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Butyrate and its analogs have been shown to induce fetal hemoglobin in humans and primates and in erythroid cell cultures. To obtain insights concerning the cellular mechanisms of butyrate action, we analyzed the effects of butyrate on human globin gene expression in hybrids produced by fusing mouse erythroleukemia cells (MEL) with human fetal erythroid cells (HFE). These hybrids initially express human fetal hemoglobin but subsequently switch to adult globin expression after several weeks in culture. We found that alpha-aminobutyric acid, a butyrate analog which does not induce terminal maturation, strikingly delays the rate of the gamma- to beta-globin gene (gamma-to-beta) switch in the HFE x MEL hybrids. The effect of butyrate on globin expression is transient, with the result that the delay of globin gene switching requires the continuous presence of this compound in culture. Furthermore, butyrate fails to induce fetal hemoglobin expression in hybrids which have switched, suggesting that the effect of this compound on gamma-globin expression is due to inhibition of gamma gene silencing rather than to induction of gamma gene transcription. Since in other cellular systems, glucocorticoids antagonize the action of butyrate, the effect of dexamethasone on the gamma-to-beta switch in HFE x MEL hybrids was examined. Dexamethasone strikingly accelerated the gamma-to-beta switch, and its effect was irreversible. The effects of dexamethasone and butyrate on the gamma-to-beta switch of the HFE x MEL hybrids appear to be codominant. These results indicate that steroids can have a direct effect on globin gene switching in erythroid cells.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.

The purpose of this pilot study was to determine the effect of recombinant hirudin (r-hirudin) on coagulopathy and the relationship between concentrations of thrombin-antithrombin III (ATIII) complex (TAT) and thrombin-hirudin complex (THC) in patients with disseminated intravascular coagulation (DIC). Five patients with haematological malignancy associated with DIC were studied. r-Hirudin was administered by continuous intravenous infusion at a dose of 0.005 mg/kg/h for 4-9 days to each patient. Fibrin/fibrinogen degradation products (FDP), D-dimer, TAT and plasmin-alpha 2 antiplasmin complex (PAP) concentrations decreased after treatment with r-hirudin in four patients studied. However, in one patient, serum creatinine increased to 1.7 mg/dl and aPTT was prolonged to 74.4s. Statistical analysis disclosed significant positive correlations between plasma concentrations of hirudin and THC, and between concentrations of THC and TAT. The concentrations of THC were much higher than those of TAT. In conclusion, these findings indicate that r-hirudin more strongly inhibited thrombin than did ATIII without heparin, and that administration of r-hirudin to renal insufficiency required individual adjustment of dosage. The present findings also suggest that r-hirudin can be considered a new agent for the treatment of DIC.

Estrogen and progesterone receptor content was determined in 34 patients with synchronous and 23 patients with asynchronous bilateral breast cancer. Steroid receptor content was measured quantitatively by DCC method. It was shown that progesterone receptor content could not be predicted, as well as, that steroid receptor content of the second tumor significantly influenced the development of asynchronous bilateral breast cancer. The high discordance rate concerning histologic type between two tumors within synchronous as well as asynchronous biopsies was observed. The obtained results indicate that both synchronous and asynchronous bilateral breast tumors may be considered as biologically different tumors whose both steroid receptor levels should be determined whenever possible.

Genetic factors have been implicated in the etiology of affective disorders but due to the complex inheritance patterns of these disorders, identification of the responsible gene(s) has so far been unsuccessful. Decreased platelet serotonin (5-HT) transport and reduced binding of imipramine or paroxetine to brain and platelet 5-HT uptake sites/transporters in patients with depression and suicide victims define the 5-HT transporter (5-HTT) as a candidate gene. The primary structure of the 5-HTT was analyzed in 17 patients meeting DSM-III-R diagnostic criteria for major depressive or bipolar disorder and in 4 healthy controls using polymerase chain reaction (PCR-) amplification and sequencing of complementary deoxyribose nucleic acid (cDNA) synthesized from platelet 5-HTT messenger ribose nucleic acid (mRNA). Direct PCR sequencing of the protein coding region failed to reveal changes in the deduced amino acid sequence of the platelet/brain 5-HTT (40,000 base pairs sequence screened), although a conservative single-base substitution representing a silent polymorphism was found. The results provide preliminary evidence that alterations in the primary structure of 5-HTT are not generally involved in the pathogenesis of unipolar depression and manic-depressive illness.

The gene deleted in colorectal cancer (DCC) is a candidate tumor suppressor gene encoding a neural cell adhesion molecule like transmembrane protein. Over the past year, data supporting DCC inactivation in multiple tumor types have continued to accumulate. Functional studies suggest that DCC may participate in signaling pathways that regulate cell proliferation and/or differentiation, two cellular processes that often go awry during tumorigenesis.

