Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12

Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [³H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells. The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Stimulating effects of foliar K-fertilizer applied at the appropriate stage of development of maize: A new way to increase yield and improve quality

A series of eight experiments was conducted using large pots to (1) find the most effective date, site, concentration of K-solution and K-salt for foliar K-fertilization of maize plants (Zea mays, L.) grown with sufficient K-supply in soil, (2) explain why maize responded to the K-treatment, and (3) examine the influence of various levels of N and P supplies on the effectiveness of K-fertilizer via the leaves. A single spraying on sweet maize and field maize on any day between 50% tasselling date to 10 days after tasselling shortened maturity date, increased grain yield, stover yield, grain-stover ratio, absorption of N, P, K, Ca and Mg, sweetness of young grain (of sweet maize), and crude protein content of grain. However spraying on the third day after 50% tasselling was most effective. The second application later than 7 days after the 50% tasselling date suppressed the effects of spraying on the most effective date. In application of many repetitive sprayings covering the most effective date, a spraying program with late spraying could reduce grain yield. KNO₃, 2.5% KNO₃-solution, and applications on all aerial parts were found to be the most effective. Increases in grain yield for spraying on all aerial parts, spraying on ear leaf only, spraying on all leaves above ear leaf and applying K to soil were 74%, 51%, 41% and 23%, respectively. The foliar K-fertilization affected maize by stimulating chlorophyll synthesis and not by increasing leaf area. A balance in N and K supplies was determined to be effective for the K-fertilization.     

Analysis of phylogenetic relations of durum,carthlicum and common wheats by means of comparison of alleles of gliadin-coding loci

Summary Polymorphism and inheritance of wheat storage protein, gliadin, of durum (macaroni) and carthlicum wheats have been studied. Analysis of gliadin in 78 cultivars and in F₂ seeds of intercultivar crosses of durum wheat revealed three different chromosome 1A-encoded blocks of components similar to those found in common wheat (GLD1A2, GLD1A18, GLD1A19). Most of the durum cultivars studied had these three blocks; GLD1A2 was also frequent in common wheat. In contrast, all chromosome 1B-encoded blocks of durum clearly differed in component composition from those found in common wheat. Therefore, durum could not be an ancestor or a derivate of recent bread wheat. Analysis of gliadin in the collection of carthlicum wheat (14 accessions) revealed several suspected chromosome 1A, 1B, and 6A-controlled blocks, some of which were similar to those in common wheat, while others were different. Therefore, carthlicum is likely to be an ancestor or a derivate of some forms of bread wheat. There were also chromosome 1A and 6A-, but not 1B-encoded blocks which were identical in durum and carthlicum wheats. The results confirm that all three wheats share the same genome A, but emphasize the heterogeneity of genotypes among donors of this genome. Discovery of identical blocks in tetraploids and hexaploids indicates polyphyletic [from different genotypes of donor (s)] origin of these wheats.     

Use of the cell-attached patch clamp technique to examine regulation of single cardiac K channels by cyclic GMP

Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10^-4 to 10^-3 M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged ( 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10^-5 M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells.     

Algesia and local responses induced by neurokinin A and substance P in human skin and temporal muscle

Neurokinin A (NKA), substance P (SP) and the two peptides combined (SP + NKA) were injected intracutaneously on the forearm and into the temporal muscle of healthy volunteers. Pain intensity, cutaneous wheal and flare responses and tenderness of the temporal muscle were quantitated. SP but not NKA induced cutaneous pain. This relates the algesic effect of SP to the specific N-terminal amino acid sequence of the peptide, not shared by NKA. NKA, however, potentiated the algesic effect of SP as SP + NKA induced a significantly prolonged cutaneous pain sensation. Both peptides induced wheals, but only SP induced flare. These results confirm previous studies relating wheal formation to the identical C-terminal amino acid sequence of the two peptides and flare reaction to the N-terminal part of SP. Injections into the temporal muscle did not cause pain or tenderness.