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J Velasco

Publications and source records attributed to J Velasco.

At least 37 records · Page 2Linked to original sources
PubMed2000

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans.

Two protein bands with strong esterase activity are present in broths of Nocardia lactamidurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed Km values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C.

Amino Acid Sequence↗
PubMed2000

The gene encoding gamma-actin from the cephalosporin producer Acremonium chrysogenum.

The nucleotide sequence of a 3240-bp genomic fragment including the gamma-actin-encoding gene from Acremonium chrysogenum has been determined, showing an open reading frame of 1691 bp, interrupted by five introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, pyrimidine stretches and the polyadenylation sequence AATAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (> 90%). Gene phylogenies constructed using DNA and protein sequences support the grouping of A. chrysogenum actin close to those from the majority of the filamentous fungi. The actA gene is present as a single copy in the genome of A. chrysogenum; and its expression level, opposite to pcbC and cefEF cephalosporin biosynthetic genes, was steady during cephalosporin fermentation, showing a single 1.4-kb transcript.

Acremonium↗
PubMed2000

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum.

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.

Acremonium↗
PubMed2000

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance.

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity.

Alphaproteobacteria↗
PubMed1999

Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits.

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative alpha- and beta-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

Acetyltransferases↗
PubMed1999

Postneoadjuvant hormone PSA levels and prognosis in locally advanced prostate cancer.

OBJECTIVES: To determine whether the response to hormonal therapy before radiation predicts the rate of biochemical relapse in patients with locally advanced prostate cancer. METHODS: Between October 1991 and December 1997, 105 patients with locally advanced adenocarcinoma of the prostate received radiotherapy in two dose-escalation studies. Sixty-seven patients received neoadjuvant hormonal therapy. The mean and median duration of hormonal therapy before radiotherapy was 4 months each. All treatments were designed using three-dimensional conformal therapy. The total dose to the gross tumor volume ranged from 73 to 87 Gy in 2 Gy per fraction photon equivalent dose. The median follow-up time was 30 months (range 1 to 66). RESULTS: The median prostate-specific antigen (PSA) nadir after neoadjuvant hormonal therapy but before radiotherapy was 1.7 ng/mL (range less than 0.05 to 71.2). The median nadir after radiation for patients who did and did not receive neoadjuvant androgen deprivation was 0.25 ng/mL (range less than 0.05 to 6.2) and 1.35 ng/mL (range 0.08 to 10), respectively. Median time to achieve nadir was 6 months (range 1 to 42) with and 12 months (range 1 to 48) without hormonal therapy. There was no significant difference in the rate of biochemical failure for patients with a posthormone (before irradiation) PSA nadir less than 1 ng/mL versus 1 ng/mL or greater (overall P = 0.9). However, there was a significant difference in biochemical no evidence of disease rates between those with a PSA nadir less than 1 ng/mL and those with a PSA nadir of 1 ng/mL or greater after radiation (63% versus 22% at 3 years, overall P <0.001). CONCLUSIONS: Our data showed that the initial response to hormonal therapy before radiation, as indicated by the PSA level, did not impact on the rate of recurrence. However, the time to reach nadir and the absolute nadir level achieved were lower in patients who did receive hormonal therapy.

Actuarial Analysis↗
PubMed1999

PHA/IL2: an efficient mitogen cocktail for cytogenetic studies of non-Hodgkin lymphoma and chronic lymphocytic leukemia.

This study documents the utility of a mitogen/cytokine cocktail composed of phytohemagglutinin and Interleukin 2 (PHA/IL2) used to stimulate cultures from patients with chronic lymphoproliferative disorders. We report the results of a selected series of 57 patients with non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL), in which only the culture stimulated with PHA/IL2 demonstrated the presence of an abnormal clone. On average, cells in the abnormal clone comprised 40% of the mitotic cells in this culture. The most common abnormalities observed in these patients were trisomy 12, present in 39% of the cases, and t(14;18), seen in 14% of cases.

