Biomedical subjects

T Requena

Publications and source records attributed to T Requena.

17 recordsLinked to original sources

PubMed2006

Discrepancies between the phenotypic and genotypic characterization of Lactococcus lactis cheese isolates.

AIMS: The use of randomly amplified polymorphic DNA (RAPD)-PCR fingerprinting and plasmid profiles to determine at the strain level, the similarity of Lactococcus lactis isolates obtained during sampling of traditional cheeses and to verify its correspondence to the selected phenotypic characteristics. METHODS AND RESULTS: A total of 45 L. lactis isolates were genotypically analysed by RAPD-PCR fingerprinting and plasmid patterns. Phenotypic traits used to compare strains were proteolytic, acidifying, aminotransferase (aromatic and branched chain aminotransferase) and alpha-ketoisovalerate decarboxylase (Kivd) activities. The results show that 23 isolates could be grouped in clusters that exhibited 100% identity in both their RAPD and plasmid patterns, indicating the probable isolation of dominant strains during the cheese sampling process. However, there were phenotypic differences between isolates within the same cluster that included the loss of relevant technological properties such as proteinase activity and acidifying capacity or high variation in their amino acid converting enzyme activities. Likewise, the analysis of a specific attribute, Kivd activity, indicated that 7 of 15 isolates showed no detectable activity despite the presence of the encoding (kivd) gene. CONCLUSION: Phenotypic differences found between genotypically similar strains of L. lactis strains could be linked to differences in enzymatic expression. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic analysis of L. lactis isolates should be considered when selecting strains with new cheese flavour forming capabilities.

Amino Acid Sequence↗

PubMed2005

Enzymatic ability of Bifidobacterium animalis subsp. lactis to hydrolyze milk proteins: identification and characterization of endopeptidase O.

The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide alpha s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.

Bacterial Proteins↗

PubMed2004

[Evaluation of a pharmaceutical care program to improve adherence to antiretroviral therapy].

OBJECTIVE: To establish the impact of a pharmaceutical care program on the improvement of adherence to antiretroviral therapy, and on patient immunologic and virologic outcome. MATERIALS AND METHODS: A multicenter, observational, prospective study in a HIV-infected patient cohort under treatment with antiretrovirals selected by random sampling in 19 Spanish hospitals. The study lasted 12 months, in which the program was applied through a baseline preprocedural visit and 4 quarterly visits. Adherence estimation was based on pill counting. An adherence > or = 90, or > or = 95% was considered adequate (in two time points). RESULTS: 541 patients were included, most of them were males (68.8%) between 20 and 78 years of age. Major risk groups included injecting drug users (43.4%) and heterosexuals (29.4%). Sixty percent had already received treatment for more than 3 years. Mean baseline viral load and CD4 count values were 32,866 copies/ml and 485 cells/mm3, respectively. Throughout the study a slight increase in the percentage of adherent patients was seen; however, statistical significance was not reached (64.3 and 79.2% of patients showed an adherence > 95 and > 90%, respectively, during the fourth quarter, versus 59.8 and 75.5% at baseline). A statistically significant decrease in viral load and increase in CD4 cells was seen following program application. The percentage of patients with a viral load < 200 copies/ml was 72.2, 76.7, and 75.0% at the 2nd, 3rd, and 4th quarters, respectively, versus 64.2% at baseline. CD4 cell counts increased by 50 cells/mm3 on average from the start to the end of follow-up. CONCLUSIONS: Patients included in the program had a good immunologic and virologic outcome, and a trend towards an increased percentage of patients with good adherence was also seen. These results confirm the need to implement follow-up programs for patients receiving antiretrovirals in order to ensure maximum therapeutic benefits.

Adult↗

PubMed2001

Use of a bacteriocin-producing transconjugant as starter in acceleration of cheese ripening.

The non-conjugative 46 kb plasmid that encodes the biosynthesis of lacticin 3147 in Lactococcus lactis IFPL105 has been transferred to the starter L. lactis IFPL359, used in goat's milk cheesemaking. The accelerating effect exerted on proteolysis and development of sensory characteristics of semi-hard cheese by the bacteriocin-producing transconjugant L. lactis IFPL3593 (Lac+ Bac+ Imm+), which is able to induce cell lysis in starter adjuncts with high peptidase activity, has been studied. It has been demonstrated that the use of IFPL3593 as starter accelerates cheese ripening as it increases the level of amino nitrogen correlated with early cell lysis of adjuncts. The fact that the bacteriocin-producing microorganism used is immune to the bacteriocin. allowed proper acidification of the curd without altering the cheese-making process.