The aim of this study was to evaluate the effects of dietary iron on hepatocarcinogenesis in an animal model mimicking noncirrhotic genetic hemochromatosis. Iron overload may lead to liver cirrhosis and an increased risk of developing primary hepatocellular carcinoma. It is unknown if iron is of pathogenic importance for the carcinogenic process, or whether the increased cancer risk results solely from the cirrhotic process. We investigated the initiating, promoting, and mitogenic properties of carbonyl iron in the Solt-Farber model of chemical hepatocarcinogenesis. A diet supplemented with 2.5% to 3.0% carbonyl iron was either added to, or replaced, the initiating and promoting events in the model. None of the animals developed hepatic fibrosis. Hepatic iron was increased 6- to 13-fold in iron-treated animals, and predominantly located in periportal hepatocytes. Iron as an initiator did not increase the number of glutathione-S-transferase-Yp-positive foci. Iron reduced the number of foci when added to low-dose diethylnitrosamine plus partial hepatectomy, which may be explained by a delayed hepatic regeneration in iron-loaded liver. As a promoter, iron did not selectively induce proliferation of initiated cells. Added to a complete promotive regimen, iron decreased the volume density of preneoplastic nodules, possibly because of a mitostimulatory effect of iron on normal hepatocytes surrounding the nodules. Iron increased the hepatocyte labeling index and counteracted the mitoinhibitory effect of 2-acetylaminofluorene on regenerating liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemochromatosis (HC), an inherited disorder of iron metabolism, shows a very strong founder effect in Australia, with the majority of patients being of Celtic (Scots/Irish) origin. Australian HC patients thus provide an ideal group in which to examine HC-gene-region haplotypes, to analyze the extent of linkage disequilibrium and genetic heterogeneity in HC. We have analyzed chromosomes from 26 multiply affected HC pedigrees, and we were able to assign HC status unambiguously to 107 chromosomes--64 as affected and 43 as unaffected. The haplotypes examined comprise the following highly polymorphic markers: the serological marker HLA-A and the microsatellites D6S248, D6S265, HLA-F, and D6S105. All show highly significant allelic association with HC and no evidence of separation from the disease locus by recombination. Analysis identified a predominant ancestral haplotype comprising alleles 5-1-3-2-8 (marker order: D6S248-D6S265-HLA-A-HLA-F-D6S105), present in 21 (33%) of 64 affected chromosomes, and exclusively associated with HC (haplotype relative risk 903). No other common haplotype was significantly associated with HC. Haplotype analysis in Australian HC patients thus provides strong evidence for (a) the introduction of HC into this population on an ancestral haplotype, (b) a common mutation associated with HC in Australian patients, and (c) a candidate HC-gene region extending between and including D6S248 and D6S105.

Huntington's disease (HD) is an autosomal dominant disorder characterized by involuntary movements, dementia, and progressive, global, but regionally accentuated, brain atrophy. The disease affects the striatum most severely. An expansion of a trinucleotide repeat on chromosome 4p16.3 within the coding region of a gene termed IT15 has been identified as the mutation causing HD. The normal function of IT15 and the mechanisms by which the presence of the mutation causes HD are unknown. Although IT15 expression has been detected in the brain, as well as in other organ tissues, by Northern blot and in situ hybridization, it is not known whether a preferential regional or cellular expression of IT15 exists within the central nervous system of normal, affected, and presymptomatic individuals. Using quantitative in situ hybridization methods, we examined extensively the regional and cellular expression of IT15. In controls, IT15 expression was observed in all brain regions examined with the highest levels seen in cerebellum, hippocampus, cerebral cortex, substantia nigra pars compacta, and pontine nuclei. Expression in the striatum was intermediate and expression in the globus pallidus was low. IT15 was expressed predominantly in neurons; a low but significant level of expression was seen in glial cells. Analysis of grain counts per square micrometer in neurons showed that the regional differences in the level of mRNA expression were related to density and size of neurons in a given region and not primarily to differences in levels of mRNA expression in individual cells after correction for cell size. Neurons susceptible to degeneration in HD did not selectively express high levels of IT15 mRNA. In HD brains (grades 2-4), the distribution and levels of IT15 mRNA were comparable with controls in all areas except in neostriatum where the intensity of labeling was significantly reduced. Presymptomatic HD brains had a striatal expression similar to controls and surviving striatal neurons in more advanced HD had an expression of IT15 within normal limits. It is apparent from these results that the presence of expanded trinucleotide repeats in HD does not result in the absence of IT15 mRNA expression or in altered patterns or levels of expression. The lack of correlation between the levels of IT15 mRNA expression and susceptibility to degeneration in HD strongly suggests that the mutant gene acts in concert with other factors to cause the distinctive pattern of neurodegeneration in HD.

