Inhibition of rabbit lung angiotensin-converting enzyme by N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline

Two novel peptide analogs, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline and the corresponding L-lysyl-L-proline derivative, have been demonstrated to be potent competitive inhibitors of purified rabbit lung angiotensin-converting enzyme: Ki = 2 and 1 X 10(-10) M, respectively, at pH 7.5, 25 degrees C, and 0.3 M chloride ion. Second-order rate constants for addition of these inhibitors to enzyme under the same conditions are in the range 1-2 X 10(6) M-1 s-1; first-order rate constants for dissociation of the EI complexes are in the range 1-4 X 10(-4) s-1. The association rate constants are similar to those measured for D-3-mercapto-2-methylpropanoyl-L-proline, captopril, but the dissociation rate constants are severalfold slower and account for the higher affinity of these inhibitors for the enzyme. The dissociation constant for the EI complex containing N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline is pH-dependent, and reaches a minimum at approximately pH 6: Ki = 4 +/- 1 X 10(-11) M. The pH dependence is consistent either with a model for which the protonation state of the secondary nitrogen atom in the inhibitor determines binding affinity, or one for which ionizations on the enzyme alone influence affinity for these inhibitors. The affinity of this inhibitor for the zinc-free apoenzyme is 2 X 10(4) times less than for the zinc-free apoenzyme is 2 X 10(4) times less than that for the holoenzyme. If considered as a "collected product" inhibitor, N alpha-[(S)-1-carboxy-3-phenylpropyl]L-alanyl-L-proline appears to derive an additional factor of 375 M in its affinity for the enzyme compared to that of the two products of its hypothetical hydrolysis, a consequence of favorable entropy effects.     

Energy conservation in whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Respiratory chain phosphorylation has been investigated in the methylotrophic bacterium Methylophilus methylotrophus following the addition of oxidisable substrates to aerobic, whole cell suspensions. Initial-rate experiments showed that ATP synthesis occurred at the overall expense of AMP and inorganic phosphate via the sequential action of the ATP phosphohydrolase and adenylate kinase; some of the nascent ATP was rapidly used to synthesis nonadenine nucleoside triphosphates. After being corrected for ATP turnover, Pi/O quotients of 0.46 to 0.54, 0.77 and 1.37 nmol/ng-atom O were obtained for the oxidation of methanol dehydrogenase-linked substrates (methanol, ethanol and acetaldehyde), duroquinol and formate (NAD⁺-linked) respectively. These values were proportional to the H⁺/O and/or K⁺/O quotients exhibited by these substrates, and yielded an average H⁺/ATP (H⁺/Pi) quotient of 4.2 ng-ion H⁺/nmol. Steady-state experiments showed that the extent of cellular energisation varied with the respiration rate but was always in the order methanol > duroquinol > acetaldehyde, thus indicating that under these longer-term conditions methanol was completely oxidised to yield PQQH₂ and 2NAD(P)H. These results are discussed in terms of the various reactions which lead to the generation or utilisation of the protonmotive force in this organism.Abbreviations FCCP carbonylcyanide p-trifluoromethyxyphenyl-hydrazone - bulk phase, transmembrane electrochemical potential difference of protons ( ) - pH bulk phase, transmembrane pH difference (pH_in–pH_out) - bulk phase, transmembrane electrical potential difference (_in - _out) - [P] concentration of anhydride phosphate bonds in adenine nucleotides (2[ATP]+[ADP]) - FPLC fast protein liquid chromatography - PQQ pyrroloquinoline quinone - Gp phosphorylation potential     

Transport of glycine-betaine by Listeria monocytogenes

Uptake of [¹⁴C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4–0.5 M. The glycine-betaine transport system had a K _m of 22 M and a V _max of 11.7 nmol^-1 min^-1 mg^-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the V _max increased to 27.0 nmol^-1 min^-1 mg^-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K⁺ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus

