Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes.

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor

Summary 80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR ⁺ strain but not by corresponding preparations from an argR ^- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on -galactosidase synthesis.     

Utilization of cellulosic materials through enzymatic hydrolysis. II. Preliminary assessment of an integrated processing scheme

An integrated processing scheme is described for the conversion of a cellulosic waste (newsprint) to sugars by enzymatic hydrolysis and then to ethanol and yeast by fermentation. The unconverted solids are burned to produce process energy requirements and surplus electrical power. Preliminary designs and cost studies are developed to provide a rough perspective on the potential economic feasibility of this method of cellulose utilization.     

Generation of ATP by chloroplasts through solvent perturbation.

A two-stage method was discovered for generating ATP by chloroplasts in the dark at constant pH through solvent perturbation. With cold acetone as the perturbing solvent, the yield of ATP was found to increase with the volume percent of acetone in the first stage medium. The results are difficult to explain in term of the proton gradient model, but is consistent with the conventional model of prior water formation and subsequent ATP generation.     

Neutral lipids of Aedes aegypti and Aedes albopictus cells cultured in vitro.

Aedes aegypti feeding on chickens infected with Plasmodium gallinaceum take less blood and lay fewer eggs than those feeding on uninfected hosts. Both activities show an inverse correlation with the degree of parasitemia. Mosquitoes feeding on infected chickens ingest blood in amounts directly proportional to the length of time spent on the hosts, whereas there is no relationship between host contact and blood meal size for mosquitoes feeding on uninfected hosts. Feeding and probing choice experiments demonstrate that infected chickens are less attractive to Aedes aegypti than uninfected chickens.     

Interactions of Concomitant Species of Nematodes and Fusarium oxysporum f. sp. vasinfectum on Cotton

Meloidogyne incognita, Hoplolaintus galeatus, and North Carolina and Georgia populations of Belonolaimus longicaudatus were introduced singly and in various combinations with Fusarium oxysporum f. sp. vasinfectum on wilt-susceptible ''Rowden'' cotton. Of all the nematodes, the combination of the N. C. population of B. longicaudatus with Fusarium promoted greatest wilt development. H. galeatus had no effect on wilt. With Fusarium plus M. incognito or B. longicaudatus, high nematode levels promoted greater wilt than low levels. The combination of either population of B. longicaudatus with M. incognita and Fusarium induced greater wilt development than comparable inoculum densities of either nematode alone or where H. galeatus was substituted for either of these nematodes. Nematode reproduction was inversely related to wilt development. Without Fusarium, however, the high inoculum level resulted in greater reproduction of all nematode species on cotton. Combining M. incognita with B. longicaudatus or H. galeatus gave mutually depressive effects on final nematode populations. The interactions of H. gateatus with B. longicaudatus varied with two populations of the latter.     

Optical rotatory dispersion and circular dichroism of silver(I):Polyribonucleotide complexes

Optical rotatory dispersion (ORD) and circular dichroism (CD) spectra of single- and multistranded polyribonucleotides undergo extensive changes on binding of the silver ion. These changes are consistent with the proposition that Ag(I) binds to the heterocyclic bases and not to the phosphate groups of polynucleotides. ORD and CD of silver complexes of poly(A)·poly(U) and double-helical rice dwarf viral RNA display negative Cotton effects when there is more than one Ag(I) per two nucleotide residues in solution. These observations suggest a significant distortion of the double-helical conformation as a result of Ag(I) binding. Silver(I) binding sites of pyrimidine polynucleotides are apparently saturated when there is one Ag(I) per two nucleotide residues and those of purine polynucleotides at one Ag(I) per nucleotide in solution. These data are consistent with the supposition that some Ag(I) binding sites exist on the pyrimidine ring and additional sites on the imidazole ring of polynucleotides. The sedimentation coefficient of poly(A) increases by severalfold when one Ag(I) is present per nucleotide residue. Silver(I) may introduce intra- and interstrand cross-links (through bidentate chelates) in single-stranded polynucleotides, resulting in structures with high sedimentation coefficients. Among the polynucleotides studied, poly(U) was an exception. Silver(I) did not affect the optical properties (absorbance, ORD, and CD) of poly(U) at neutral pH.     

Studies of trachoma in families on Taiwan.

This study was undertaken to clarify the natural history and pathogenesis of trachoma. A group of families who live in a formerly trachoma hyperendemic area of Southern Taiwan were placed under continuous surveillance. The development in recent years of the micro immunofluorescence test for trachoma antibody, along with improved cell culture isolation methods, have allowed this surveillance to include repeated effective laboratory studies in addition to clinical observations. After four years' study of one group of families and three years of another, a number of interesting findings have been obtained. Evidence is presented supporting our hypothesis that trachoma is a disease of immumopathology and results from repeated reinfections with the trachoma organisms. The clinical findings of papillae, especially those of an acute nature, has been the clinical finding most closely associated with the isolation of the organism and the demonstration of antibody. Evidence is presented that transmission of the organism is usually within the family group. Although only trachoma immunotypes B and C previously had been associated with trachoma infection on Taiwan, data is presented from one family in which type D infections occurred. While a series of new and reinfections with trachoma organisms were demonstrated in some of the families under observation, the majority of the families not only showed no new infections but showed spontaneous healing or disappearance of clinical and laboratory evidence of trachoma infection. This tendency of active trachoma infection to disappear from a family in the absence of transmission of the organism parallels the rapid fall and prevalence of active trachoma on Taiwan during the past decade.     

Purification of bull sperm hyaluronidase by concanavalin-A affinity chromatography.

A new method for obtaining highly purified hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1.25) in high yield is described. Bull seminal plasma was fractionated with (NH4)2 SO4 and the 30 to 65% saturation fractions were applied to a DEAE-cellulose column. The first protein peak contained hyaluronidase, beta-N-acetylglucosaminidase and beta-glucuronidase. The latter two enzymes were separated by gel filtration on Sephadex G-200. The hyaluronidase was further purified by a Concanavalin-A Sepharose 4B affinity column. By gradient elution with alpha-methyl-D-glucoside a fraction which had a specific activity of 1998 units/mg protein (57 942 National Formulary Standard units/mg protein) was obtained. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 4.3. The purified hyaluronidase did not show any beta-glucuronidase or beta-N-acetylglucosaminidase activities. The percent yield of purified hyaluronidase calculated on the basis of total activity was ten times higher than by any pervious method [Yang, C.H. and Srivastava, P.N. (1975) J. Biol. Chem. 250, 79-83].     

Purification and properties of hyaluronidase from bull sperm.

Hyaluronidase from bull sperm was fractionated by ammonium sulfate and further purified by DEAE-cellulose and Sephadex chromatography. The highly purified hyaluronidase preparation showed 2,370 units per mg of protein (68,730 N.F. units per mg of protein), i.e. 182-fold purification. Disc gel electrophoresis showed one major component. The molecular weight of bull sperm hyaluronidase was 62,000 by sodium dodecyl sulfate gel electrophoresis. Hyaluronidase from bull sperm has optimum activity at pH 3.8 and an absolute requirement for cations. Kplus and Naplus have a greater effect than Ca2plus, Mg2plus, and Mn2plus, whereas Co2plus, Cu2plus, and Zn2plus do not affect the enzyme activity. Purified preparations are less stable than crude extracts stored frozen at minus 15 degrees. Km of hyaluronidase with hyaluronic acid as substrate is 3.7 mg per ml and Vmax is 2.4 mumol per min by Hofstee plot.