Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

Analysis of the UL36 open reading frame encoding the large tegument protein (ICP1/2) of herpes simplex virus type 1.

Using peptide antisera specific for regions within the N terminus and C terminus of the predicted UL36 gene product, immunoblotting experiments were performed to demonstrate definitively that ICP1/2 is encoded by the UL36 gene. These data also suggest that both the cell- and the virion-associated forms of ICP1/2 are colinear with the complete predicted amino acid sequence of the UL36 gene. Computer-assisted analyses of the predicted amino acid sequence of the UL36 gene revealed the presence of two putative leucine zipper-type motifs and a potential ATP-binding domain. The possible functions of these consensus domains will also be discussed.     

Reactivity of alpha 1-antitrypsin mutants against proteolytic enzymes of the kallikrein-kinin, complement, and fibrinolytic systems

Increased extracellular proteolysis because of unregulated activation of blood coagulation, complement, and fibrinolysis is observed in thrombosis, shock, and inflammation. In the present study, we have examined whether the plasma kallikrein-kinin system, the classical pathway of complement, and the fibrinolytic system could be inhibited by alpha 1-antitrypsin reactive site mutants. Wild-type alpha 1-antitrypsin contains a Met residue at P1 (position 358), the central position of the reactive center. It did not inhibit plasma kallikrein, beta-factor XIIa, plasmin, tissue-type plasminogen activator (t-PA), or urokinase. In contrast, these serine proteases were inhibited by alpha 1-antitrypsin Arg358. For the inhibition of C1s, a double mutant having Arg358 and a Pro----Ala mutation at P2 (position 357) was required. This double modification was made because C1-inhibitor, the natural inhibitor of C1s, has Arg and Ala residues at positions P1 and P2. Plasminogen activator inhibitor 1, the natural inhibitor of t-PA, also has Arg and Ala residues at positions P1 and P2. In a purified system, alpha 1-antitrypsin Ala357-Arg358 was 150-fold less efficient against C1s than C1-inhibitor and 27,000-fold less efficient against t-PA than plasminogen activator inhibitor-1. In plasma, 2.3 microM alpha 1-antitrypsin Ala357-Arg358 reduced by 65% the formation of a complex between kallikrein and C1-inhibitor following activation of the intrinsic pathway of blood coagulation by kaolin. Furthermore, after supplementation by 2.0 microM alpha 1-antitrypsin Ala357-Arg358, zymographic analysis showed that the majority of the free t-PA of normal plasma formed a bimolecular complex with the double mutant. In contrast, 3.4 microM alpha 1-antitrypsin Ala357-Arg358 did not prevent the activation of the classical pathway of complement observed when normal serum is supplemented with anti-C1-inhibitor F(ab')2 fragment. These results demonstrate that alpha 1-antitrypsin Ala357-Arg358 has therapeutic potential for disorders with unregulated activation of the intrinsic pathway of blood coagulation and the fibrinolytic system; however, the double mutant is not an efficient inhibitor for the classical pathway of complement.     

Epidermis generated in vitro: practical considerations and applications

The technology for culture of epidermis is one of the most advanced to date for generation of a tissue in vitro. Cultured epidermis is already used for a number of applications ranging from use as a permanent skin replacement to use as an organotypic culture model for toxicity testing and basic research. While simple epidermal sheets have been grafted successfully, more advanced models for skin replacement consisting of both dermal and epidermal components are in development and being tested in a number of laboratories. One of the most advanced in vitro models is the living skin equivalent, an organotypic model consisting of a collagen lattice contracted and nourished by dermal fibroblasts overlaid with a fully formed epidermis.     

Metabolism of [14C]4-chlorobiphenyl by hepatic microsomes isolated from razorbills, pigeons and rats.

1. Analysis of individual PCB-isomers and congeners in extracts of adipose tissue from N = 12 razorbills suggested that 4-chlorobiphenyl was subjected to metabolism. 2. In vitro metabolism studies using [14C]-4-chlorobiphenyl as substrate showed that razorbills metabolise this substrate to [14C]4-chloro-4'-hydroxybiphenyl at an average rate of 20 pmol/mg microsomal protein/min. For comparison, the metabolism of [14C]-4-chlorobiphenyl by pigeons and rats was also studied, and average rates in the formation of [14C]-4-chloro-4'-hydroxybiphenyl of 12 pmol/mg microsomal protein min and 342 pmol/mg microsomal protein min were estimated. 3. A comparison of the hepatic drug metabolising enzyme system of razorbills and pigeons showed similar concentrations of cytochrome P-450, cytochrome b5 and comparable catalytic activities of cytochrome P-450-dependent monooxygenase, when assessed for HHDN epoxidase, PROD, EROD and the Phase II enzymes glutathiones-S-transferase, but were significantly lower, when compared with rats. The results obtained suggest fundamental differences in the catalytic activities of cytochrome P-450-dependent monooxygenase between avian and mammalian species. 4. The present study, however, provides evidence that fish-eating seabirds have the ability to metabolically dispose of certain PCB isomers and congeners, which are amongst the most ubiquitously distributed pollutants in the ecosystem.     

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.     

Production of antibody to individual polypeptides derived from purified hepatitis B surface antigen.

Purified preparations of hepatitis B surface antigen (HBsAg) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE). Pools of the individual fractions eluted from the preparative PAGE were concentrated and purified further by analytical PAGE. Five purified polypeptides were isolated from HBsAg, types adw and ayw, with molecular weights of 19,000, 24,000, 27,000, 35,000, and 40,000. Each preparations was emulsified in Freund complete adjuvant and injected into guinea pigs. Antibody to each HBsAg type was measured by radioimmunoassay. The 19,000 molecular weight polypeptide derived from ayw particles and the 27,000 molecular weight subunit obtained from both types failed to elicit an antibody response. The other three polypeptides derived from the ayw particles elicited group-specific antibody responses. Similar group-specific reactivities were observed in the testing of anti-adw 35,000 and anti-adw 40,000 molecular weight polypeptide sera. However, guinea pigs immunized with the 19,000 and the 24,000 molecular weight polypeptides of the adw type produced antibody that reacted preferentially with adw particles. This indicates that either these subunits carry predominately d determinants or that, because of the low levels of material used for inoculation, no immune response or an undetectable one was elicited to the a or w components.     

Abscisic acid and the response of the roots of Zea mays L. seedlings to gravity

Summary Exogeneous application of abscisic acid (ABA) to intact roots of LG 11 maize seedlings inhibits root elongation and induces bending of the root in response to gravity in darkness, even though the roots of these seedlings are not normally positively geotropic in the dark. ABA cannot, however, induce geotropic curvature in dark-exposed decapped roots, thus confirming that the root cap is the site of graviperception in the intact root.Abbreviation ABA abscissic acid     

Acid-induced growth and the geotropic response of the wheat node

Summary Growth is initiated in segments of the leaf sheath base of Triticum aestivum by gravitational stimulation and by incubation in buffer solutions of pH 3. Both responses involve only an increase in segment length, are rapidly terminated on removal of the stimulus, have a similar Q₁₀ and are dependent upon cell turgor. They differ, however, in that the response to acid solutions is rapid and unaffected by anoxia. Acid-induced growth can be stopped and started repeatedly by changing the pH, an increase in pH from 3 to 5 or 7 being sufficient to terminate the response. The maximum growth induced by low pH is not increased by simultaneous stimulation by gravity.