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Biomedical subjects

G Bestetti

Publications and source records attributed to G Bestetti.

At least 37 records · Page 2Linked to original sources
PubMed1997

Production of substituted naphthalene dihydrodiols by engineered Escherichia coli containing the cloned naphthalene 1,2-dioxygenase gene from Pseudomonas fluorescens N3.

Naphthalene dioxygenase, a key enzyme in the dihydroxylation of naphthalene, is encoded by the plasmid pN3, responsible for naphthalene metabolism in Pseudomonas fluorescens N3. The naphthalene dioxygenase, including all the sequences for its expression and the regulatory region, has been localized on the 4.3-kb HindIII-ClaI fragment and on the 3.5-kb HindIII fragment of the plasmid pN3, by Southern analysis using as probes nahA and nahR genes, the homologous genes of the plasmid NAH7 from Pseudomonas putida G7. We cloned in Escherichia coli JM109 the dioxygenase gene and its regulatory region and developed an efficient bacterial system inducible by salicylic acid, able to produce dihydrodiols. E. coli containing recombinant plasmids carrying the dioxygenase gene were analysed for their potential as a biocatalytic tool to produce dihydrodiols from different naphthalenes with the substituent on the aromatic ring at the alpha or beta position. The dihydrodiols, identified by HPLC (high-performance liquid chromatography) and 1H-NMR (nuclear magnetic resonance) were produced with yields ranging from 50 to 94%. The degree of bioconversion efficiency depends on the nature and the position of the substituent and indicates the broad substrate specificity of this dioxygenase and its potential for the production of a wide variety of fine chemicals.

Bacterial Proteins↗
PubMed1997

Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST.

The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Base Sequence↗
PubMed1997

Simultaneous onset of primary cutaneous B-cell lymphoma and human herpesvirus 8-associated Kaposi's sarcoma.

We report the simultaneous occurrence of Kaposi's sarcoma (KS) and primary cutaneous B-cell lymphoma (CBCL) of the leg in a 79-year-old woman, seronegative for HIV-1, HTLV-1 and HTLV-2. The CBCL underwent complete clinical remission after local radiotherapy, whilst the KS became disseminated within a year following diagnosis. However, 2 years after the diagnosis of KS, the patient died with neurological symptoms. These were presumed to be due to involvement of the central nervous system by lymphoma, although in the absence of an autopsy, this could not be proven. Skin biopsies from the original KS and CBCL lesions, as well as short-term culture of spindle cells from the KS lesion and peripheral blood mononuclear cells (PBMC), were studied by semiquantitative polymerase chain reaction (PCR) using primers specific for DNA sequences of a novel gamma-herpesvirus-8 (HHV-8). PCR studies were strongly positive for the virus on KS cells and PBMC; conversely, a low viral load was found on CBCL cells. A high titre of serum IgG antibodies reacting with the nuclei of the HHV-8 positive cell line BCP-1 was found. These data suggest that reactivation of latent infection with HHV-8 had occurred in this patient, and that HHV-8 is directly involved in KS, but not in CBCL of the leg, an aggressive variant of CBCL.

Aged↗
PubMed1996

Organisation of the tmb catabolic operons of Pseudomonas putida TMB and evolutionary relationship with the xyl operons of the TOL plasmid pWW0.

In Pseudomonas putida (Pp) TMB the genes involved in the catabolism of methyl-substituted aromatic hydrocarbons 1,2,4-trimethylbenzene, m- and p-xylene (tmb operon), are functionally and genetically homologous to the xyl genes of the plasmid pWW0, but are chromosomally encoded. We have analysed by cloning. Southern blotting and sequencing of selected regions the organisation of the tmb cluster. This analysis shows that the structural and regulatory genes of the tmb and xyl systems exhibit a high degree of homology and are similarly organised in operons. However the operons are differently arranged on the Pp TMB chromosome and on the pWW0 plasmid. Comparison of the two systems suggests that the operon arrangement found in pWW0 may have originated from that found in Pp TMB via cointegration mediated by replicative transposition or by intermolecular recombination between two copies of the insertion element IS1246.