Animals↗

PubMed2001

Conversion of methionine to methional by Lactococcus lactis.

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.

Aldehydes↗

PubMed2001

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination.

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.

Carbon-Sulfur Lyases↗

PubMed2000

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105.

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

Amino Acid Sequence↗

PubMed2000

Requirement of autolytic activity for bacteriocin-induced lysis.

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.

Bacteriocins↗

PubMed2000

[Clinical ethics and nutritional support].

Artificial nutrition is a modern technique enjoying a great boom of late. Regulations on the use of important clinical and technological advances in connection with nutritional support in certain groups of patients represent one of the ethical problems most often posed in daily medical practice. Terminal patient and those with irreversible neurological damage are population groups at risk of these ethical problems when it comes to the prescription, withdrawal or omission of artificial nourishment and hydration. The establishment of certain guidelines in order to resolve the ethical problems arising seems to be a complex process. So far, no consensus has been reached among doctors, with no clear legal framework and no universally accepted ethical principles. It is necessary to ensure the consideration of all the circumstances and parties coming into conflict in order to find the best solution for all concerned by means of shared decision-making.

Ethics, Medical↗

PubMed1999

Effect of different membrane separation technologies (Ultrafiltration and microfiltration) on the texture and microstructure of semihard low-fat cheeses.

Semihard low-fat cheeses made from ultrafiltered (UF) or microfiltered (MF) milk were compared. The use of MF membranes and milder pasteurization of the milk reduced the retention of whey proteins in the retentate to 35%, compared with approximately 100% retained in the UF process. Microbiological development, physicochemical composition, and cheese ripening were not altered by the concentration processes. The lower retention of whey protein in MF cheeses accounted for their higher hardness, which correlated with higher firmness values in the textural analysis. Microstructure showed a protein matrix with open spaces through the protein network, although micrographs of UF cheeses showed the presence of spongy structures linked to the casein, which did not appear in MF cheeses and which correspond to the denatured whey protein bound to the casein. Firmness was scored better in MF cheeses, although when MF membranes were used, the optimum yields achieved using UF membranes were not attained.

Cheese↗

PubMed1999

Treatment of malignant melanoma.

OBJECTIVE: To review the current treatments for cutaneous melanoma and discuss treatment approaches for each patient population. DATA SOURCES: MEDLINE and IOWA database search from January 1990 to December 1998. DATA EXTRACTION: Clinical trials and review articles were selected and classified to answer questions considered of clinical relevance. RESULTS: Patients with stage I, II, and III melanoma should undergo excision after biopsy. In patients with stage IV melanoma, surgical excision of metastatic melanoma is not considered curative but can provide palliation and improve quality of life. Therapeutic lymph node dissection should be performed in patients with melanoma in stages III and IV once pathologic confirmation is obtained. Patients at high risk for recurrence or metastasis may also be considered for elective node dissection. Adjuvant therapy after surgery excision is not a standard of care in patients with stage I and IIa melanoma. In patients with stage IIb and III melanoma, the best results have been obtained with high doses of interferon alfa-2b, although toxicity is of concern. Isolated limb perfusion with melphalan adjuvant to surgery has demonstrated clinically significant benefit in patients with locally recurrent melanoma and in-transit metastases. Studies comparing efficacy and quality of life with this technique or with high doses of interferon alfa-2b are needed. The technique cannot be recommended for high-risk primary melanoma of an extremity with no clinical evidence of metastatic disease. CONCLUSIONS: To date, dacarbazine still appears to be the treatment of first choice in metastatic melanoma, outside of a clinical trial. The combination of chemotherapy with interferon alfa-2b or interferon alfa-2a enhances toxicity without a significant survival advantage. Aldesleukin may be an alternative in selected patients when other treatments have failed, but the higher toxicity and cost must be considered.

Animals↗

PubMed1998

Salt balance and rennet clotting properties of cow's, ewe's, and goat's milks preserved with carbon dioxide.

Cow's, ewe's, and goat's milk samples were treated with carbon dioxide gas until a pH of 6.1 was reached and stored at 4 degrees C to determine the resulting modifications in the mineral balance. The amounts of calcium and phosphorus dissolved during the acidification were similar in the three species. The acidification with CO2 produced the dissolution of phosphorus and magnesium in concentrations similar to those attained by acidification with lactic acid or hydrochloric acid. Still, the contents of soluble calcium and ionic calcium were higher with the CO2 treatment. The increase of ionic calcium due to the addition of CO2 could explain why milk subjected to such treatment is better suited for coagulation. Removal of added CO2 by shaking the milk for several hours at atmospheric pressure resulted in a higher concentration of ionic-calcium than was found in control milks to which no CO2 had been added. Thus the addition of CO2 improved milk's technological suitability for cheesemaking.

