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K H Falchuk

Publications and source records attributed to K H Falchuk.

At least 37 records · Page 2Linked to original sources
PubMed1986

Inhibition of Euglena gracilis and wheat germ zinc RNA polymerases II by 1,10-phenanthroline acting as a chelating agent.

Copper complexes of 1,10-phenanthroline (OP-Cu) hydrolyze DNA [D'Aurora, V., Stern, A. M., & Sigman, D. S. (1978) Biochem. Biophys. Res. Commun. 80, 1025-1032; Marshall Pope, L., Reich, K. A., Graham, D. R., & Sigman, D. S. (1982) J. Biol. Chem. 257, 12121-12128]. This reaction has been studied to determine whether the 1,10-phenanthroline (OP) inhibition of the activity of RNA and DNA polymerases is the result of template hydrolysis or the chelation of a metal associated with and essential to the function of these enzymes. Addition of 4',6-diamino-2-phenylindole dihydrochloride (DAPI) to DNA generates a fluorescence signal with a linear increase of the intensity over a broad range of DNA concentrations from 0 to 100 micrograms/mL. The progress of hydrolysis of DNA by DNase I or OP (2 mM) is monitored by the time-dependent decrease in DAPI-induced fluorescence. In the presence of OP, the rate of hydrolysis increases as the Cu2+ concentration in the reaction mixture rises from 10(-8) to 10(-5) M. The rate differs for each nucleic acid template used; hydrolysis of poly(dA-dT) greater than denatured DNA greater than double-stranded DNA. However, millimolar amounts of OP do not hydrolyze the template even in the presence of Cu2+ (10(-6) M) when DNA is complexed with either Escherichia coli DNA polymerase I or Euglena gracilis or wheat germ RNA polymerase II. Under the same conditions, OP inhibits the activity of both varieties of RNA polymerase II with pKi's of 3.4 and 3.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Chelating Agents↗
PubMed1986

Testosterone allosterically regulates ethanol oxidation by homo- and heterodimeric gamma-subunit-containing isozymes of human alcohol dehydrogenase.

Testosterone and its physiologically active metabolite 5 alpha-dihydrotestosterone are selective, allosteric inhibitors of the gamma subunit-containing isozymes of class I human alcohol dehydrogenase (ADH) with apparent Ki values for testosterone at pH 7.4 between 3.5 and 16 X 10(-6) M. Testosterone inhibition is noncompetitive with respect to ethanol, NAD+, 1,10-phenanthroline, and 4-methylpyrazole, identifying a regulatory site distinct from the catalytic site. Testosterone does not inhibit the class I isozymes composed only of alpha and/or beta subunits and only weakly inhibits the class II and III isozymes. Importantly, none of these human ADH isozymes oxidize or reduce the steroids with the delta 4 double bond or 5 alpha configuration. The allosteric effect of testosterone, restricted to the gamma subunits of human ADH, suggests unique metabolic specificities and pathways for these isozymes, apart from all others. This inhibition may ultimately be critical to an identification of their function(s). Analogous considerations of other metabolic effectors might further lead to similar insights regarding the alpha and beta subunit-containing isozymes as well as the class II and III ADH.

Alcohol Dehydrogenase↗
PubMed1985

Zinc deficiency and the Euglena gracilis chromatin: formation of an alpha-amanitin-resistant RNA polymerase II.

Both the single DNA-dependent RNA polymerase found in zinc-deficient (-Zn) Euglena gracilis and the RNA polymerase III from zinc-sufficient (+Zn) cells have been isolated by methods previously used to purify polymerases I and II [Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1976) Biochemistry 15, 4468; Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1977) Biochem. Biophys. Res. Commun. 74, 1206]. Like class II polymerases, the enzyme from -Zn organisms elutes from DNA-cellulose and phosphocellulose with 0.6 M NaCl and 0.35 M NH4Cl, respectively. It is inhibited by 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, alpha,alpha'-bipyridyl, dipicolinic acid, and 1,10-phenanthroline (OP); 4,7-phenanthroline, the nonchelating analogue, does not inhibit. The pKI(OP) of this enzyme is identical with that of polymerase II but distinct from those of polymerases I and III. Elemental analysis confirms that zinc is the functional metal while copper, manganese, iron, and magnesium are absent. However, the -Zn enzyme is at least 4 orders of magnitude more resistant to alpha-amanitin (alpha-A) than the class II polymerase. Further, its response to alpha-A is unlike that of either polymerase I or polymerase III. Thus, -Zn cells contain a single, alpha-amanitin-resistant (alpha-Ar) RNA polymerase, whose behavior otherwise resembles that of the alpha-amanitin-sensitive polymerase II.

