBRA as a set of 29, 22-residue peptides with an overlap of 14 amino acids. The ZEBRA peptides were tested in enzyme-linked immunosorbent assay (ELISA) for IgG binding in sera from 37 selected NPC patients who had IgG antibodies to the native ZEBRA protein. The most immunogenic epitope was peptide 1 at the amino-terminal end with 36 of the sera reactive against it. Further analysis of peptide 1, using the multipin peptide-scanning technique, defined a 10-amino-acid sequence FTPDPYQVPF, which was strongly bound by IgG. Two other regions of ZEBRA were also identified as immunodominant IgG epitopes, namely peptide 11 (amino acids 82-103) and peptide 19/20 (amino acids 146-175) with 8-13 of the NPC sera reactive against the peptides. The number of peptides reactive with individual NPC serum varies from 1 to 6 or more and there is some correlation between a greater number of peptide (at least 4) bound and a higher (at least 1:40) titre of serum IgA to viral capsid antigen. The immunodominant ZEBRA peptide 1 could be utilised in IgG ELISA for the detection of NPC.

We investigated the intrapulmonary metabolism of prostaglandin E1 (PGE1), hemodynamics, oxygenation indexes (OI) and intrapulmonary shunt ratios (QS/QT) in patients with pulmonary hypertension (PH group, n = 7) and those with normal pulmonary artery pressure (N group, n = 5). PGE1 at a concentration of 20 approximately 60 ng.kg-1.min-1 was continuously administered intravenously to the patients in both groups for treatment. The intrapulmonary metabolism of PGE1 was calculated as 100 x (1--the concentration ratio of PGE1 in radial/pulmonary artery) %. The concentrations of PGE1 were measured by radioimmunoassay (DCC method) with the specific anti-PGE1 antibodies supplied by Ono Pharmaceutical Co. Hemodynamics were measured using a Swan-Ganz catheter, and OI and QS/QT were calculated from blood gas analyses. In the PH group, the intrapulmonary metabolisms of PGE1 decreased significantly compared with the N group. In addition, mPAP/mSAP in the PH group increased significantly compared with the N group regardless of PGE1 administration. There were no significant differences in OI and QS/QT between the two groups in spite of PGE1 administration. We concluded that the intrapulmonary metabolisms of PGE1 in patients with pulmonary hypertension were significantly decreased compared to the patients with normal pulmonary artery pressure. For this reason, it is important to monitor hypotension more carefully whenever we administer PGE1 to patients for the treatment of pulmonary hypertension.

This study reports on the bioavailability and pharmaco kinetics of a new low molecular weight heparin (RO-11), with a mean molecular weight of 3,600-3,800 daltons. It was administered to 12 healthy individuals in an open, cross-over study. Each subject was randomly assigned to three experimental treatments: a) 30 mg by subcutaneous route, b) 60 mg subcutaneously and c) 60 mg intravenously, leaving a wash-out period of one week between treatments. The pharmacokinetic profile was calculated by means of the anti-FXa effect, measured on serial samples taken before and during the 24 h period after each treatment. The effects of the drug on global coagulation tests, Antithrombin-III levels, tissue-factor pathway inhibitor activity and anti-FIIa activity were also assessed. After the intravenous injection, a maximal anti-FXa effect of 1.30 +/- 0.18 IU/ml was measured at 0.05 h and was not measurable after 12 h. After the subcutaneous injection of 30 and 60 mg, the maximal anti-FXa effect (0.34 +/- 0.08 IU/ml and 0.54 +/- 0.06 IU/ml) was reached after 2-4 h, and was not measurable after 12-18 h. The magnitude of this effect was dose-dependent and the absorption and elimination processes followed a first-order pattern for every dose and route. RO-11 showed a biological half-life of about 5 h and a high subcutaneous bioavailability (96%).

The level of DCC mRNA expression was evaluated in tissue specimens from lung cancer patients by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blot analysis. Obvious reduction of DCC gene expression was observed in 4 of 7 specimens (55%). In two specimens DCC transcript could only be detected after Southern blot hybridization of RT-PCR product. The average level of DCC expression in cancer tissue was about 45% of normal tissue as estimated by laser densitometer. We also studied DNA samples for loss of heterozygosity (LOH) at DCC locus at two polymorphic sites. Among the 15 specimens including 7 samples for RT-PCR, 9 (60%) were informative at either of two polymorphic sites. LOH was observed in 5 (55%). Two at the MspI-RFLP (restriction fragment length polymorphism) site and 3 at the site of VNTR (variable number of tandem repeat). These results suggest that allele loss and decreased expression of DCC gene are frequent events and the possible involvement of DCC gene in the pathogenesis of human lung cancer.

<h4>Background</h4>Necrolytic migratory erythema (NME) is an uncommon inflammatory dermatosis with a distinct clinical and histologic appearance. NME is usually associated with glucagonoma. Only a few cases of NME in the absence of glucagonoma have been previously reported.<h4>Objective</h4>We sought to understand further the pathogenesis of NME by analyzing data from three patients.<h4>Methods</h4>Three patients were examined both clinically and histopathologically.<h4>Results</h4>Each patient had an extensive erythematous scaling eruption in intertriginous, perioral, and acral areas, and a markedly red, smooth tongue. Skin biopsy specimens showed confluent parakeratosis, epidermal pallor, papillary edema, and a lymphohistiocytic infiltrate. Two patients had alcoholic liver disease and one had liver dysfunction as a result of hemochromatosis. Serum albumin level was depressed, and liver enzyme values were increased in all three patients. Glucagonoma was undetectable in these patients.<h4>Conclusion</h4>In the absence of glucagonoma, hepatocellular dysfunction and hypoalbuminemia appear to be the most common factors associated with NME.

1. The antiinflammatory actions of glucocorticoid steroids are thought to occur through induction of the protein lipocortin-1 (LC-1; annexin-1). The purpose of the current study was to investigate whether astrocytic LC-1 content was increased in the presence of a synthetic glucocorticoid, dexamethasone. 2. Steroid-induced changes in cellular levels of LC-1 in astrocytes were determined by electrotransfer and immunoblotting techniques. Separate cell fractions were investigated to study the influence of dexamethasone on astroglial LC-1 content. The effect of culture state on LC-1 expression was also examined. 3. Intracellular LC-1 content was found to decrease after initiation of culture, with a substantial rise in both cell proliferation and LC-1 expression occurring after the replenishment of medium containing steroid-free serum. A further increase in intracellular LC-1 occurred upon incubation with dexamethasone. The glucocorticoid-induced change in intracellular LC-1 was a time-dependent event and coincided with an increase in membrane-associated LC-1. 4. The findings in this study indicate that astrocytic LC-1 content is influenced by cell culture conditions and, in the presence of glucocorticoid steroids, the cellular localization of LC-1 is altered. This may indicate that LC-1 has functions at more than one cellular locality.