Purification, separation, and characterization of two molecular forms of D-1-amino-2-propanol:NAD+ oxidoreductase activity from extracts of Escherichia coli K-12.

D-1-Amino-2-propanol:NAD+ oxidoreductase activity, which catalyzes the second step in a pathway wherein L-threonine is converted to D-1-amino-2-propanol via the intermediate formation of aminoacetone, has been purified 500-fold from Escherichia coli K-12. Although the enzyme catalyzes the oxidation of certain diols as well as 1-amino-2-propanol, it is completely specific for the D-isomer of the amino alcohol and for NAD+. Two molecular forms (designated Form L and Form S) of the oxidoreductase, both of which are catalytically active, have been separated by gel filtration on Sephadex G-200; apparently, Form L is converted to Form S by dissociation (Form L leads to Form S). Molecular weight determinations indicate that the two forms of the enzyme are different not only in size but also in shape; Form L apparently is an asymmetric tetramer of Form S. The two molecular species have similar catalytic properties. Both exhibit the same pH optimum of 8.6, have nearly identical apparent Km values for substrate and cosubstrate, are equally sensitive to inhibition by p-mercuribenzoate and N-ethylmaleimide, and show the same specificity for cosubstrate. Neither form of the enzyme has an absolute requirement for added thiol compounds or divalent metal ions.     

Transport of methotrexate in basolateral membrane vesicles from rat liver.

Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by initial uptake studies and by varying medium osmolarity with increasing concentrations of sucrose. Methotrexate transport was linear for the first 20 s of incubation. Transport was not affected by imposition of a Na+ gradient across the vesicular membrane. Transport of methotrexate displayed a broad pH optimum: at an intravesicular pH of 7.5, the initial rate of uptake was not significantly different at extravesicular pH values ranging from 5.5 to 7.5, but uptake was less at extravesicular pH of 5.0 or 8.0. Methotrexate transport was saturable: Km = 0.15 +/- 0.05 microM and Vmax = 11.4 +/- 1.1 pmol 10 s-1 mg-1 protein. Methotrexate uptake into BLMVs was not inhibited by 5-methyltetrahydrofolate nor by 5-formyltetrahydrofolate but was weakly inhibited by folic acid in a concentration-dependent manner. Uptake was also inhibited by anion-exchange inhibitor 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), and by the structurally unrelated anions ATP, ADP, Cl-, SO4(2-), and oxalate2-. Adenosine (no negative charge) had no effect on transport. When vesicles were preloaded with anions (ADP, SO4(2-), oxalate2-) such that an anion gradient existed from the intra- to the extravesicular compartment, and methotrexate uptake was measured, no stimulation of uptake was seen. Methotrexate uptake into rat liver BLMVs was electrogenic as shown by stimulation of the initial rate of uptake by a valinomycin-imposed K+ diffusion potential across the vesicular membrane. These results suggest that methotrexate is transported into the hepatocyte across the basolateral membrane by an electrogenic, multispecific anion carrier system.     

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.     

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.     

Reconstitution of urea-dissociated subunits of a pig heart phosphoprotein phosphatase

Pig heart phosphoprotein phosphatase [phosphoprotein phosphophydrolase, EC 3.1.3.16] of Mr 224,000 was dissociated by gel-filtration on Sephacryl S-300, into an active subunit (alpha subunit) of Mr 31,000 and inactive subunits of higher molecular weight in the presence of 6 M urea. After the removal of urea, these subunits reassociated, forming two enzyme forms of Mr 237,000 (Form 1) and Mr 123,000 (Form 2). Form 2 was produced by association of the alpha subunit with an inactive subunit (beta subunit) of Mr 80,000, while Form 1 was formed by combination of the alpha subunit with a complex of inactive subunits which was eluted from a Sephadex G-150 column in fractions of molecular weight range greater than 80,000. The dissociation and reassociation of the subunits of Form 1 by the same urea method produced not only Form 1, but also significant amounts of Form 2, indicating that the inactive subunits of Form 1 were a complex of the beta subunit with another inactive subunit(s). The molecular parameters and other properties of Form 1 were very close to those of the original enzyme. By the conversion of Form 1 to Form 2, the activities of Form 1 towards phosphorylase a and glycogen synthetase b were enhanced 2-3 fold with no significant change in activity towards P-H1 histone or in response to the stimulatory effect of Mg(CH3COO)2 on the dephosphorylation of P-H2B histone. However, removal of the beta subunit from From 2 resulted in strong suppression of activity towards P-H1 histone and response to the salt effect with lesser effects on the activities of Form 2 towards phosphorylase a and glycogen synthase b.     

