Manuel Nieto

[51] Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces

Publisher Summary Strains R39 and R61 are soil isolates. Their designations are arbitrary. In strain R39, the cross-link between the peptide units of the wall peptidoglycan extends from the C-terminal D-alanine of one unit to the amino group at the D-center of meso-diaminopimelic acid of another unit (peptidoglycan of chemotype I). The interpeptide bond is in position to a free carboxyl group. In strain R61, the cross-link extends from a C-terminal D-alanine of a peptide unit to a glycine residue attached to the amino group of LL-diaminopimelic acid of another peptide unit (peptidoglycan of chemotype II). The exocellular DD carboxypeptidases-transpeptidases produced by both strains catalyze hydrolysis, react with β-lactam antibiotics. This chapter explains the assay methods for DD-Carboxypeptidase activity like the standard reaction, chemical estimation of free Alanine, as well as, assay method for β-Lactamase. It also discusses the Excretion of DD-Carboxypeptidase-Transpeptidase and β -Lactamase by Streptomyces R39, Excretion of DD-Carboxypeptidase-Transpeptidase and β-Lactamase by Streptomyces R61, purification of the DD-Carboxypeptidase-Transpeptidase from Streptomyces R39 (for 500 Liters of Culture Fluid), Purification of the DD-Carboxypeptidase-Transpeptidase from Streptomyces R61 (for 400 Liters of Culture Fluid), Physicochemical Properties of DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61, Interaction between DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 and β-Lactam Antibiotics , Titration of DD-Carboxypeptidases-Transpeptidases from Streptornyces R39 and R61 by β-Lactam Antibiotics, Hydrolysis Reactions Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61, Concomitant Hydrolysis and Transfer Reactions Involving Distinct Donor and Acceptor Peptides, Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptonayces R39 and R61, Concomitant Hydrolysis and Transfer Reactions Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 and in Which the Same Peptide Acts as Donor and Acceptor and Inhibition of DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 by β-Lactam Antibiotics in the Presence of Substrates.

A stratigraphical framework for Miocene (MN4-MN13) continental sediments of Central Spain

New bio- and magnetostratigraphic data from the Miocene continental sediments of Central Spain are used to update the existing stratigraphical framework. Our revised record is based on the study of more than two hundred mammal faunas, ranging from the Late Ramblian (ca 18 Ma) to the Late Turolian (ca 6 Ma).

dd-Carboxypeptidase-Transpeptidase and Killing Site of β-Lactam Antibiotics in Streptomyces Strains R39, R61, and K11

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of β-lactam antibiotics. With strain R61, it was found that the exocellular enzyme has a sensitivity towards some antibiotics different from that of the membrane-bound enzyme. Under the growth conditions used in the present investigations, β-lactamase activity was not involved in susceptibility to β-lactam antibiotics.

Changes in Western European Rhinocerotidae related to climatic variations

Abstract The good record of Spanish Neogene Rhinocerotidae is used as the base of the study of their evolutionary patterns and their correlation with climatic changes. The sample has a temporal range from Lower Miocene to Lower Pliocene. From a taxonomical point of view, 15 species (and three indeterminate forms) of five main groups of Rhinocerotidae have been considered. General diversity and first and last records of the species have been signaled for each stage or biozone. Other features here analyzed are relative body size and weight and the gracility index. Rhinocerotid diversity in Spain varies through the Miocene, with a maximum of seven species in the Lower Aragonian falling down to one during the Middle Aragonian. Afterwards three or four species are present until the Upper Turolian and Alfambrian when a new minimal representation is reached. A general trend to size increase is established with an interesting change in the minimal values of each biozone at MN9. This change is supposed to represent the end of the small rhinos that were dominant before. Gracility indexes show a similar trend for all metapodials. There is a slight tendency towards robustness related to size increase, but some changes in gracility within the same sizes are also observed. Turnovers in rhinocerotid associations are here related to changes in environmental conditions. Small and gregarious species disappear in Upper Vallesian, and only large, solitary and more scarce forms remain later on.