Amanitins↗
PubMed1985

Microparticulate-induced phlebitis. Its prevention by in-line filtration.

We carried out a double-blind prospective study of the effect of a filter on the incidence of phlebitis associated with intravenous infusion in 541 patients. A total of 277 patients received infusions through intravenous sets with 0.22-micron IVEX-HP filters, and 264 received infusions without filters. Each infusion was evaluated daily for a maximum of three days. The incidence of phlebitis on Days 1, 2, and 3 of the study was 14.3, 31.1, and 27 per cent for patients receiving infusions without filters and 6.8, 9.7, and 11.3 per cent for those receiving infusions through the filters (P less than 0.001). Thus, the incidence was reduced by approximately two thirds in the patients who received infusions through the IVEX-HP filters. We conclude that infusion-related phlebitis is a pervasive problem in hospitalized patients, and that it is usually caused by microparticulate components that are present in the infusion fluids and can be removed by in-line filtration.

Adolescent↗
PubMed1984

Histone formation, gene expression, and zinc deficiency in Euglena gracilis.

Histones, and other basic proteins, have been isolated from zinc-sufficient (+Zn) Euglena gracilis by standard chromatographic methods. These cells contain 2.46 micrograms of histones and 1.96 micrograms of DNA per 10(6) organisms. Each of the histones, H1, H3, H2A, H2B, and H4, is present in both log- and stationary-phase +Zn cells and has been characterized according to its electrophoretic mobility and molecular weight. H1 has been further identified on the basis of its amino acid composition and its cross-reactivity with calf thymus histone H1 antibodies. Similarly, H3 has been recognized as well by its specific reaction with an H3 antibody. In contrast, log-phase zinc-deficient (-Zn) cells contain H1 and H3 while H2A, H2B, and H4 are absent. All of the histones vanish in stationary-phase-Zn organisms. The DNA content increases as the -Zn cells progress from log to stationary phase, reaching a value of 4.40 micrograms/10(6) cells, double that of comparable stationary-phase +Zn organisms. A 2000-3000-dalton polypeptide whose electrophoretic behavior differs from that of the known histones constitutes over 90% of the total basic proteins of -Zn cells. On addition of zinc to stationary -Zn cells, cell division resumes, and all the histones and other basic proteins reappear. Together with previous results, the data demonstrate that zinc significantly affects the metabolism of all major chromatin components, i.e., the RNA polymerases, DNA, and histones of E. gracilis [Vallee, B.L., & Falchuk, K.H. (1981) Philos. Trans. R. Soc. London, Ser. B 294, 185-197]. The implications of these effects of zinc on chromatin structure and function are discussed.

Animals↗
PubMed1983

Composition and structure of zinc-deficient Euglena gracilis chromatin.

The histone content of zinc-deficient (-Zn) Euglena gracilis decreases while, concomitantly, DNA content increases and the transcription rate is reduced markedly [Mazus, B., Falchuk, K. H., & Vallee, B. L. (1983) Biochemistry (in press); Falchuk, K. H., Fawcett, D. W., & Vallee, B. L. (1975) J. Cell Sci. 17, 57-78]. The effects on major constituents of the genome have been examined by studying the rate and extent of hydrolysis of +Zn and -Zn chromatin by micrococcal nuclease, DNase I, or DNase II. The size of hydrolyzed DNA fragments suggests similarity of the +Zn E. gracilis chromatin organization to that of other eukaryotes. The major protein constituent of -Zn chromatin is a polypeptide of less than 3000 daltons whose electrophoretic mobility differs from that of any known histone components of chromatin, the latter described elsewhere (K. H. Falchuk et al., unpublished results). This protein profoundly affects the structure of -Zn chromatin, which is about 10-30-fold more resistant to micrococcal nuclease hydrolysis than +Zn chromatin. Moreover, the resultant DNA fragments [2000 base pairs (bp)], are much larger than those of +Zn cells. Under conditions which hydrolyze +Zn chromatin into DNA fragments smaller than 50 bp, only 50% of -Zn chromatin is digested into fragments less than 2000 bp, i.e., in the range of those expected for oligonucleosomes. Removal of the low molecular weight protein from -Zn chromatin reverses its enhanced resistance to nucleolysis and results in extensive hydrolysis. Conversely, addition of the low molecular weight protein to +Zn chromatin increases the resistance of this complex to digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1983

Spontaneous obliteration of pancreaticoduodenal artery aneurysm after retroperitoneal hemorrhage.