Aminotransferases for aromatic amino acids and aspartate in Bacillus subtilis.

Two proteins (form A and form B2) with aromatic-amino-acid aminotransferase activity were detected in extracts of Bacillus subtilis. A histidinol phosphate aminotransferase (protein B1) with aminotransferase activity for the aromatic amino acids was also present. The aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) (protein C) also displayed similar activity. Each of the four proteins was isolated free from the others by the successive application of DEAE-cellulose column chromatography and flat-bed isoelectric focusing at pH range 4-6. Form B2 is the major form of the aromatic-amino-acid aminotransferase (aromatic-amino-acid:2-oxoglutarate amino-transferase, EC 2.6.1.57) and the Km values of tyrosine and phenylalanine with this form are somewhat lower than with the minor form A. The Km of tyrosine with histidinol phosphate aminotransferase (protein B1) is in the same range, but the Km of phenylalanine with this enzyme is 12-20 times higher than the corresponding values with the two forms of the aromatic-amino-acid amino-transferase. Apparent molecular weights were estimated with Sephadex gel filtration to be approx. 73 000, 64 000, 54 000 and 66 000 for form A, form B2, histidinol phosphate aminotransferase and aspartate aminotransferase, respectively. Form B2 is being reported for the first time in this communication.     

Human thymidylate synthetase derived from blast cells of patients with acture myelocytic leukemia. Purification and chracterization.

Thymidylate synthetase (EC 2.1.1.B.) from blast cells of patients with acute myelocytic leukemia has been purified more than 1470-fold by affinity column chromatography. Methotrexate was the affinity ligand. dUMP was found to be a necessary additive for retention of the enzyme by the affinity column. Disc electrophoresis and sucrose density gradient centrifugation revealed a single enzyme species with a molecular weight of 76,000. The enzyme exhibits a temperature-dependent conformational change with activation energies of 5.3 +/- 0.4 and 17.3 +/- 1.9 kcal/mol, respectively, above and below a transitional temperature of 35 degrees. This conformational change is reflected in the binding affinity of dUMP but not of 5,10-methylenetetrahydrofolate. The enzyme displays a broad pH maximum in the range of pH 7.4 to 8.8. The Michaelis constants for dUMP and (+/--L-5,10-methylenetetrahydrofolate are 1.8 +/- 0.2 and 31 +/- 8.3 micrometer, respectively. Initial velocity and product inhibition studies reveal the enzymic mechanism to be ordered sequential. dUMP binds before 5,10-methylenetetrahydrofolate and dihydrofolate is released before TMP. 5-Fluoro-2'-deoxy-5'-uridylate (FdUMP) behaves as in irreversible inhibitor with a Ki of 1.68 +/- 0.45 nM. The enzyme has a turnover number of 6 min-1 per FdUMP binding site. Methotrexate inhibits noncompetitively with respect to dUMP and binds tighter to the enzyme in the presence of dUMP. Methotrexate antagonizes inactivation of the enzyme by FdUMP.     

