Relaxation of supercoiled phosphorothioate DNA by mammalian topoisomerases is inhibited in a base-specific manner

The nucleotide preferences of calf thymus topoisomerases I and II for recognition of supercoiled DNA have been assessed by the relaxation and cleavage of DNA containing base-specific phosphorothioate substitutions in one strand. The type I enzyme is inhibited to varying degrees by all modified DNAs, but most effectively (by approximately 60%) if deoxyguanosine 5'-O-(1-thiomonophosphate) (dGMP alpha S) is incorporated into negatively supercoiled DNA. A DNA in which all internucleotide linkages of one strand are phosphorothionate is relaxed, most probably via the unsubstituted strand. The type II enzyme is inhibited when deoxyadenosine 5'-O-(1-thiomonophosphate) (dAMP alpha S) or deoxyribosylthymine 5'-O-(1-thiomonophosphate) is incorporated into the DNA substrate, and the course of the relaxation reaction changes from a distributive mode to a predominantly processive mode. A fully substituted DNA is very poorly relaxed by the type II enzyme, illustrating the strict commitment of the enzyme to relaxation via double-strand cleavage. The sense of supercoiling does not affect the inhibition profile of either enzyme. DNA strand breaks introduced by type II topoisomerase in a normal control DNA or deoxycytidine 5'-O-(1-thiomonophosphate)-substituted DNA on treatment with sodium dodecyl sulfate at low ionic strength are prevented by pretreatment with 0.2 M NaCl. In contrast, breaks in DNA having either dAMP alpha S or all four phosphorothioate nucleotides incorporated in one strand are prevented only with higher NaCl concentrations. Thus indicating activity at the phosphorothioate linkage 5' to dA but not 5' to dC. We conclude that topoisomerase II activity occurs preferentially at sites possessing dAMP or dTMP, and that dGMP is involved in DNA recognition by topoisomerase I.     

Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain

Three types of protein kinase C, designated types I, II, and III, were purified from rat brain cytosol, and have been shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. Their relative activities in the whole brain tissue were roughly 26, 49, and 25% with H1 histone as a substrate. Type II enzyme was an unequal mixture of two subspecies (roughly 1:7) encoded by beta I and beta II sequences which differ from each other only in a short range of their carboxyl-terminal end regions. Although the three types have closely similar structures, they showed slightly different modes of activation and kinetic properties. Type I enzyme was less sensitive to diacylglycerol but was significantly activated by low concentrations of free arachidonic acid. Type II enzyme exhibited substantial activity without elevated Ca2+ levels, and responded well to diacylglycerol and, to some extent, arachidonic acid. The type III enzyme responded to diacylglycerol as well as to arachidonic acid. The mode of activation of the enzyme by arachidonic acid required elevated levels of Ca2+ but not phospholipid. In the presence of phospholipid, phorbol esters could activate all three types in a manner similar to diacylglycerol. Among various phospholipids tested, phosphatidylserine was the most effective for all three types. Type III enzyme was most sensitive to 1-stearoyl-2-arachidonylglycerol for activation. Conversely, type I enzyme was activated most efficiently by synthetic permeable diacylglycerols, such as 1,2-didecanoylglycerol and 1,2-dioctanoylglycerol. Many heavy metal ions exerted variable and distinct effects on the catalytic activities of these three types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype

Three clonal strain types (I, II, and III) of Toxoplasma gondii predominate worldwide. The outcome of infection in mice is highly dependent on the parasite genotype with type I strains being uniformly virulent, while types II and III are nonvirulent. Interactions with the innate immune response play a major role in determining the outcome of infection in the murine model. To identify key early differences in the innate immune response that contribute to pathogenesis, we examined the cytokine production of macrophages after in vitro infection with parasites of virulent type I and nonvirulent type II genotypes. Infection with type II strain parasites stimulated the production of proinflammatory cytokines, and particularly high levels of the Th1-polarizing cytokine, IL-12. Infection with type II strain parasites stimulated NF-kappaB nuclear translocation at early time points and led to the up-regulation of mRNA levels of IL-12 and other proinflammatory cytokines that was dependent on the myeloid differentiation factor 88 signaling pathway. Induction of IL-12 required active invasion by live parasites and was not blocked by infection with virulent type I strain parasites, arguing against an active inhibition of signaling. Our findings suggest that early induction of high levels of IL-12 by macrophages infected with type II strain parasites may contribute to more effective control.     

Deoxyadenosine deamination and phosphorylation in rat liver mitochondria

1. Deoxyadenosine metabolism was measured in freshly isolated mitochondria; these organelles took up the deoxynucleoside and formed three detectable products: deoxyinosine, dAMP and dIMP. 2. Enzyme extracts prepared from sonicated mitochondria exhibited deoxyadenosine deaminase, deoxyadenosine kinase, dAMP deaminase and deoxyinosine kinase activities. 3. These data suggest that deoxyadenosine was initially altered in mitochondria by at least two metabolic reactions--deamination and phosphorylation. Deoxyinosine and dAMP were produced. 4. These two products were subsequently phosphorylated and deaminated, respectively to produce dIMP.     

