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Biomedical subjects

G Pozzi

Publications and source records attributed to G Pozzi.

At least 55 records · Page 3Linked to original sources
PubMed1999

The type 2 capsule locus of Streptococcus pneumoniae.

The type 2 capsule locus of Streptococcus pneumoniae was characterized in Avery's strain D39, which is the parent strain of the standard transformation recipients currently used in pneumococcal research and is largely used as a virulent strain in studies on the pathogenesis of pneumococcal infections. The capsule locus was sequenced by using a 21.7-kb PCR fragment from the D39 genome as a template. Sequence data analysis showed the presence of 18 open reading frames, 17 of which have the same direction of transcription and all of which are potentially involved in capsule biosynthesis. It was also shown that R36A and R6, which are unencapsulated (rough) derivatives of D39, carry a 7,504-bp deletion involving nine capsule genes.

Bacterial Capsules↗
PubMed1999

rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy.

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser-->Leu) and 11 (29.7%) had GAC in position 526 (His-->Asp).

Alleles↗
PubMed1998

Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis.

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.

Bacillus subtilis↗
PubMed1998

Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells.

Here, we show that bacteria induce de novo synthesis of both major histocompatability complex (MHC) class I and II molecules in a mouse dendritic cell culture system. The neo-biosynthesis of MHC class I molecules is delayed as compared with that of MHC class II. Furthermore, bacteria stabilize MHC class I molecules by a 3-fold increase of their half-life. This has important consequences for the capacity of dendritic cells to present bacterial antigens in the draining lymph nodes. In addition, a model antigen, ovalbumin, expressed on the surface of recombinant Streptococcus gordonii is processed and presented on MHC class I molecules. This presentation is 10(6) times more efficient than that of soluble OVA protein. This exogenous pathway of MHC class I presentation is transporter associated with antigen processing (TAP)-dependent, indicating that there is a transport from phagolysosome to cytosol in dendritic cells. Thus, bacteria are shown to be a potentially useful mean for the correct delivery of exogenous antigens to be presented efficiently on MHC class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Direct sequencing of long polymerase chain reaction fragments.

Direct sequencing of polymerase chain reaction (PCR)-generated templates is a commonly used technique in molecular biology laboratories. We describe an improved method for direct sequencing of PCR fragments longer than 20 kb obtained with a commercial mixture of Taq and Pwo DNA polymerases. The sequencing protocol was optimized for an automated infrared DNA sequencer, consistently yielding long reads (500-600 bases).

DNA↗
PubMed1998

Vaginal immunization of Cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens.

Cynomolgus monkeys (Macaca fascicularis) were immunized by intravaginal administration of live recombinant Streptococcus gordonii. The vaccine strains of S. gordonii expressed the V3 domain of the gpl20 of human immunodeficiency virus type 1 (HIV-1), and the E7 protein of human papillomavirus type 16 (HPV 16). Multiple inocula of recombinant bacteria were used, since S. gordonii could persist for no longer than 3 days in the monkey vagina. Vaginal immunization was found to induce a local and systemic immune response specific for the heterologous antigen expressed by the bacteria. This antigen-specific immune response consisted of vaginal IgA, serum IgG, and a T-cell proliferative response measured on PBMCs. Vaginal IgG and serum IgA were not detected.

Animals↗
PubMed1998

Vaginal immunization with recombinant gram-positive bacteria.

PROBLEM: Many viral and bacterial pathogens enter the body through the genital mucosa. Therefore, one of the major goals of a vaccine against sexually transmitted diseases (STDs) should be to induce an immune response in the genital mucosa capable of controlling the entry of the pathogen. Our approach for the development of vaccines against STDs is based on the use of nonpathogenic Gram-positive bacteria as live vaccine vectors. METHOD OF STUDY: Recombinant Gram-positive bacteria expressing vaccine antigens were constructed using genetic systems developed in our laboratory. Balb/c mice and Cynomolgus monkeys were inoculated by the vaginal route and vaginal samples were collected using absorbent wicks. Colonization was evaluated by the presence of recombinant bacteria in the vaginal samples. Local and systemic immune responses were studied. RESULTS: We have developed genetic systems for the expression of heterologous antigens on the surface of the human commensals Streptococcus gordonii and Lactobacillus spp. Both S. gordonii and L. casei stably colonized the murine vagina after a single inoculum. Vaginal colonization of mice with recombinant strains of S. gordonii, expressing human papillomavirus (HPV) and human immunodeficiency virus (HIV) antigens, induced antigen-specific vaginal immunoglobulin A (IgA) and serum IgG. Local and systemic immune responses also were detected in monkeys immunized intravaginally with recombinant S. gordonii. CONCLUSION: The results obtained indicated that the approach of using colonizing Gram-positive bacteria as live vectors has a great potential for the development of vaccines against STDs.