D-1-amino-2-propanol:NAD+ oxidoreductase. Purification and general properties of the large molecular form of the enzyme from Escherichia coli K12

Growth of Escherichia coli K12 under relatively anaerobic conditions in a medium containing casein hydrolysate, 0.8% glycerol, and 0.8% hydroxyacetone has been found to induce the level of D-1-amino-2-propanol oxidoreductase activity 50- to 100-fold over that in cells grown in casein hydrolysate alone or with 0.8% glycerol added. A large molecular weight form of this oxidoreductase (designated Form L) has been purified to apparent homogeneity in good yield by three simple steps designed to obviate its conversion to a smaller species. The molecular weight of native Form L and its basic subunit are 417,000 +/- 20,700 and 50,500 +/- 2,770, respectively; hence Form L would appear to consist of eight identical subunits. The pH activity profile for Form L shows one optimum in the range of 8.3 to 8.6 and another at pH 10.0 to 10.2. This form of the oxidoreductase has no apparent requirement for added metal ions (rather, numerous divalent transition metal ions are strongly inhibitory) or thiol compounds; it catalyzes the oxidation of several vic-glycols but is completely stereospecific for the D-isomer of 1-amino-2-propanol, utilizes only NAD+ as cosubstrate in the oxidation reaction (Km for NAD+ with DL-1-amino-2-propanol = 1.23 mM), but both NADH and NADPH serve as cosubstrate in the reduction of hydroxyacetone. Oxidoreductase activity of Form L is highly sensitive to inhibition by Hg2+, p-mercuribenzoate, or dithiodipyridine; inhibition by the latter two compounds is completely reversed by adding a thiol in excess.     

Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution

Intramolecular G-quadruplexes formed by human telomere sequences are attractive anticancer targets. Recently, four-repeat human telomere sequences have been shown to form two different intramolecular (3 + 1) G-quadruplexes in K(+) solution (Form 1 and Form 2). Here we report on the solution structures of both Form 1 and Form 2 adopted by natural human telomere sequences. Both structures contain the (3 + 1) G-tetrad core with one double-chain-reversal and two edgewise loops, but differ in the successive order of loop arrangements within the G-quadruplex scaffold. Our results provide the structural details at the two ends of the G-tetrad core in the context of natural sequences and information on different loop conformations. This structural information might be important for our understanding of telomere G-quadruplex structures and for anticancer drug design targeted to such scaffolds.     

Regulation of beta-D-galactoside alpha 2----3 sialyltransferase activity. The effects of detergents and lysophosphatidates

Peptide maps of Form A and Form B of porcine submaxillary gland beta Gal alpha 2----3 sialyltransferase were essentially identical, consistent with the view that the two forms are not different enzyme species but that one, the B form (Mr = 44,000) is derived from the A form (Mr = 49,000). Analysis of the sialyltransferase activity in subcellular fractions from homogenates of porcine submaxillary glands reveals that 85% of the total activity of the transferase is bound to membranes, mostly in the Golgi apparatus, and that the remainder is soluble. The relative amounts of the membrane-bound and soluble forms as well as their response to detergents suggests that they are the cellular counterparts to the A and B forms of the transferase. The activity of Form A and the membrane-bound enzyme is stimulated to similar extents by various detergents. Triton-type detergents are more effective than Brij-type. Lysophosphatidylcholine is a potent stimulator of the activity of Form A but lysophosphatidylethanolamine is without effect and lysophosphatidylserine and lysophosphatidylglycerol are inhibitory. C16-18 acyl derivatives of lysophosphatidylcholine stimulate the activity more extensively than the C14 acyl derivative, and the C12 acyl derivative is without effect. In contrast, Form B is fully active in the absence of all detergents tested although it is inactivated just as Form A by lysophosphatidylglycerol and octylglucoside. Kinetic analysis of Forms A and B reveal that detergents stimulate the activity of Form A by lowering the KD and KM of CMP-NeuAc and increasing the Vmax of the reaction. Form B in contrast, which is fully active in the absence of detergents, has kinetic parameters like those of Form A in the presence of detergent. Taken together, these results suggest that Form A of the sialyltransferase, but not Form B, contains a lipid-binding domain, and that binding of detergents or lipids to the domain modulates the activity of the enzyme.     

