Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Persistence and expression of the plasmid pBR322 inEscherichia coli K12 cultured in complex medium

Summary The stability and gene expression of plasmid pBR322 in a chemostat with complex non-selective medium at different dilution rates were studied. It was observed that pBR322 was eventually lost from the population after a long lag period. The rate of plasmid loss decreases with decreasing dilution rate. This result is different from those obtained with cells grown in defined medium, where plasmid loss was observed to decrease with increasing dilution rate. In addition, it was observed that the -lactamase activity per ml per optical density of cell culture, independent of the dilution rates, increases with time and reaches a maximum around 4.5 units after 100 hrs of continuous culture.     

Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla)

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.     

Primary structure and functional properties of the hemoglobin from the free-tailed bat Tadarida brasiliensis (Chiroptera). Small effect of carbon dioxide on oxygen affinity

The hemoglobin of the Free-Tailed Bat Tadarida brasiliensis (Microchiroptera) comprises two components (Hb I and Hb II) in nearly equal amounts. Both hemoglobins have identical beta-chains, whereas the alpha-chains differ in having glycine (Hb I) or aspartic acid (Hb II) in position 115 (GH3). The components could be isolated by DEAE-Sephacel chromatography and separated into the globin chains by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman degradation with the film technique or the gas phase method (the alpha I-chains with the latter method only), using the native chains and tryptic peptides, as well as the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The comparison with human hemoglobin showed 18 substitutions in the alpha-chains and 24 in the beta-chains. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1-contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are substituted. A comparison with other chiropteran hemoglobin sequences shows similar distances to Micro- and Megachiroptera. The oxygenation characteristics of the composite hemolysate and the two components, measured in relation to pH, Cl-, and 2,3-bis-phosphoglycerate, are described. The effect of carbon dioxide on oxygen affinity is considerably smaller than that observed in human hemoglobin, which might be an adaptation to life under hypercapnic conditions.     

Analysis of teleost hemoglobin by Adair and Monod-Wyman-Changeux models

Summary The allosteric effects of the erythrocytic nucleoside triphosphates (NTP) and of proton concentrations were investigated by precise measurement of Hb–O₂ equilibria of tench hemoglobin (including extreme, high and low saturation ranges) and analysed in terms of the MWC two state model and the Adair four step oxygenation theory.At low concentrations (NTP/Hb ratio=1.0, and pH>7.3) ATP, GTP and protons decrease Hb–O₂ affinity by increasing the allosteric constantL and reducingK _T, the association constant¹ of the deoxy, tense state of the Hb, without significantly affecting that (K _R) of the oxy state, increasing the free energy of cooperativity (G). High concentrations of these effectors, however, also reduceK _R. The greater sensitivity of the half-saturation O₂ tension (P ₅₀) of the Hb to GTP than to ATP at the same concentration, correlates with greater effects of GTP on bothK _T andK _R. The pH and NTP dependence of the four Adair association constants and the calculated fractional populations of Hb molecules in different stages of oxygenation show that the autochthonous NTP effectors and protons stabilize the T structure and postpone the TR transition basic to cooperativity in fish Hb.The possible implications of the findings for aquatic respiration are discussed.Abbreviations ATP adenosine triphosphate - DPG 2,3-diphosphoglycerate (glycerate-2,3-bisphosphate) - GTP guanosine triphosphate - IHP inositol hexaphosphate - NTP nucleoside triphosphates In this paperK _T andK _R are defined as theassociation equilibrium constants instead of dissociation constants (as originally defined by Monod et al. 1965) to facilitate comparison with the Adair constants     

Thermodynamic analysis of precisely measured oxygen equilibria of tench (Tinca tinca) hemoglobin and their dependence on ATP and protons

Summary Precise oxygen equilibria including extreme, high and low saturation values were determined for hemoglobin (Hb) from the freshwater teleostTinca tinca at three temperatures, each at two pH levels and in the presence and absence of the erythrocytic cofactor ATP, at twofold molar excess over Hb.Analysis of the data in terms of Adair's successive oxygenation theory shows that in the absence of ATP, each of the four oxygenation steps are exothermic, but that net heat release decreases as pH falls from 8.2 to 7.4. ATP greatly depresses the temperature sensitivity of oxygenation particularly at physiological erythrocytic pH, where endothermic cofactor dissociation finds expression in a reverse temperature sensitivity for binding of the 3^rd oxygen molecule to the tetrameric Hb.Enthalpy (H _i) and entropy (S _i) changes of oxygenation vary with oxygenation step, i, as well as with pH and ATP addition, but the variations of H _i are similar to those of S _i reflecting enthalpy-entropy compensation.The data show that the cooperative effects in tench Hb can be dominated either by entropic or enthalpic contributions, depending on the experimental condition and the oxygenation step.     

Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.