Base Sequence↗
PubMed1996

Restricted tissue distribution of extralesional Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS patients with Kaposi's sarcoma.

Specific herpesvirus-like DNA sequences have been found in Kaposi's sarcoma (KS) lesions of AIDS patients, suggesting that a novel gamma herpesvirus, homologous to Epstein-Barr virus and herpesvirus saimiri, could be implicated in the pathogenesis of KS. To better understand the role of this putative etiological agent, named Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), we investigated by the polymerase chain reaction (PCR) the presence of viral DNA sequences in various organs obtained at autopsy from seven AIDS patients with KS and six without KS. For each sample, to exclude positive results due to visceral KS dissemination, the presence of microscopic foci of KS cells was rules out by histology and CD34 immunohistochemistry on serial frozen sections immediately adjacent to those employed for DNA extraction. PCR and nested PCR were performed with primers specific for the HIV-8 330 Bam fragment originally described by Chang et al. (Science 1994;266:1865-1869). As quality control, the extracted DNA was amplified with primers for human beta-globin. All KS legions were HHV-8 positive. In addition, extralesional KSHV DNA sequences were detected in seven of seven lymphoid organs and in five of five prostate glands of KS patients. Normal skin was positive in three of five cases and bone marrow in two of three tested cases, all other tissues being negative by PCR and nested PCR. By contrast, no virus was detected in tissue samples of AIDS cases without KS. The restricted organ distribution here documented argues for a selective tissue tropism of HHV-8 in vivo in AIDS patients and suggests that in the infected host lymphoid organs and the prostate gland may represent privileged sites of viral latency and persistence.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST.

A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.

Acetaldehyde↗
PubMed1995

Insulin-like growth factor I absorption in the jejunum of neonatal calves.

The absorption of recombinant human insulin-like growth factor I(rhIGF-I), [125I]rhIGF-I, xylose and [3H]lysine, administered into a clamped jejunal segment in anesthetized neonatal calves, was studied by measuring their appearance in the mesenteric vein draining the gut segment. Only trace amounts of IGF-I and of [125I]rhIGF-I and/or 125I were absorbed following dissolution in saline, buffer or colostrum. The absorption rate of both [3H]lysine and/or 3H and of xylose was much greater than the absorption of rhIGF-I, [125I]rhIGF-I and/or 125I but was also transient. Biological effects of ingested IGF-I in neonatal calves should barely occur as a consequence of intestinal absorption even in 1-day-old calves, known to absorb various peptides and proteins.

Absorption↗
PubMed1993

Feeding of rapeseed presscake meal to pigs: effects on thyroid morphology and function and on thyroid hormone blood levels, on liver and on growth performance.

Effects of feeding soybean meal (SBM) or a mixture of SBM and 5%, 10% and 15% of either 0- and 00-rapeseed presscake meal (RPM) on thyroid, liver and growth performance were studied in castrated boars. Goitrin (G) intake was closely correlated with total glucosinolate intake and blood thiocyanate concentration. In pigs fed 10% and 15% 0-RPM refused feed was enhanced and daily gain decreased. Liver weight and DNA/total liver increased with increasing G intake. Thyroid weights increased up to a G intake of 4.6 mmol/d and then decreased with higher G intake. DNA content/g dry matter (DM) increased continuously and total thyroidal DNA content up to a G intake of 4.6 mmol/l, indicating hyperplasia. Epithelial thickness increased up to a G intake of 13.3 mmol/d, whereas follicular area only up to an intake of 4.6 mmol G/d and then decreased, mirrored by a decrease of DM content. Thyroidal thyroxine (T4) and triiodothyronine (T3) content decreased with increasing G intake. Blood serum levels of T4 decreased with increasing G intake, whereas T3 levels only tended to decrease. Therefore, measured parameters differently reacted to different G intake. In groups fed high amounts of G, hyperplasia was not sufficient to maintain physiological circulating T4 levels, but enhanced thyroidal secretion T3 and/or extrathyreoidal T4 to T3 conversion obviously allowed maintenance of near normal circulating T3 concentrations and barely reduced growth rate despite marked hypothyroidism.