Pathogen-induced calmodulin isoforms in basal resistance against bacterial and fungal pathogens in tobacco

Thirteen tobacco calmodulin (CaM) genes fall into three distinct amino acid homology types. Wound-inducible type I isoforms NtCaM1 and 2 were moderately induced by tobacco mosaic virus (TMV)-mediated hypersensitive reaction, and the type III isoform NtCaM13 was highly induced, while the type II isoforms NtCaM3-NtCaM12 showed little response. Type I and III knockdown tobacco lines were generated using inverted repeat sequences from NtCaM1 and 13, respectively, to evaluate the contribution of pathogen-induced calmodulins (CaMs) to disease resistance. After specific reduction of type I and III CaM gene expression was confirmed in both transgenic lines, we analyzed the response to TMV infection, and found that TMV susceptibility was slightly enhanced in type III CaM knockdown lines compared with the control line. Resistance to a compatible strain of the bacterial pathogen Ralstonia solanacearum, and fungal pathogens Rhizoctonia solani and Pythium aphanidermatum was significantly lower in type III but not in type I CaM knockdown plants. Expression of jasmonic acid (JA)- and/or ethylene-inducible basic PR genes was not affected in these lines, suggesting that type III CaM isoforms are probably involved in basal defense against necrotrophic pathogens in a manner that is independent of JA and ethylene signaling.     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

Three deoxyribonucleic acid-dependent adenosine triphosphatases from Bacillus subtilis.

We have isolated from Bacillus subtilis three deoxyribonucleic acid (DNA)-dependent adenosine triphosphatases (ATPases) (gamma-phosphohydrolases). The enzymes were extensively purified, and their physicochemical and functional properties were determined. The three enzymes (ATPases I, II, and III) were shown to be different by several criteria. ATPases II and III showed an absolute requirement for single-stranded DNA as a cofactor, whereas ATPase I had some residual activity also with double-stranded DNA. They required Mg2+ and had a pH optimum of 6.5 to 7. Only adenosine 5'-triphosphate and deoxyadenosine 5'-triphosphate were hydrolyzed. The molecular weights of ATPases I, II, and III were 108,000, 115,000, and 148,000, respectively. Km values for adenosine 5'-triphosphate and DNA were also evaluated and shown to be different for each enzyme. All three enzymes formed physical complexes with single-stranded DNA. We present evidence that ATPases I and II might migrate along DNA during adenosine 5'-triphosphate hydrolysis. On the other hand, this effect was not observed with ATPase III, which exhibited the highest affinity for single-stranded DNA.     

Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes

Heat treatment of poly(deoxycytidylic acid)-[poly(dC)] induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III. The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC). The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase. The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions. However, the alteration of E. coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites. There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites. The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B. The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro. Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.     

Growth and Respiratory Response of Fig (Ficus carica L. cv. Mission) Fruits to Ethylene

Growth in diameter of the fig (Ficus carica L. cv. Mission) fruit takes place in three distinct periods; two periods (I and III) of rapid growth are separated by a period (II) of slow growth. With respect to exposure to ethylene, the fruit exhibits a two phase response. Ethylene inhibits fruit growth in phase A (period I), the period of cell division, stimulates growth in early phase B (early period II), and stimulates both growth and ripening during the remainder of phase B (late period II and period III). The adverse effect of exogenous ethylene on fruits during phase A is thought to be due to inhibition of cell division. The gradual transition occurring in the response of fruits during phase B was interpreted in terms of carbohydrate level in the fruits.     

Applications of fluorescent antibody for detecting capsular substances in Staphylococcus epidermidis

A fluorescent antibody technique was developed for the determination of the type of capsule of strains of Staphylococcus epidermidis. Many mouse virulent and avirulent strain populations were investigated. Of 300 fresh isolates of Staph. epidermidis, 27 were mouse virulent strains and of these 74.1% and 25.9% were mono- and polyvalent, respectively. The frequency of capsular type antigens I, II and III, produced by the 27 virulent strains was found to be 18.5%, 88.9% and 18.5%, the majority being capsular type II. In the mouse avirulent strains, capsular type antigen production was demonstrated in 263 out of 273 strains examined and mono- and polyvalent capsular types comprised 52.3% and 44.0%, respectively. Capsular type I, II, III strains and non-typable strains occurred at frequencies of 15.0%, 95.2%, 34.9% and 3.7% respectively, the majority of mouse avirulent strains also being capsular type II. These results indicate that a majority of ordinary Staph. epidermidis produced capsular type antigens although the capability is quantitatively different according to strain.     