High-affinity 3,3',5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3',5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3'5'-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).

Colorectal carcinomas demonstrate extensive molecular genetic alterations throughout the genome. The genetic changes in cancer of the colon and rectum are among the best understood of any common human cancer. The genetic abnormalities include both dominant-acting oncogenes (Ki-ras, c-src) and tumor-suppressor genes which undergo inactivation or loss (APC, DCC, p53). The evolution of the cancer is a complicated and multistep process. At the various steps of this phenomenon we can recognize specific molecular genetic alterations. These particular genetic changes may be useful as improved markers to predict those patients who have an aggressive cancer of the colon, with occult metastases or increased metastatic capability and this selection of patients could lead to improved surgical and medical management.

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema. Equivalent substitutions have been described in alpha 1-antitrypsin and antithrombin III. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional. Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor. A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val451-->Met and Pro476-->Ser mutant proteins, and to a lesser extent Phe455-->Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val451-->Met and Pro476-->Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val451-->Met or Pro476-->Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxylterminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented.

The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with the prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)ethylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Soc. 110, 7560-7561], was characterized. CyHEA undergoes facile DBM-catalysed allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic rearrangement, and with stereochemistry consistent with that of DBM-catalysed benzylic hydroxylation and sulphoxidation. The kcat. of 90 s-1 for CyHEA oxygenation is about 75% of the kcat. for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA also results in mechanism-based inactivation of DBM, with the inactivation reaction yielding kinact. = 0.3 min-1 at pH 5.0 and 37 degrees C, and a partition ratio of 16,000. Although both CyHEA turnover and inactivation exhibit normal kinetics, CyHEA processing also results in gradual depletion of copper from DBM; however, mechanism-based irreversible DBM inactivation occurs independent of this copper depletion when sufficient copper is present in the assay solution. A likely mechanism for turnover-dependent DBM inactivation by CyHEA involves initial abstraction of an allylic hydrogen to form a resonance-stabilized allylic radical, which can then either partition to product or undergo attack by an active-site residue. Acyclic, non-conjugated olefinic analogues exhibit diminished substrate activity toward DBM. Thus, kcat. for oxygenation of cis-2-hexenylamine, which also produces only allylic alcohol product, is only 14% of that for CyHEA. Similarly, kinact./KI for turnover-dependent inactivation by the acyclic olefin 2-aminomethyl-1-pentene is more than an order of magnitude smaller than that for benzylic olefins. Our results establish that DBM catalyses allylic oxygenation of a number of non-conjugated olefinic substrate analogues with neither epoxidation nor allylic rearrangement occurring. The absence of epoxide products from non-conjugated olefinic substrates implies an inability of the activated copper-oxygen species of DBM to effect radical cation formation from a non-conjugated olefinic moiety. The striking contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substrates, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.

We report the isolation and sequencing of MTJ1, a 1792-bp cDNA from an M27 murine lung carcinoma cell line. The largest ORF within MTJ1 encodes a 63,869-Da protein, containing a 73-amino-acid (aa) sequence (the J domain) that is conserved in proteins of the DnaJ family of chaperonins. The J domain of MTJ1 is bracketed by potential transmembrane domains in a similar configuration to the J domain of the yeast DnaJ-like protein, SEC63. Polyclonal antibodies raised against deduced aa sequences within MTJ1 recognized antigens of 62, 42 and 41 kDa that were enriched in the nuclear and heavy microsome subcellular fractions of murine tumor cells. Northern analysis detected a major 3.2-kb transcript that was present in all murine organs examined, but was relatively underexpressed in brain and heart.

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Asp132Asn/Ala), an exterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity. Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Asp132 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-III loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steady-state mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.

Hemolytic uremic syndrome (HUS) is an uncommon complication of chemotherapy that contributes to the morbidity of oncology and bone marrow transplant patients. The pathogenesis is not well understood and no established clinical animal model exists. We studied four rhesus monkeys (RM) that developed fatal HUS following high-dose chemotherapy. Microangiopathic hemolytic anemia (pre-Hct 40% and day 5-8 Hct 31% (P < .05), increased BUN (168 mg/dl), creatinine (8.2 mg/dl), and lactate dehydrogenase (1458 IU/L) (mean day 5-8 measurements) were observed. Platelets counts decreased to 39 +/- 15 x 10(9)/l from a mean of 397 +/- 31 x 10(9)/L (P < .0001). vWF, ATIII, thrombin:anti-thrombin complex (T:AT) and prothrombin fragment F1.2 levels were not different from a control group (N = 2). The data presented describe chemotherapy-induced HUS with typical clinical and laboratory features which may provide an animal model for the study of this important syndrome.