We present a rare case of aneurysm of the pancreaticoduodenal artery that bled into the region of the head of the pancreas. Computed tomography (CT) demonstrated the presence and extent of the resultant mass (hematoma) but the angiographic examination allowed a specific diagnosis via visualization of the aneurysm. Unlike previously reported cases, the patient was treated conservatively. Follow-up arteriogram and CT showed spontaneous closure of the pancreaticoduodenal artery and aneurysm and complete resolution of the mass in the pancreatic head. These findings suggest that in some cases a conservative approach to a bleeding pancreaticoduodenal artery aneurysm may be indicated.

Aneurysm↗
PubMed1981

Zinc and gene expression.

During the last two decades, zinc has been shown to be a functionally essential component of more than 120 enzymes and concurrently has been recognized as indispensable to normal cellular growth, development and differentiation of all species. However, the manner in which it exerts its effects are still unknown. Studies of the phytid E. gracilis have demonstrated that zinc is critical for transcription and translation, affecting the metabolism of RNA polymerases, mRNA and proteins, among which are histones and arginine-containing peptides. These and other data lead to the suggestion that this element has a fundamental role in gene activation and/or repression, accounting for its pervasive effects on cell metabolism.

Amino Acyl-tRNA Synthetases↗
PubMed1978

RNA metabolism, manganese, and RNA polymerases of zinc-sufficient and zinc-deficient Euglena gracilis.

The three major RNA classes from zinc-sufficient [(+Zn)] and zinc-deficient [(=Zn)] Euglena gracilis have been separated by affinity chromatography on oligo(dT)- and N-[N'-[m-(dihydroxyboryl)phenyl]succinamoyl]aminoethyl (DBAE)-celluloses. The total RNA content and the ribosomal and transfer RNA fractions are the same in (+Zn) and (=Zn) cells. IN (-Zn) cells, the messenger RNA fraction increases, and its altered base composition reveals additional bases and a 2-fold increase in the (G+C)/(A+U) ratio. Since the intracellular content of manganese increases in (-Zn) cells, we have examined its role in determining these changes in RNA composition. An increase in the Mn2+ content from 1 to 10 mM in assays with RNA polymerases I and II from (+Zn) cells and those with the single RNA polymerase from (-Zn) cells decreases the ratio of UMP to CMP incorporated from 1.7 to 1.0, 2.1 to 0.8 and 3.5 to 0.4, respectively. Thus, Mn2+ concentration can significantly alter the products of the enzymatic action of RNA polymerases from both (+Zn) and (-Zn) E. gracilis cells.

Base Sequence↗
PubMed1977

Effect of acute disease and ACTH on serum zinc proteins.

The effect of acute disease and ACTH infusion on serum zinc proteins was studied in serums from 156 healthy and diseased subjects. The mean (+/-2 S.D.) zinc content of 20 normal serums was 96 +/- 20 microng per 100 ml. In 87 serums from acutely ill patients the zinc ranged from 92 to 40 microng per 100 ml. The mean values for nearly all categories of disease studied were lower than normal (P is less than 0.001). Chromatography of normal serum on Sephadex G-100 separates two protein fractions, I and II, containing 37.8+/-8.8 and 76+/-10 microng of zinc per 100 ml, respectively. In serum from diseased patients the zinc in fraction I is unaltered whereas that in fraction II decreases to 29.8+/-7.5 microng per 100 ml (P is less than 0.001). ACTH administration reduces secrum zinc from 10 to 60 microng per 100 ml, the decrements being due to changes in the zinc content of fraction II. Thus, ACTH may have an important role in the reduction of zinc content associated with pathologic states.

Acute Disease↗
PubMed1977

1,10-Phenanthroline inhibition of lymphoblast cell cycle.

The effects of the metal-chelating agent, 1,10-phenanthroline (OP), on the cell cycle progression of CCRF-CEM lymphoblasts has been studied by flow microfluorometry. Lymphoblasts were incubated with 2,3-dihydro-1H-imidazo[1,2-b]pyrazole in order to block them in G1-early S. The block was then reversed by incubating the cells in fresh media. Within 2 to 4 hr, approximately 90% of the cells were in S phase and, by 6 hr, approximately 80% were in late S. Aliquots of these latter cells then were incubated with podophyllotoxin to block them in G2-M. These cells divided within 4 hr of reversal of the podophyllotoxin block. Lymphoblast populations in G1-early S, mid-S, or G2-M were then incubated with 4 micronM OP. OP blocked entry of G1 cells into S as well as progression through S but had no effect on progression from G2-M to G1. The OP effects were reversed by addition of Zn2+, Cu2+, or Fe2+ or by dilution of the chelating agent in the growth media. Incubation of CCRF-CEM lymphoblasts with 1,7-phenanthroline, a nonchelating analog of OP, did not affect the cell cycle. The data indicate that OP reversibly blocks progression of cells from G1 to S and through S by chelation of metals involved in processes essential for those cell cycle events.