Applications of fluorescent antibody for detecting capsular substances in Staphylococcus epidermidis

I chiman , Y. 1984. Applications of fluorescent antibody for detecting capsular substances in Staphylococcus epidermidis, Journal of Applied Bacteriology 56 , 311–316.
A fluorescent antibody technique was developed for the determination of the type of capsule of strains of Staphylococcus epidermidis . Many mouse virulent and avirulent strain populations were investigated. Of 300 fresh isolates of Staph. epidermidis , 27 were mouse virulent strains and of these 74.1% and 25.9% were mono- and polyvalent, respectively. The frequency of capsular type antigens I, II and III, produced by the 27 virulent strains was found to be 18.5%, 88.9% and 18.5%, the majority being capsular type II. In the mouse avirulent strains, capsular type antigen production was demonstrated in 263 out of 273 strains examined and mono- and polyvalent capsular types comprised 52.3% and 44.0%, respectively. Capsular type I, II, III strains and non-typable strains occurred at frequencies of 15.0%, 95.2%, 34.9% and 3.7% respectively, the majority of mouse avirulent strains also being capsular type II. These results indicate that a majority of ordinary Staph. epidermidis produce capsular type antigens although the capability is quantitatively different according to strain.     

Polymerase-catalyzed synthesis of DNA from phosphoramidate conjugates of deoxynucleotides and amino acids

Some selected amino acids, in particular L-aspartic acid (L-Asp) and L-histidine (L-His), can function as leaving group during polymerase-catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. Although L-Asp-dAMP and L-His-dAMP bind, most probably, in a different way in the active site of the enzyme, aspartic acid and histidine can be considered as mimics of the pyrophosphate moiety of deoxyadenosine triphosphate. L-Aspartic acid is more efficient than D-aspartic acid as leaving group. Such P-N conjugates of amino acids and deoxynucleotides provide a novel experimental ground for diversifying nucleic acid metabolism in the field of synthetic biology.     

Ulnar polydactyly

A retrospective review of 148 patients with ulnar polydactyly was conducted to analyze the types, patterns of involvement, associated anomalies, treatments, and outcomes of this malformation. The hands only were involved in 123 patients, both hands and feet in 20 patients, and five patients had mixed radial and ulnar polydactyly. Ulnar polydactyly was more prevalent among males. Among African Americans, the condition was often bilateral. When unilateral, ulnar polydactyly occurred more often on the left side. The racial distribution was 103 African Americans (70 percent), 37 Caucasians (25 percent), four Native Americans, three Latin Americans, and one Asian. Five types were encountered: type I cutaneous nubbin, type II pedunculated digit, type III articulating digit with fifth metacarpal, type IV fully developed digit with sixth metacarpal, and type V polysyndactyly. The distribution of types in order of frequency was type II, III, V, I, and IV. Types I and II ulnar polydactyly combined were more prevalent (82 percent) than types III, IV, and V (18 percent). Types I and II were more common among African Americans. Types III, IV, and V ulnar polydactyly occurred more frequently among Caucasians, but these were slightly less prevalent than types I and II in this racial group. Five patients were syndromic; four were Caucasians, and one Asian. Most cases of ulnar polydactyly of the hand were treated by ligation (71 percent) in the nursery, whereas polydactyly of the foot was more often referred to a specialist to be treated by surgical ablation (92 percent). Treatment complications occurred more frequently in the hands than in the feet. The complication rate after ligation of ulnar polydactyly of the hand was 23.5 percent. The two main complications were tender or unacceptable nubbins and infections.     

Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.     

ATP-sensitive and ATP-insensitive phosphofructokinase in Escherichia coli K-12.

The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150,000 (subunit mol. wt 73,000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350,000 (subunit mol. wt 90,000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP-insensitive, whereas type II and the anaerobic enzyme are ATP-sensitive. The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer. The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.     

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.     

Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from S?o Paulo, Brazil: unexpected findings.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding S?o Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.     

Forming and Removing Stain Precipitates on Ultrathin Sections

Stain precipitates resulting from the use of lead or uranyl salts, or both, on ultrathin sections can be classified as belonging to one of three morphological types: I) extremely electron-dense particles caused by prolonged use of lead salts only, II) amorphous networks formed following double staining with either aqueous or alcoholic uranyl and lead salts, and III) crystalline needles sometimes resulting from double staining with alcoholic uranyl and lead salts. It has been found, however, that either acetic acid or aqueous uranyl acetate can be used to remove type I and type II precipitates from sections, and that oxalic acid and alcoholic uranyl solution will remove type II precipitates. Unfortunately, type III precipitates are unaffected by any agents tested so far.     

Forming and removing stain precipitates on ultrathin sections

Stain precipitates resulting from the use of lead or uranyl salts, or both, on ultrathin sections can be classified as belonging to one of three morphological types: I) extremely electron-dense particles caused by prolonged use of lead salts only, II) amorphous networks formed following double staining with either aqueous or alcoholic uranyl and lead salts, and III) crystalline needles sometimes resulting from double staining with alcoholic uranyl and lead salts. It has been found, however, that either acetic acid or aqueous uranyl acetate can be used to remove type I and type II precipitates from sections, and that oxalic acid and alcoholic uranyl solution will remove type II precipitates. Unfortunately, type III precipitates are unaffected by any agents tested so far.