Structural variety of copper(II)-peroxide adducts and its relevance to DNA cleavage

The reactivity of copper (II) compounds with several tetradentate ligands towards some spin-trapping reagents was studied in the presence of hydrogen peroxide. The compounds used in this study are roughly divided into two groups based on the reactivity towards 2,2,6,6-tetramethyl-4-piperidinol (and also 2,2,6,6-tetramethyl-4-piperidone), which are trapping agents for singlet oxygen. 1O2(1deltag); The A-group compounds exhibited a high activity to form the corresponding nitrone radical, which was detected by ESR spectroscopy, but corresponding activity of the B-group compounds was very low. The A-group compounds defined as above exhibited high activity for cleavage of DNA (supercoiled) Form I) in the presence of hydrogen peroxide, yielding DNA Form II (relaxed circular) or Form III (linear duplex) under our experimental conditions ([Cu (II)] = 0.1 approximately 0.5 mM). On the other hand, the B-group compounds effected complete degradation of the DNA (double-strand scission) under the same experimental conditions, formation of Form II or Form III DNA was negligible. Two different DNA cleavage patterns observed for A- and B-group compounds were elucidated by the different structural property of the copper (II)-peroxide adducts, which is controlled by the interaction through both DNA and the peripheral group of the ligand system.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.

Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.     

Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity.

The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.     

Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 E · m^–2 · s^–1) and then shifted to high light (750 E·m^–2·s^–1). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.     

The design and use of the bioethics consultation form

The emergence of the ethics consultation as a means to resolve moral crises in clinical medicine has revealed the need for a worksheet that would facilitate intake and analysis. The author developed the "Bioethics Consultation Form" as an attempt to remedy this need. The form is arranged in an outline format and is a useful asset to ethics committee discussions and record keeping. The first section covers basic intake data concerning the patient's medical and personal information, advance directives, and values, as well as the values of the physician and family. After the intake section is completed with the above data, the ethics consultant then turns to the analysis section. This second section allows for (1) the discussion of conflicting values, (2) the identification of priorities, and (3) the elucidation of ethical norms relevant to the case. The Bioethics Consultation Form was adopted by the Patient Care Advisory Committee of the Franklin Square Hospital Center in Baltimore, Maryland in 1986. The methodology in the use of the form will be discussed. Further, the potential spectrum of consultative cases that can be analyzed using the form will be highlighted.     

Evidence for two activated forms of pyruvate kinase from Bacillus stearothermophilus in the presence of ribose 5-phosphate

Allosteric activation of pyruvate kinase from a thermophilic bacterium, Bacillus stearothermophilus, by ribose 5-phosphate (R5P) was kinetically examined. Two activated forms of this enzyme could be distinguished, depending on the R5P concentration. One form (Form I) was observed at about 10(-5) M R5P. It showed a slightly negative cooperativity for phosphoenolpyruvate (PEP). The other form (Form II) was observed at more than 10(-3) M R5P and showed Michaelis-Menten kinetics for PEP. The PEP and ADP concentrations that yield half-maximal velocity were essentially identical for the two forms (about 0.1 and about 0.5 mM, respectively), but Form I had a larger Vmax value than Form II. In the absence of R5P, the enzyme showed a homotropic positive cooperativity for PEP; the concentration required for the half-maximal velocity was about 2 mM and that of ADP was about 1.6 mM. The enzyme was more susceptible to protease digestion in the presence of R5P than in the absence of it. The concentration of R5P required for the enzyme to be susceptible to protease digestion was approximately identical with that required to generate Form I. With more than 10(-3) M R5P, the thermostability of the enzyme was greatly increased. The concentration of R5P required for the enzyme to be thermostable was in good agreement with that required to generate Form II. These results indicate that the two activated forms distinguished kinetically differ in their conformations, too. The saturating level of PEP did not cause such a change in the thermostability or the susceptibility to protease.     