Cell Division↗
PubMed1976

Euglena gracilis DNA dependent RNA polymerase II: a zinc metalloenzyme.

Zinc is essential for cellular proliferation. Zinc deficiency of Euglena gracilis results in arrest of cell division and deranges nucleic acid and protein metabolism pointing to a decisive role of zinc in transcription and translation. We have, therefore, investigated the role of zinc in the function of the DNA-dependent RNA polymerases of this organism. Two RNA polymerases from zinc sufficient organisms were purified first by affinity chromatography on a DNA cellulose column and subsequently separated on diethylaminoethyl (DEAE)-Sephadex A-25. The two fractions were characterized as polymerase I and II by their elution pattern from DEAE-Sephadex and sensitivity to alpha-amanitin. RNA polymerase II has a provisional molecular weight of 700 000 and contains an average of 2.2 g=atoms of zinc per mol of enzyme, but not Mn, Cu, or Fe, as measured by microwave emission spectroscopy. Chelating agents, such as 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, and lomofungin, inhibit activity. In contrast, the nonchelating analogues, 1,7-and 4,7-phenanthroline, do not affect activity. Inhibition by 1,10-phenanthroline is instantaneous and fully reversible by dilution. 1,10-Phenanthroline also inhibits RNA polymerase I, suggesting a role of zinc in its function. The demonstration that RNA polymerase II is a zinc enzyme indicates the involvement of zinc in eukaryotic RNA synthesis and serves as a further basis for the definition of the role of this element in eukaryotic cell growth, division, and differentiation.

Amanitins↗
PubMed1975

DNA distribution in the cell cycle of Euglena gracilis. Cytofluorometry of zinc deficient cells.

Analysis of DNA content of intact cells by laser induced cytofluorometry permits dynamic studies of the cell cycle in the synchronously dividing eukaryote Euglena gracilis. In this manner, the effects of zinc deficiency and cadmium toxicity on in situ DNA synthesis and cell division of this organism have been studied. In the G-1 hase of the cell cycle, prior to initiation of DNA synthesis, the DNA content of synchronously growing E. gracilis is characteristic of cells with an ureplicated genome. In S phase there is a progressive increase in DNA content which leads to genome duplication as the cells enter G2. In the subsequent mitosis all cells divide. Cytofluorometric definition of DNA content serves as a standard of reference to study variables which alter or block each stage of the cell cycle. Growth in zinc dificient media inhibits cell division. The DNA content of such zinc dificnet cells is characteristic of a population of cells blocked in S/G2 with a small fraction in G1. Moreover, cells synchronized in G1 and placed in zinc deficient media do not progress into S phase. Cadmium also inhibits cell division, and the DNA content of these blocked cells is three to four times greater than that of cells in G1. Zinc is essential for the biochemical events of the premitotic state which include initiation of DNA synthesis, DNA synthesis, and progression from G-2 to mitosis. Cadminum-induced derangements of the cell cycle include alterations in regulation cellular DNA content.

Animals↗
PubMed1975

Role of zinc in cell division of Euglena gracilis.

Euglena gracilis is a suitable model system to investigate the role of zinc in the process of cell division. In zinc-deficient organisms there is a characteristic arrest of cellular proliferation, the DNA content of the cells doubles, whereas RNA and protein contents decrease. The present investigations include the growth characteristics, changes in cellular morphology at various stages in the growth cycle, quantitation of zinc uptake and incorporation of tritium-labelled precursors into RNA by organisms grown in zinc sufficient (Zn+), (Zn2+ content 1 times 10-minus 5 M) or zinc-deficient (Zn minus), (Zn2+ content 1 times 10-7 M) medium. Cell division ceases on depletion of zinc from the medium. There are 20-fold less cells in (Zn minus) medium than in control cultures. The size of (Zn+) cells decreases during log phase due to a reduction in the paramylon content of the cytoplasm. The size of (Zn minus) cells, however, increases, due to an accumulation of paramylon. This results in a 13-fold increment in dry weight compared to control. Other cytoplasmic organelles, including Golgi bodies, mitochondria, etc. are normal. Nuclear morphology also is unchanged. There is a reduction in the rate of incorporation of labelled precursors into RNA by (Zn minus) cells. The DNA content of (Zn minus) E. gracilis, the absence of morphologic evidence to indicate that cell division has followed the doubling of the DNA, and the arrest in proliferation suggests that a critical zinc-dependent step in the cell cycle, localized to G2, is blocked in zinc deficiency.

Cell Division↗