Biomedical subjects

E García

Publications and source records attributed to E García.

At least 217 records · Page 12Linked to original sources

PubMed1988

Modulation of macrophage lysosomal pH by Mycobacterium tuberculosis-derived proteins.

Macrophage lysosomal pH was significantly (greater than 1 pH unit) increased in a reversible, concentration-dependent manner characterized by a saturable and cyclic kinetics after exposure to culture filtrate protein extract derived from Mycobacterium tuberculosis. Lysosomal alkalinization peaked 30 min after administration of culture filtrate protein extract to cells of the macrophagelike cell line J774A.1. The alkalinization was reversible, and a second peak was observed approximately 60 min after incubation. Maximum lysosomal alkalinization increased as a function of culture filtrate protein extract concentration, reaching an apparent saturation level around 700 to 1,000 micrograms/ml, although the time course for this process was not significantly dependent on antigen concentration. The alkalinizing agent(s) was heat labile and produced a similar effect in cells which had a different lysosomal enzyme composition. Our observations are consistent with the presence of one or more mycobacterial antigens which have a pH-dependent affinity for lysosomal structures essential for lysosomal acidification and which are able to inhibit this lysosomal acidification.

Animals↗

PubMed1987

Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae.

A pneumococcal recombinant plasmid, pRG2, containing the lytA gene that codes for the pneumococcal N-acetylmuramoyl-L-alanine amidase has been constructed using the pneumococcal plasmid pLS1 as a vector. pRG2 was introduced by genetic transformation into a mutant of Streptococcus pneumoniae (M31) that has a complete deletion of the lytA gene. The transformed strain (M51) grew at a normal growth rate as 'diplo' cells and underwent autolysis at the end of the exponential phase of growth, two properties that had been lost in the deleted mutant M31. M51 lysed very rapidly at the end of the exponential phase when the cells were grown in choline-containing medium probably because of the higher level of amidase activity present in this strain as compared to the lysis-prone strain M11. These findings show that the expression of the plasmid-linked gene was placed under the mechanism(s) of control of the cell during the exponential phase. Our results demonstrate that the physiological role of the pneumococcal amidase was to catalyze the separation of the daughter cells at the end of the cell division to produce diplo cells; in addition we have also confirmed the basic role of this autolysin in the bacteriolytic nature of beta-lactam antibiotics.

Amidohydrolases↗

PubMed1987

Overproduction and rapid purification of the amidase of Streptococcus pneumoniae.

Oligonucleotide-directed mutagenesis of a plasmid containing the lytA gene coding for the pneumococcal amidase has allowed the separation of the coding sequence of the gene. This sequence has been placed in plasmid pIN-III(lppP-5)-A3 downstream from both a modified lipoprotein promoter and the lactose promoter to construct the recombinant plasmid pGL100. When Escherichia coli RB 791 (pGL100) was grown in the presence of lactose, the pneumococcal amidase accounted for 7% of the total protein present in this strain after 18 h incubation at 37 degrees C. The overproduced amidase was purified in a single-step procedure using a choline-Sepharose 6B column taking advantage of the fact that this enzyme was the unique protein with affinity for choline present in extracts obtained from E. coli RB791 (pGL100). The development of the above design opens up the possibility of studying the mechanism that regulates the activity of this important autolysin by using physiochemical techniques that require the availability of high amounts of purified amidase.

Amidohydrolases↗

PubMed1987

3'-end modifications of the Streptococcus pneumoniae lytA gene: role of the carboxy terminus of the pneumococcal autolysin in the process of enzymatic activation (conversion).

Plasmids containing modifications at the 3' end of the lytA gene encoding the pneumococcal amidase were constructed by DNA recombinant techniques. Several deleted and fused amidases were obtained. These modified amidases were capable of degrading cell walls containing choline residues in their teichoic acid components without need of conversion (i.e., change of the inactive E form of amidase to the active C form). The reintroduction of as few as the terminal 11 amino acid (aa) residues present in the wild-type (wt) amidase into the sequence of the most extensively deleted form of the autolysin obtained in this work (E-520) partially restored the need of conversion. Our results demonstrate the importance of the C terminus for the catalytic activation of the wt amidase.

Amidohydrolases↗

PubMed1987

Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin.