The penicillin receptor in Streptomyces

Penicillin kills bacteria by suppressing or decreasing the efficiency of the membrane-bound transpeptidase, which during the last steps of the wall synthesis catalyzes cross-linking between the peptide units of the nascent peptidoglycan and makes the polymer insoluble.l, 2 In addition to this specific receptor, other penicillin-binding sites also occur within the bacterial membrane~.3-~ These sites, or at least most of them, seem to be irrelevant as far as peptidoglycan synthesis is concerned.4 Although involved in antibiotic specificity, they do not appear to be the killing target of penicillin. At present, no transpeptidase has been isolated from bacterial membranes and characterized. For a long time, the technical limitation to such an achievement has resided in the lack of a suitable assay for transpeptidase activity. Cell-free particulate multienzyme preparations obtained from various bacteria were shown to catalyze the in vitro utilization of the nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl pentapeptide for the entire sequence of peptidoglycan synthesis, which includes the peptide crosslinking.g-15 These assays, however, were devised in such a way that they did not allow measurement of the transpeptidation reaction per se. Another approach was undertaken through a joint effort between our laboratories and was based on the development of well-defined systems of peptide donors and acceptors that could be used by bacterial transpeptidases for transpeptidation reactions and, hence, would allow these enzymes to be operative and tested independently of the preceding biosynthetic sequential reactions. Streptornyces sp were chosen as a model, because they had the property, probably unique in the bacterial world, of spontaneously excreting an enzyme that appeared to be a soluble form of the membrane-bound transpeptidase.

Milk fat globule membranes. Inhibition by sucrose of the alkaline phosphomonoesterase

Sucrose, a widely used agent in the preparation of membranes, inhibited the alkaline phosphomonoesterase of the milk fat globule membrane in both its membrane-bound and detergent-solubilized forms. The inhibition was kinetically competitive and reversible by dialysis. However, its mechanism was more complex than simple competition with substrate because: (a) sucrose induced the appearance of prolonged time-lags in the progress curves of the enzyme; (b) the extent of inhibition and of the time-lags depended on the age of the membrane preparation, the period of pre-exposure of the membranes to sucrose, and the temperature of pre-exposure. On the other hand the acid phosphomonoesterase and the phosphodiesterase activities also present in the membrane preparations were unaffected by the disaccharide.

Historical Determinants of Mammal Diversity in Africa: Evolution of Mammalian Body Mass Distribution in Africa and South America During Neogene and Quarternary Times

Local mammalian communities in Africa present the highest species richness in the world, only paralleled by some communities in the Oriental biogeographic region. Differences in mammalian species richness are especially outstanding when compared with South American communities, despite their similar latitudinal position and regional species richness. Recent study has shown that these differences are not only related to contemporary determinants but also to biogeographic-historic factors, which acted on the composition of the regional pool of species. One of the main differences in composition between the two regions relates to the high diversification of large mammals in Africa, which greatly contributes to the high values of local community richness in this region. The absence of extant large mammals in the South American region has been proposed to result from Pleistocene-Holocene extinctions, which affected large mammals all over the world. However, a gradual pattern of decrease in the abundance of large mammal species can be appreciated in almost all regions except Africa since the late Miocene and through the Pliocene. To test these hypotheses we compare the patterns of macromammal body mass distribution — at regional and local scales — in the two regions over the past 20 million years and relate the observed changes to major geological events.