Adult↗
PubMed1999

A group of previously not recognized cytogenetic abnormalities in myeloid hematological malignancies.

We have identified a group of previously not reported chromosome abnormalities related to myeloid hematological malignancies. Cases 1 and 2 were observed to have an additional i(4)(p10) as the sole anomaly with similar clinical features of myeloid disorders; that is, acute nonlymphocytic leukemia (ANLL-M2) and myelodysplastic syndrome (MDS)-refractory anemia with an excess of blasts in transformation, respectively. Fluorescence in situ hybridization studies with the use of a 4p-specific microdissection probe further confirmed the presence of an i(4)(p10) in these patients. Case 3 was diagnosed with ANLL-M1 and had an additional i(8)(p10) as the only change, also confirmed by a whole-chromosome painting procedure. In cases 4-6, deletions of 18q at breakpoints q12, q23, and q21 were identified as the sole anomaly in a myeloproliferative disorder (MPD), MPD, and MDS, respectively. X-autosome translocations other than t(X;10)(p11;p11) and t(X;11)(q13;q23) have not been reported as recurrent or primary changes in hematological disorders. In the present study, a t(X;9)(q26;q22) and t(X;5)(q13;q33) as the sole anomaly were found in cases 7 and 8, respectively. Both cases had the same diagnosis of MDS. Considering that trisomies 4 (+4) and 8 (+8) are common anomalies in MDS and ANLL, our findings strongly indicate that amplification of genes on 4p and 8p, but not on 4q and 8q, may play a crucial role in the pathogenesis of MDS and ANLL. In addition, genes on 18q12-23 and on Xq13-26 may be involved in the pathogenesis of myeloid disorders.

Adult↗
PubMed1999

Accelerated extraction of the fat content in cheese using a focused microwave-assisted soxhlet device.

A fast method for hydrolysis and extraction of fat in cheese based on the use of a Soxhlet extractor assisted by microwaves is proposed. The use of this type of energy dramatically reduces the time required for both steps (namely, the hydrolysis time was decreased from 1 h to 10 min with avoidance of the neutralization step, and the extraction time was decreased from 6 h to 40 min), providing similar efficiency and reproducibility comparable to or better than that of the conventional Soxhlet methodology. In addition, the proposed method is cleaner than conventional Soxhlet, as 80-85% of the extractant is recycled. The determination of the fatty acid composition by gas-liquid chromatography and quantification of both polymer formation by high-performance size exclusion liquid chromatography and nonpolar triacylglycerol formation by thin-layer chromatography with flame ionization detection in the extracts obtained by the proposed method, conventional Soxhlet and Weibull-Berntrop procedures (Soxhlet with a prior hydrolysis step), showed that the quality of the extracts obtained by the proposed procedure is better than that of the conventional alternatives, possibly due to the shorter time required by the accelerated method.

Cheese↗
PubMed1999

Stenting in a patient with a single coronary artery and myocardial infarction.

The single coronary artery is an uncommon congenital defect. We report on a patient admitted to our hospital with an acute myocardial infarction and a single coronary artery arising from the left sinus of Valsalva who was treated with coronary angioplasty and stent implantation. We have not found an identical report of stenting in such an anomaly in the medical literature.

Adult↗
PubMed1998

Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase.

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

Aldehyde Oxidoreductases↗
PubMed1998

Structural studies on the lipopolysaccharide from a rough strain of Ochrobactrum anthropi containing a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide lipid A backbone.