Cp-1, a small virulent bacteriophage infecting Streptococcus pneumoniae, encodes its own lytic enzyme (CPL). A fragment of Cp-1 DNA containing the gene cpl coding for CPL was cloned and expressed in high amounts in Escherichia coli. CPL was purified to electrophoretic homogeneity by using affinity chromatography on choline-Sepharose (T. Briese and R. Hakenbeck, Eur. J. Biochem. 146:417-427, 1985), and the enzyme showing a Mr of 39,000 was characterized as a muramidase. This muramidase required for in vivo and in vitro activity the presence of choline in the teichoic acids of the pneumococcal cell walls. Free choline or lipoteichoic acid noncompetitively inhibited the activity of CPL.

Bacteriophages↗

PubMed1987

Influence of the qualitative caloric composition of enteral diets on liver protein synthesis in normal and operated rats.

Protein synthesis L-(l-14C) leucine in liver was studied. Normal (n = 40) and operated (n = 40) Sprague-Dawley rats with an initial body weight of 161.18 +/- 12 g were divided into four groups (n = 10), respectively. Each group, subjected to the same conditions of temperature, light, noise and movement received a different commercial formula for human enteral nutrition and water for 8 days (normal rats) and six days (operated rats). All rats received the same caloric and protein intake per ml (l kcal; 6-7.10(-3) g protein), with no statistically significant differences among groups. The differences in diets were merely qualitative. The tolerance to all diets was similar, with rats attaining the expected weight. Losses of protein, alpha aminic nitrogen, total fat and non sterified fatty acid in faeces were negligible. In normal rats, different results in liver protein synthesis were obtained. No mass/synthesis rate correlation was found. Under these conditions it would appear that these results are due to the different diets used. In operated rats, a significantly different liver protein synthesis per gram of body weight was obtained, but not per gram of organ weight. These results suggest that the influence of the qualitative caloric intake, in the stress phase, is less than in the normal state.

Animals↗

PubMed1986

Molecular characterization of an autolysin-defective mutant of Streptococcus pneumoniae.

The mutant gene lyt-4 of the autolysin-defective mutant R6ly4-4 of Streptococcus pneumoniae, which synthesized a temperature-sensitive autolytic enzyme, has been cloned in Escherichia coli. The nucleotide defect of the lyt-4 mutation has been characterized as a CG to TA transition. This transition causes the appearance of a glutamic acid instead of a glycine in the amino acid sequence of the autolysin, altering the hydropathic profile of the protein. This alteration might explain the observed thermosensitivity of the mutated autolytic enzyme. The present work represents the first molecular characterization of a mutation in the structural gene of a bacterial autolysin.

Amidohydrolases↗

PubMed1986

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae.

A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.

Base Sequence↗

PubMed1986

Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli.

Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [García et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [Höltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.

Amidohydrolases↗

PubMed1986

Dendritic development in the neocortex of adult rats following a maintained prenatal and/or early postnatal life undernutrition.

The Golgi-Cox method was used to study the maturation of the large pyramidal cells of the Vth cortical layer in three groups of adult rats: one subjected to undernutrition during the first month of life, another throughout the first 2 mth of life, and the last one during gestation and the suckling period. The main alterations consist of a decrease in the number and span of dendritic basilar processes of large pyramidal cells. In animals malnourished during prenatal life and the suckling period the reduction of the basal dendritic arborization was more apparent. It is postulated that the vulnerable period for the basal dendritic development occupies the period from the end of pregnancy until the first 3 wk of postnatal life in the rat (suckling period). Noxious influences acting during this phase induce sequelae that cannot be reversed by subsequent refeeding. A maintained nutritional insult during prenatal and early postnatal life induces the most severe changes in dendritic arborizations, compared to those resulting from a prolonged postnatal malnutrition.

Animals↗

PubMed1986

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin.

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.

Amidohydrolases↗

PubMed1986

Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP.

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.

Bacteriophages↗

PubMed1986

Studies on the replication of bacteriophage Cp-1 DNA in Streptococcus pneumoniae.

The DNA of bacteriophage Cp-1 replicates at optimal conditions when Cp-1-infected Streptococcus pneumoniae was incubated at 30 degrees C. The in vitro formation of the initiation complex between the terminal protein and 5'-dAMP was only partially inhibited at 37 degrees C whereas an almost complete inhibition of the DNA replication was found at this temperature in vivo. Aphidicolin inhibited the multiplication of phage Cp-1 but not that of Dp-4. This drug did not affect the in vitro formation of the initiation complex but seems to affect extensive Cp-1 DNA replication in vivo.

