Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase

The CphA metallo--lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.Received 3 March 2003; received after revision 4 August 2003; accepted 25 August 2003     

Axillary 5α-androst-16-en-3-one in men and women: Relationships with olfactory acuity to odorous 16-androstenes

Summary Axillary 5-androst-16-en-3-one (5-androstenone) levels were found to be significantly higher in men than in women but do not vary between left and right axillae, are not related to age, handedness or degree of hirsutism (in women) nor to anosmia to this steroid. In men (but not in women), levels are related linearly to axillary cholesterol concentrations but not to squalene. Olfactory thresholds for 5-androstenone varied widely, the lowest recorded being 0.2 ppb, but there was no difference in thresholds between men and women. Women (70%) found the smell repellant but anosmia did not differ greatly between men and women (9–20%). Anosmia to the smell of 5-androst-16-en-3-ol was most marked in women (90%) rather than in men (45%). Axillary 5-androstenone values were generally consistent with the musky or strong smells of male axillary extracts, compared with the sweet smell of those from female subjects.Supported by the Herbert Dunhill Trust.     

Localisation of acetylcholinesterase in rat myotubes in the presence of β-endorphin and β-endorphin-(1-27)

Summary In rat embryo skeletal myotubes, acetylcholinesterase is present, as multiple forms, and can be detected in deposits at the cell surface. Myotubes cultured in the presence of -endorphin, exhibit an increased predominance of the globular (precursor) forms of the enzyme, which are largely restricted to intracellular sites associated with nuclei. In the presence of -endorphin-(1-27), the relative proportions of the different forms of acetylcholinesterase is similar to that seen in the controls, but the enzyme is intracellular and has a characteristic focal localisation in the myotube.     

Free intracellular and protein bound amino acids in tissues as affected by a mixedβ-adrenergic agonist

The administration of metaproterenol induced an increase in gastrocnemius muscle weight without change in body growth rate or tissue protein concentrations, while epididymal fat was reduced. This effect was accompanied by an enhancement in the levels of intracellular amino acids in muscle. By contrast, liver amino acids were unaffected by treatment with the mixed -adrenergic agonist.     

Structure of the porcine thyrotropin receptor: a 200 kilodalton glycoprotein heterocomplex

Summary We have determined that the porcine thyroidal TSH receptor is a glycoprotein heterotetramer composed of two M_r 35,000 () covalently linked subunits which interact noncovalently with two copies of (M_r 66,000) chains.Acknowledgments. This work was supported by grants from the Medical Research Council of Canada.     

Age distribution of circulatingα-interferon

Summary A sensitive radioimmunoassay showed that circulating -interferon in the plasma of healthy individuals was low in children and reached the highest level in the young adult, then declined gradually with age. Circulating -interferon was 0.201±0.059 ng/ml in males (n=19) and 0.184±0.076 ng/ml in females (n=14) at ages 30–39 years old. It was noted that circulating -interferon was maintained up to a certain level even in elderly individuals.     

Antiproliferative effects of 4′, 5′-unsaturated adenine nucleosides on leukemia L 1210 cells in vitro

Summary Several 4, 5-unsaturated adenine nucleosides were shown to have antiproliferative activity against L 1210 leukemia cells in vitro. The active nucleosides were cytotoxic to the L 1210 cells as demonstrated by Trypan Blue uptake. The cytotoxicity was not induced by alterations in the ribonucleoside and deoxyribonucleoside triphosphate levels of the L 1210 cells.     

Evidence for the extranuclear localization of thymosins in thymus

A new radioimmunoassay has been developed for thymosin ₄ by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1–15 coupled to KLH. The synthetic analogue [Tyr¹²]-thymosin ₄ (1–15) was used as tracer. This radioimmunoassay, with a useful range of 10–1000 pmoles, showed cross-reactivity with the second homologous -thymosin of man and rat (thymosin ₁₀) but not of calf (thymosin ₉). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin , was employed for the measurement of the levels of thymosin ₄ and parathymosin in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of - and -thymosins.     

Noradrenergic control of ascorbic acid and glutathione concentrations in brown fat of cold-exposed rats

Summary Ascorbic acid and glutathione concentrations increase in brown fat of cold-exposed rats. This phenomenon can be reproduced by noradrenaline or isoproterenol administration, and thus seems to be under sympathetic control. Histological study shows ascorbic acid storage in brown adipocyte nuclei.     

Ethanol and opioid receptor signalling

Summary Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate -opioid receptor binding by increasing receptor B_max, and, in some cases, chronic ethanol exposure decreases the density or affinity of the -opioid receptors. By contrast, high concentrations of ethanol acutely decrease -opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the -opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the -opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the -opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of G_s and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the -opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in G_s or G_i. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the -opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in G_s and increases in G_i. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.     