The effects of EDTA and related metal-chelating agents on the respiratory system of the methylotrophic bacterium Methylophilus methylotrophus have been investigated. EDTA completely inhibited whole cell methanol oxidase activity and concomitantly decreased the aerobic steady-state reduction level of cytochrome c, but only partially inhibited methanol dehydrogenase activity. Analysis of the inhibition kinetics of EDTA and other chelating agents on methanol oxidase activity indicated that they were effective in the order CDTA greater than EDTA greater than HEDTA greater than EGTA greater than EDDA, and that they inhibited in an uncompetitive manner. Inhibition by EDTA varied as a function of the ambient cell mass in the assay, and could be partly reversed by the addition of divalent cations. EDTA had no effect on the activity of solubilised methanol dehydrogenase. These and other results indicate that EDTA binds a divalent metal ion, probably Mg2+, which is involved in the functional association of methanol dehydrogenase with the respiratory membrane. This concept is discussed in terms of the electron transfer properties and spatial organisation of the terminal respiratory chain of M. methylotrophus.     

Excitation of System I and System II in Photosynthesis as a Possible Control Mechanism of Glycolate Metabolism in the Blue-Green Alga Anacystis nidulans

Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Eradicating Helicobacter pylori and symptoms of non-ulcer dyspepsia.

OBJECTIVE--To examine the effect of eradication of Helicobacter pylori on symptoms of non-ulcer dyspepsia. DESIGN--Four week prospective study. SETTING--One hospital outpatient and endoscopy department. PATIENTS--90 adults with persistent symptoms typical of non-ulcer dyspepsia but no clinical or endoscopic evidence of other peptic, biliary, pancreatic, or malignant disease; all had histological and microbiological evidence of infection with H pylori. 83 patients completed the treatment regimen. INTERVENTION--Colloidal bismuth subcitrate 120 mg four times a day for four weeks (27 patients); metronidazole 400 mg and amoxycillin 500 mg each three times a day for one week (27); and bismuth subcitrate 120 mg four times a day for four weeks, metronidazole 400 mg three times a day for one week, plus amoxycillin 500 mg three times a day for the first week (29). MAIN OUTCOME MEASURES--Change in symptom scores determined with questionnaire; histological evidence of gastritis and microbiological evidence of presence of H pylori in biopsy specimens. RESULTS--Overall, H pylori was eradicated in 41 (49%) patients. Although gastritis scores improved significantly in only patients in whom H pylori had been eradicated (from 1.56 to 0.61, p less than 0.01 v from 1.83 to 1.07, p = 0.52) mean symptom scores after treatment were similar in patients in whom H pylori had or had not been eradicated (3.0 v 2.3, NS). Similarly the mean symptom score improved whether or not gastritis improved (2.8 v 3.1 respectively, p = 0.72). The observations were similar for treatment groups analysed individually. CONCLUSION--Antral infection with the organism does not seem to have an important aetiological role in non-ulcer dyspepsia short term.     

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods

Lectins from Helix pomatia, Canavalia ensiformis, Agaricus bisporus and Triticum vulgaris agglutinated cultures of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. This agglutination was specific as it was inhibited (except with A. bisporus lectin) by the competing sugar substrates. The ability of three of these lectins, immobilized on a variety of supports, to separate these micro-organisms from pure cultures was investigated. Immobilization of the lectins on magnetic microspheres was the most effective method. Immobilized T. vulgaris lectin bound 87–100% of cells from cultures of L. monocytogenes , 80–100% of Staph. aureus , 33–45% of Salmonella spp. and 42–77% of E. coli. The A. bisporus lectin bound 31–63% of cells in cultures of L. monocytogenes , 83% of Staph. aureus but only 3–5% of the salmonella cells. Similarly H. pomatia lectin bound >92% of Staph. aureus and 64% of L. monocytogenes cells but was poor at binding the Gram-negative organisms. This preference for binding Gram-positive organisms was confirmed when mixed cultures were studied. The T. vulgaris lectin was effective in removing L. monocytogenes (43%) and Staph. aureus (26%) from diluted milk and Salmonella (31–54%) from raw egg. Agaricus bisporus lectin removed L. monocytogenes from undiluted milk (10–47%) or ground beef (32–50%).