Aphidicolin↗

PubMed1985

Cloning and expression of the pneumococcal autolysin gene in Escherichia coli.

A 7.5 kb BclI-fragment of Streptococcus pneumoniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the inactive form (E-form) of the pneumococcal N-acetyl-muramyl-L-alanyl amidase (EC 3.5.1.28). Our results demonstrate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be converted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme production in the homologous system. The production of this protein in E. coli HB101 was 500-fold higher than in the homologous host. E. coli CSR603 containing pGL30 and labeled with [35S]methionine synthesized a 35 kd protein. pGL30 can transform at high frequency an autolysin-defective mutant of S. pneumoniae to the lyt+ phenotype.

Amidohydrolases↗

PubMed1985

Inverted terminal repeats and terminal proteins of the genomes of pneumococcal phages.

The nucleotide (nt) sequence at the ends of the genomes of the Streptococcus pneumoniae phages Cp-5 and Cp-7 has been determined and compared with the corresponding sequence of phage Cp-1. The genomes of phages Cp-5 and Cp-7 have inverted terminal repeats (ITRs) 343 and 347 bp long, respectively. In Cp-1 DNA the ITR is 236 bp long and the following 116 bp are 93% homologous. Some regions within the ITRs are conserved in the three genomes although the complete sequence of the ITRs is no more conserved than the rest of their genomes. The chromatographic behavior of their tryptic peptides suggests that the terminal proteins (TPs) of at least two of the phages are similar and that the TPs of the three pneumococcal phages differ markedly from that of the Bacillus subtilis phage psi 29.

Bacteriophages↗

PubMed1985

Dendritic development in the neocortex of adult rats subjected to postnatal malnutrition.

The Golgi-Cox method was used to study the maturation of the large pyramidal cells of the Vth cortical layer in two groups of adult rats, one subjected to early postnatal malnutrition and another malnourished only during the second month of life. The main alterations were observed in the pyramidal cells of cortical layer V of rats malnourished during the first month of life. They consist of a decrease in the number and span of dendritic basilar processes. In animals malnourished during the second month of life, the number and span of basilar dendritic processes in pyramidal cells of layer V, were normal. It is postulated that early postnatal malnutrition induced immediately after birth, profoundly disturbs the process of neuronal maturation in the neocortex of the rat brain, probably with permanent effects.

Age Factors↗

PubMed1985

[Phenotypical curing of Streptococcus pneumoniae treated with amidase induced by the Dp-1 bacteriophage].

A phage-associated murein hydrolase activity (PAL) induced in an autolysis-defective mutant of Streptococcus pneumoniae infected with the bacteriophage Dp-1 has been recently isolated and purified to electrophoretic homogeneity as well as biochemically characterized as an endo-N-acetyl-muramyl-L-alanine amidase (1, 3, 4). The PAL and the inactive form (E-form) of the host cell autolysin show a remarkable biochemical similarity, although they differ in their immunological characteristics. The PAL was adsorbed onto a live, defective mutant of pneumococcus (cwl) and such cells reverted to the wild type phenotype ("cured" cells) in some important characteristics present in the wild type strain (R6), as: i) lysis of the culture in the stationary phase, ii) protoplast formation by hypertonic sucrose, and iii) bacteriolytic response against the penicillin in contrast with the bacteriostatic response of the "non-cured" cwl. The adsorbed enzyme segregates during growth of the "cured" cells. Our results demonstrate that PAL acts in the phenotypically "cured" cells in a similar way to that previously described for the host enzyme, and also confirm the finding that the autolysins play a direct role in the irreversible effects produced in S. pneumoniae by the betalactamic antibiotics.

Amidohydrolases↗

PubMed1985

[Schönlein-Henoch syndrome: review of a series of 142 cases].

Clinical and laboratory findings in 142 children with Schönlein-Henoch syndrome are studied. The children were diagnosed during a ten-year period (1974-1984). Purpura, as more significant clinical finding, was observed in all patients, followed by articular signs (61.2%), abdominal complaints (57.7%), renal signs (30.2%) and testicular (4.2%). IgA, IgG, IgM, complement and cryoglobulin alterations, are remarkable factors to assume an immunologic origin for the syndrome.

Child↗