Tissue-specific induction of intestinal glutathione S-transferases by αβ-unsaturated carbonyl compounds

Summary Glutathione S-transferase activity in rat intestinal mucosa was increased by the injection of -unsaturated carbonyl compounds such as phorone and diethylmaleate, but that in the liver and kidney was not. Since the administration of cycloheximide completely blocked the increase of the enzyme activity by phorone, the increase of the activity may be due to de novo synthesis rather than enzyme activation.     

Changes of the level of proteinase inhibitors in rat plasma during turpentine-induced inflammation

Summary The levels of rat plasma -macroglobulins, -cysteine proteinase inhibitor, haptoglobin and antipapain activity were studied during the acute-phase reaction after an injection of -pinen. An increase in concentration of all the compounds examined was observed.     

Resistance to β-lactam antibiotics

-lactams have a long history in the treatment of infectious diseases, though their use has been and continues to be confounded by the development of resistance in target organisms. -lactamases, particularly in Gram-negative pathogens, are a major determinant of this resistance, although alterations in the -lactam targets, the penicillin-binding proteins (PBPs), are also important, especially in Gram-positive pathogens. Mechanisms for the efflux and/or exclusion of these agents also contribute, though often in conjunction these other two. Approaches for overcoming these resistance mechanisms include the development of novel -lactamase-stable -lactams, -lactamase inhibitors to be employed with existing -lactams, -lactam compounds that bind strongly to low-affinity PBPs and agents that potentiate the activity of existing -lactams against low-affinity PBP-producing organisms.Received 9 February 2004; received after revision 30 March 2004; accepted 19 April 2004     

Efficient cell proliferation and predominant accumulation of ɛ-globin mRNA in human leukemic K562 cells which produce mostly Hb Gower 1

Summary Long-term cultures of K562(S) cells in 50–75 M hemin allow the selection of hemin-resistant K562 cells together with cells which proliferate efficiently while fully induced to express the human embryonic globin genes, as the hemoglobin Gower 1 (₂₂) is the predominant hemoglobin produced. Our experiments demonstrate that these K562 cells accumulate mostly -globin mRNA (-globin mRNA/-globin mRNA=2.9) suggesting that the control of hemoglobin expression is at a pretranslational level.We thank Dr Irene Bozzoni (Centro degli Acidi Nucleici, Università di Roma) for the pXCR7 probe. Address for reprint request: R.G. Centro Studi Biochimici sul Morbo di Cooley, Via Borsari 46, I-44100 Ferrara.     

β-Endorphin in normozoospermic and pathologic human semen

Summary -Endorphin was estimated in normozoospermic, oligozoospermic and azoospermic human semen. The mean amount in normozoospermic specimens was 278.6±43.6 (SE) pg/ml while in the others only 191.1±25 pg/ml. Both values are significantly higher than those present in the blood.     

Molecular evidence for increased hematopoietic proliferation in the spleen of the b/b laboratory rat

The splenomegaly and the appearance of a significant number of CFU-E (erythroid colony-forming units) and BFU-E¹ (erythroid burst-forming units) in the Belgrade laboratory rat (b/b) spleen prompted us to analyse further the molecular evidence for increased hematopoietic proliferation in the b/b spleen. Messenger RNAs (mRNAs) specific for globins, proteins for iron transport and deposition and the band 3 protein were used in rat erythropoietic tissues as markers for proliferation and erythroid differentiation. In the b/b spleen, all mRNAs analysed display an erythroid-specific pattern of expression. This analysis also revealed an enhanced level of mRNA for ferritin in the +/b spleen, whereas erythrocyte-specific mRNA production was normal.     

Effect of glucose and phloretin-2′-β-D-glucose (phloridzin) on in vitro fertilization of mouse ova

Summary The fertilization ratio of mouse ova in vitro decreased when glucose concentration in the medium was lowered. However, the addition of phloretin-2--D-glucose (phloridzin), known as a glucose uptake inhibitor, restored the fertilization ratio back to the control level. The glucose moiety of the phloridzin seemed to be responsible for this effect.     

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5′-AMP with hydrophobic amino acids

We have studied the chemistry of aminoacyl AMP to model reactions at the 3 terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The -branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the -branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with ademine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.     

Age- and sex-related differences in the content of prothymosinα in rat tissues

Differences in the tissue content of prothymosin during the early postnatal development of male and female rats are reported. Thymus and spleen have been found to contain significantly higher amounts of prothymosin in the newborn and prepubertal animals, as compared to adults, whereas liver has been found to contain low levels of prothymosin throughout development. These findings indicate a functional association of prothymosin with the proliferating lymphoid tissues of the young rat.