Evidence to support two conspecific cytological races on Anopheles aconitus in Thailand.

Iso-female lines (isolines) of Anopheles aconitus collected from Mae Hong Son, Phet Buri, and Chiang Mai Provinces were successfully identified to karyotypic forms. The results of identification revealed that An. aconitus Form B (X1, X2, Y2) was obtained from four and 48 isolines in Phet Buri and Chiang Mai Provinces, respectively, and Form C (X1, X2, Y3) was recovered from three and 41 isolines in Mae Hong Son and Chiang Mai Provinces, respectively. When comparing band to band on the same arm of ovarian nurse cell polytene chromosomes of An. aconitus Form B (Phet Buri: four isolines) and C (Mae Hong Son: three isolines, Chiang Mai: 20 isolines) to the standard chromosome mapping of An. aconitus Form B (Chiang Mai: 20 isolines), no major chromosomal rearrangements that related to the karyotype variations were demonstrated. The investigations on allelic frequencies of 4th stage larvae and adult females of three (Form C: Mae Hong Son), four (Form B: Phet Buri), 41 (Form C: Chiang Mai) and 48 (Form B: Chiang Mai) isolines suggested that An. aconitus Form B and C of all strains have similar allelic frequencies. This was observed at 10 isoenzymes 16 loci in 4th stage larvae, and 11 isoenzymes 13 loci in adult females. Hybridization tests among the four laboratory-raised isolines of An. aconitus Form B (Chiang Mai and Phet Buri) and C (Chiang Mai and Mae Hong Son) were employed by induced copulation. The results of crosses indicated that they were genetically compatible, providing viable progeny and completely synaptic salivary gland polytene chromosomes. The complete sequences ofrDNA internal-transcribed spacer two (ITS2) and partial sequences of mitochondrial cytochrome c oxidase subunit I and II (COI and COII) from genomic DNA of 12 isolines of An. aconitus Form B and C were identified. Total sequence lengths (ITS2+COI+COII) of An. aconitus isolines varied from 1550bp to 1556bp. Conspecific relationships between the two An. aconitus forms were well supported by low values of intraspecific distances (ranged from 0.1% to 1.0%) and genetic differentiation (d(xy): 0.01322) between the two forms. Based on evidence of no pre- and post-mating isolations, and nearly identical of DNA sequences of ITS2, COI and COII regions between An. aconitus Form B and C, we conclude that they are conspecific cytological races in the Thai population.     

Reaction of Co(II)bleomycin with dioxygen.

The reaction of Co(II)bleomycin with dioxygen has been investigated. Dioxygen binds to the Co(II) complex within the time of mixing according to electron spin resonance and uv-visible spectroscopy and dioxygen analysis. Then, two dioxygenated cobalt centers react, releasing 1 mol of O2 and forming an intermediate characterized by a few highly shifted 1H NMR resonances and loss of the ESR spectrum. This is thought to be a dioxygen-bridged dimer of cobalt bleomycin molecules. Time-dependent absorbance and dioxygen measurements yield the same second order rate constant for this step of the reaction. According to uv-visible and NMR spectral analysis, the intermediate decays into diamagnetic products in a first order rate process. High performance liquid chromatography and 1H NMR studies demonstrate that the product contains two bleomycin species of equal concentration. One component is Co(III)bleomycin, designated Form II. The other is the peroxide adduct of Co(III)bleomycin, Form I, as determined by direct determination of hydrogen peroxide, which is slowly released from the product at low pH. In contrast, hydrogen peroxide is readily detected during the reaction of Co(II)Blm with O2. In isolation, Form I is unstable at pH 7 and is converted within 24 h into a mixture of Form I and Form II.