Animal Feed↗
PubMed1992

Q fever endocarditis: diagnostic approaches and monitoring of therapeutic effects.

The scope of current diagnostic methods for Q fever endocarditis includes serology, direct demonstration of Coxiella burnetii in the resected heart valve tissue, and animal inoculation studies. Illustrated by a clinical case report, the different methods are presented and discussed. Serology represents the primary method, using the techniques of complement fixation, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). The latter two techniques allow the detection of immunoglobulins G, M, and A to the phase I and II antigens of C. burnetii. After cardiac surgery, we visualized C. burnetii on smears and specifically stained it on histologic sections of the resected heart valve by light and electron microscopic immunohistochemistry. In addition, seroconversion in animals after inoculation with valve specimens confirmed the presence of C. burnetii in the heart valve. The antibody titers determined by ELISA correlated well with the patient's clinical course during the treatment period. Therefore it is suggested that its usefulness for monitoring the efficacy of antimicrobial agents in patients with Q fever endocarditis should be further evaluated.

Endocarditis, Bacterial↗
PubMed1991

Cloning and expression of a species-specific early immunogenic 36-kilodalton protein of Mycoplasma hyopneumoniae in Escherichia coli.

Mycoplasma hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, synthesizes a 36-kDa protein which is an early and strong immunogenic factor in experimentally and naturally infected swine. The gene encoding this protein was cloned by screening a gene library of M. hyopneumoniae DNA with rabbit hyperimmune serum made against whole M. hyopneumoniae cells and convalescent-phase swine serum. Analysis of the recombinant protein expressed in Escherichia coli by immunoblot techniques showed that the protein is expressed in E. coli in its full length and does not cross-react with proteins from M. flocculare or M. hyorhinis. Genetic analysis showed that the gene was expressed from the lac promoter of the vector and seems to be translationally initiated from its own ribosome binding site. Subcloning in a transcriptional fusion vector to optimize expression resulted in production of the 36-kDa protein in E. coli at levels up to 30% of total protein.

Animals↗
PubMed1990

Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB.

The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned. We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns. The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.

Chromosome Mapping↗
PubMed1987

The preoptic-suprachiasmatic nuclei though morphologically heterogeneous are equally affected by streptozotocin diabetes.

Pituitary and gonadal disorders consistent with abnormal LHRH and LH secretion occur in streptozotocin-diabetic rats. A key role in the synthesis and regulation of LHRH and in the phasic LH release is played by the preoptic-suprachiasmatic region which is mainly formed by the medial preoptic area, the sexually dimorphic nucleus of the medial preoptic area, and the suprachiasmatic nucleus. Therefore we have studied this region by morphology and morphometry in normal and streptozotocin-diabetic rats. In normal animals, the neurons of the above mentioned nuclei were morphologically and morphometrically dissimilar. Independent of their localization, reduced cytoplasmic and nuclear areas were observed in the neurons of diabetic animals. These lesions are consistent with hypotrophied neurons. Consequently, diabetes may impair both synthesis and regulation of LHRH and may therefore account for pituitary disorders, testicular atrophy, and lacking preovulatory LH peaks. The structural differences of the neurons of the three nuclei in normal animals underline their different physiological role. Yet, the similarity of the changes found in all three nuclei suggests a generalized hypofunction of the whole preoptic-suprachiasmatic region under diabetic condition.

Animals↗
PubMed1987

Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation.

A strain of Pseudomonas putida (TMB) was found to resemble P. putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested. The ability of P. putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.

Benzene Derivatives↗