Evidence of a role for paf-acether in the pathophysiology of the shock state

The pathophysiology of the shock state includes a variety of hemodynamic changes such as systemic hypotension, pulmonary hypertension and increased vascular permeability leading to the extravasation of protein rich plasma. These changes can be initiated by different etiological factors, but many of them have been related to the stimulation of activation systems (complement, kinins, etc.) or to the generation of inflammatory mediators. The purpose of the present study has been to obtain evidence of the involvement of paf-acether in the pathogenesis of the shock state initiated in rat and mouse by Gram-negative bacteria and soluble aggregates of immunoglobulin G. The injection of 1-2 MDa aggregates of immunoglobulin G to normal Sprague-Dawley rats, induced a dose-dependent systemic hypotension which appeared about five minutes after completion of the intravenous challenge. Simultaneously, extravasation of protein-rich plasma occurred as judged from the finding of an increased clearance of 125I-BSA. In similar experiments in mice, a reduction of the vascular volume was observed using 51Cr-labelled homologous red blood cells. Under these conditions, a lipid compound analogous to paf-acether was obtained from the liver and the spleen of these animals. The generation of this compound preceded the development of blood volume depletion and could be suppressed by either quinacrine or depletion of mononuclear phagocytes by total irradiation with 700 rads. The previous treatment of the rats with the compound BN 52021 (a specific antagonist of the paf-acether receptor) at a dose of 5mg/kg, i.v., prevented the appearance of hypotension and extravasation in response to an i.v. challenge with soluble aggregates of immunoglobulin G. Interestingly, the reversal of hypotension was also observed when BN 52021 was infused after the immunoaggregates (5mg/kg). The possible involvement of paf-acether in the hemodynamic changes of Gram-negative sepsis was studied in rats which had received an intraperitoneal inoculation of E. coli. The animals inoculated with the doses of bacteria which produced mortality showed a time- and dose-dependent increase of vascular permeability as judged from the presence of abundant peritoneal exudate and the reduction of the circulating volume. Simultaneously, significant amounts of paf-acether could be obtained from the peritoneal exudate and from the spleen preceding to the development of the circulating volume depletion.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of PAF biosynthesis in human polymorphonuclear leukocytes. The role of phorbol esters and protein kinases.

The biosynthesis of PAF (1-0-hexadecyl/octadecyl2-acetyl-sn-glycero-3-phosphocholine) in human polymorphonuclear leukocytes (PMN) has been extensively studied in recent years. This has allowed the delineation of two important features of this process that are widely accepted. First, the generation of PAF only occurs after the activation of the cell by secretagogues in the presence of extracellular calcium, and, second, this generation may be modulated by interfering with the process of cell activation. A number of studies have emphasized the role of the enzyme 1-0-hexadecyl2lysos n glycero3phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) as the limiting step in the biosynthesis of PAF [1-4]. A logical consequence of the above-mentioned concept should be the correlation of the events which occur during cell activation with the stimulation of both acetyltransferase activity and PAF biosynthesis. This has been carried out in preparations other than PMN by studying the role of calcium ions and a phosphorylation-dephosphorylation mechanism in rat macrophages and spleen microsomes [5, 6]. PAF can also be synthesized from alkyl-acetyl-snglycerol and CDP-choline through DTT-insensitive cholinephosphotransferase (EC 2.7.8.16), but no evidence has been as yet provided as to the involvement of this pathway in PAF biosynthesis in PMN. Results and discussion

The significance of D-alanyl-D-alanine termini in the biosynthesis of bacterial cell walls and the action of penicillin, vancomycin and ristocetin

ABSTRACT D-Alanyl-D-alanine is a key structure in the biosynthesis of the peptidoglycans of bacterial cell walls. It is introduced as the last step in the assembly of the precursor nucleotide compound containing muramic acid and remains throughout the biosynthetic process until the terminal D-alanine residue is lost at the final transpeptidation reaction required to effect crosslinking. This transpeptidation reaction is a target of penicillin action, and soluble carboxy-peptidase-transpeptidases are inhibited by the antibiotic. The action of vancomycin and ristocetin is also tied up with the same D-alanyl-D-alanine terminus, but in a different way. These antibiotics contain an aglycone made up of phenolic amino acid residues in such a way that the resulting structure recognizes an acyl-D-alanyl-D-alanine terminus and combines with it with high affinity. By this mechanism bound vancomycin or ristocetin can inhibit reactions in the final stages of peptidoglycan synthesis. Correspondingly, in the presence of peptides that combine with vancomycin, the inhibition brought about by the antibiotic in either growing cells or in membrane preparations synthesizing peptidoglycan is reversed. At the same time some antibiotic remains bound to the preparations in such a way that it is no longer inhibitory. Studies with synthetic peptides have provided a rational basis for these observations.