A degradation protocol using de-O-acylation and subsequent alkaline de-N-acylation was applied to the lipopolysaccharide of Ochrobactrum anthropi rough strain LMG 3301. Three main oligosaccharide bisphosphates containing core-lipid A backbone structures were obtained after fractionation by anion-exchange HPLC. Using 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, and NOE spectroscopy (ROESY and NOESY), the following structures were established: [formula: see text] where Kdo is 3-deoxy-D-manno-octulosonic acid, D-GlcN3N is 2,3-diamino-2,3-dideoxy-D- glucose and R is H or alpha-D-GalpA or 4-deoxy-beta-L-threo-hex-4-enopyranuronic acid, the latter sugar being derived from alpha-D-GalpA by beta-elimination of a substituent attached to 0-4. This is the first report on the isolation from a lipopolysaccharide of an oligosaccharide containing GlcN3N in the lipid A backbone [beta-D-GlcpN3N4P-(1-->6)-alpha-D-GlcpN3N1 P]. Sugar and methylation analysis confirmed the presence of the GalA-->Kdo disaccharide and non-stoichiometric substitution of GalA. It is suggested that Glc is the substituent at 0-4 in GalA and that in the non-degraded lipopolysaccharide the amino group of GlcN is not acylated.

Carbohydrate Sequence↗
PubMed1998

Impact of postprostatectomy prostate-specific antigen nadir on outcomes following salvage radiotherapy.

OBJECTIVES: To evaluate the relationship between the postprostatectomy prostate-specific antigen (PSA) nadir and the outcome of patients treated with salvage radiotherapy. METHODS: Seventy-eight patients received definitive external beam radiation for recurrence following radical prostatectomy (RP). The PSA nadir was undetectable in 41 patients (less than 0.05 ng/mL). All patients received salvage radiotherapy (median dose 66 Gy) for a median of 19 months (range 2 to 149) following prostatectomy. The median follow-up time was 25 months (range 1 to 59) from the date of completion of radiation. RESULTS: Among patients having an undetectable or detectable postoperative PSA, 78% and 68% were free of disease, respectively, at the last follow-up. At 3 years, the disease-free survival rates were 65% and 60%, respectively (P = 0.6). Overall, the disease-free survival rate at 3 years was 78% in patients with a PSA level 2 ng/mL or less at the time of radiotherapy compared to 31% with a PSA greater than 2 ng/mL (P < 0.0001). CONCLUSIONS: Many patients who never achieve an undetectable postprostatectomy PSA level may still be salvaged with therapeutic radiotherapy. The best predictor of a favorable outcome is a low (2 ng/mL or less) PSA level at the time of radiation.

Disease-Free Survival↗
PubMed1998

Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp.

The relatedness of Brucella spp. and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and O. anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that O. anthropi LMG 3301 was closer to Brucella spp. than to O. anthropi LMG 3331T, a result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331T was in a separate cluster. The LMG 3301 and the LMG 3331T clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O. anthropi by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406-416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp. nov. is proposed (type strain is LMG 3301T=NCTC 12171T = CNS 2-75T).

Animals↗
PubMed1998

Therapeutic radiation in patients with a rising post-prostatectomy PSA level.

The optimal management of patients with an elevated post-prostatectomy prostate-specific antigen (PSA) level remains to be determined. In the pre-PSA era, many patients received immediate adjuvant radiation therapy on the basis of adverse pathologic features following prostatectomy. However, since the advent of postoperative PSA monitoring, the PSA level has become the most specific measure for determining the presence of residual disease. An elevated postoperative PSA in correlation with other factors, such as pathologic stage and imaging studies, can be a specific marker of local recurrence in most patients. Through careful case selection, treatment design, and dose delivery, up to 80% of patients can achieve a complete response (PSA < 0.1 ng/mL) following postoperative therapeutic irradiation for an elevated PSA. This treatment can be delivered with minimal toxicity to the rectum and minimal risk of increasing incontinence. Pending the results of randomized trials, an appropriate therapeutic option for high-risk patients following prostatectomy would be to wait for an elevated PSA and then proceed with a course of potentially curative radiotherapy.

Disease-Free Survival↗
PubMed1997

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-beta-1,4-xylanase of Thermomonospora alba ULJB1 with cellulose-binding ability.

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those Thermomonospora fusca. Genes encoding two different endo-beta-1,4-xylanase were cloned from T. alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48,456 Da. The protein contains a 38-amino-acid leader peptide with six Arg+ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/XylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 degrees C, and had broad pH stability in the range from pH 4.0 to pH 10.0.

Actinomycetales↗