Animals↗

PubMed1997

Autolysis of Lactococcus lactis ssp. lactis and Lactobacillus casei ssp. casei. Cell lysis induced by a crude bacteriocin.

Autolytic properties of Lactococcus lactis subsp. lactis IFPL359, its Lac Prt derivative Lc. lactis Tl and Lactobacillus casei subsp. casei IFPL731, used as starter and adjunct starter in goat's milk cheese making, have been studied. The lytic effect of a bacteriocin produced by a lactic acid bacterium isolated from raw goat's milk has also been analyzed. Lactococcal cells resuspended in phosphate buffer showed a peak of autolysis when they were harvested in the early growth phase. A more stable autolytic pattern through the exponential growth was obtained for Lb. casei IFPL731. Optimal autolysis was found in 0.1 M sodium phosphate buffer during incubation at 40 degrees C for Lb. casei IFPL731 and at 35 degrees C for the lactococci. Thermoinduction of cell lysis was not obtained in any of the cases under the conditions studied. Lytic effect of the crude bacteriocin assayed was strongest against Lc. lactis Tl. Lysis response to the bacteriocin seemed to be strain-dependent and related to growth conditions.

Bacteriocins↗

PubMed1996

Antimicrobial activity of lactic acid bacteria isolated from goat's milk and artisanal cheeses: characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL 105.

A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis, one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by alpha-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.

Ammonium Sulfate↗

PubMed1996

Proteolytic activity of lactobacilli in a model goats' milk curd system.

Mesophyllic lactobacilli cultures propagated in MRS broth were inoculated in goats' milk curd slurries and incubated at 30 degrees C for 10 d. The micro-organisms tested were Lactobacillus casei subsp. casei IFPL 731 and IFPL 99, and Lactobacillus plantarum IFPL 3. Whole cells, cell-free extracts and cell lysates were evaluated for acceleration of proteolysis in the curd slurries. Conversion of water-soluble nitrogen to non-protein nitrogen and amino acid nitrogen, reverse phase-HPLC peak areas and ratio of hydrophobic to hydrophilic peptides, were all affected by the type of inoculum used as well as the strain under study. The results suggest that the accelerated-ripening model system developed, containing cell lysates, may be suitable as a good and rapid indicator of the contribution of the strains to proteolysis during cheese ripening.

Amino Acids↗

PubMed1995

Lactococcin A overexpression in a Lactococcus lactis subsp. lactis transformant containing a Tn5 insertion in the lcnD gene.

Lactococcin A production in lactococci has recently been linked to a signal-sequence-independent secretory system consisting of a four-gene cluster. Lactococcus lactis subsp. lactis LLM23L-A1 has been obtained after Tn5 mutagenesis of pLLM23, a plasmid containing the gene cluster responsible for lactococcin A production. In contrast to other Tn5-generated mutants, strain LLM23L-Al exhibited a 12-fold increase in lactococcin A production. Overproduction of lactococcin A was not linked to an increased pLLM23 copy number. Restriction-enzyme analysis indicated the site of Tn5 insertion to be at the 3' end of lcnD, and upstream of the lcnA structural gene. From DNA sequencing, the Tn5 insertion was located -79 bp upstream of the transcription start site of the lcnA and lciA genes, eliminating eight amino acids from the C-terminal end of lactococcin D. Northern blots revealed overproduction of a 500-base transcript in strain LLM23L-A1, which corresponded to that predicted from the positions of the lactococcin A operon transcriptional start site and the termination structures. This result suggests that the overproduction of lactococcin A in strain LLM23L-A1 is at the transcriptional level and provides further impetus for elucidating the complete regulatory mechanism for lactococcin A expression.

Bacterial Proteins↗

PubMed1995

Cloning and characterization of the abortive infection genetic determinant abiD isolated from pBF61 of Lactococcus lactis subsp. lactis KR5.

A 6.3-kb fragment from pBF61 in Lactococcus lactis subsp. lactis KR5 was cloned and found to confer an abortive phage infection (Abi+) phenotype exhibiting a reduction in efficiency of plating and plaque size for small isometric- and prolate-headed bacteriophages sk1 and c2, respectively, and to produce a 10-fold decrease in c2 phage burst size. Phage adsorption was not significantly reduced. An open reading frame of 1,098 bp was sequenced and designated abiD. Tn5 mutagenesis confirmed that abiD was required for the Abi+ phenotype.

Amino Acid Sequence↗