Animals↗
PubMed1997

Comorbidity of drug dependence and other mental disorders: a two-phase study of prevalence at outpatient treatment centres in Italy.

A cross-sectional multicentre study aimed at evaluating the prevalence of DSM III-R Axis I morbidity among drug addicts seeking treatment. Consecutive outpatients of the National Health Service's treatment units (aged 18-60 years, fulfilling criteria for drug dependence and without primary diagnosis of alcohol dependence) were enrolled at intake. In the first phase of the study, professional staff members evaluated current psychiatric morbidity of 317 clients clinically. In the second phase, trained interviewers administered the Composite International Diagnostic Interview and a European adaptation of the Addiction Severity Index to 65 probands. Some current psychiatric morbidity was found in 26.2% of subjects recruited during the first phase and in 22.2% during the second; lifetime comorbidity was ascertained in 32.3% of probands recruited in the second phase. Unfortunately, remarkable rates of undetermined diagnostic assessment during the second phase are due to refusers. The distribution of categorical diagnoses is somewhat different between the two samples, but the most frequent are Anxiety and Mood disorders in both. Primary versus secondary psychiatric diagnoses were also distinguished when evaluated over a lifetime. The rates of psychiatric comorbidity are low in this study as compared with previous investigations, but suffer from a poor compliance with structured diagnostic procedures by unselected probands. Other methodological factors may also affect this kind of research. Prospective studies are needed to evaluate the validity of comorbid diagnostic constructs and their prognostic significance. Also a concurrent assessment of Axis II disorders may be helpful to explain symptomatic polymorphism in case of multiple Axis I comorbidity.

Adolescent↗
PubMed1997

The joint of Tn916 circular intermediates is a homoduplex in Enterococcus faecalis.

Tn916 is a 18-kb conjugative transposon originally identified in Enterococcus faecalis. The first step for Tn916 movement is its excision from the donor replicon with the formation of a nonreplicating covalently closed circular intermediate. Studies on formation of circular intermediates in Escherichia coli have shown that the joint between the Tn916 termini is a 6-bp heteroduplex formed by the two regions flanking the transposon before its excision (coupling sequences). In this work we studied the joint of Tn916 termini in circular intermediates formed in both E. coli and E. faecalis. Our strategy was to use direct sequencing of amplification products obtained from the joint region of single target molecules. In E. coli, 50% of circular intermediates contained a heteroduplex joint, while the remaining 50% displayed a homoduplex joint formed by one of the two coupling sequences. In E. faecalis, we could not demonstrate the presence of any heteroduplex joint. In this case 77.7% of the analyzed joints were formed by the left coupling sequence.

DNA Replication↗
PubMed1997

Angiocardiographic digital still images compressed via irreversible methods: concepts and experiments.

We defined, implemented and tested two new methods for irreversible compression of angiocardiographic still images: brightness error limitation (BEL) and pseudo-gradient adaptive brightness and contrast error limitation (PABCEL). The scan path used to compress the digital images is based on the Peano-Hilbert plane-filling curve. The compression methods limit, for each pixel, the brightness errors introduced when approximating the original image (i.e. the difference between the values of corresponding pixels as grey levels). Additional limitations are imposed to the contrast error observed when considering along the scan path consecutive pixels of both the original and the reconstructed image. After previous testing on angiocardiographic images selected as clinically significant from 35 mm films, we enlarged our experiment to a set of 38 coronary angiograms digitally acquired. BEL and PABCEL methods were experimented according to several values of the implied thresholds. Up to a compression ratio of 9:1 for the BEL method and 10:1 for the PABCEL method, no deterioration of the reconstructed images were detected by human observers. After a visual evaluation, we performed a quantitative evaluation. The visualization of pseudo-colour difference images showed the capability of BEL and PABCEL for preserving the most significant clinical details of the original images. For comparison, we applied the JPEG (joint photographic experts group) image-compression standard to the same set of images. In this case, pseudo-colour difference images showed a homogeneous distribution of errors on the image surface. Quantitative compression results obtained by testing the different methods are comparable, but, unlike JPEG, BEL and PABCEL methods allow the user to keep under his direct control the maximum error allowed at each single pixel of the original image. These different behaviors are confirmed by the values obtained for the considered numerical quality quantifiers.

Algorithms↗
PubMed1996

A host-vector system for heterologous gene expression in Streptococcus gordonii.

We have developed a host-vector system for heterologous expression in Streptococcus gordonii (Sg) Challis (formerly Streptococcus sanguis), a commensal bacterium of the human oral cavity. The system is based on (i) integration of plasmid insertion vectors into the chromosome of specially engineered recipient hosts, and (ii) the use of the M6-protein-encoding gene (emm6) as a partner for construction of translational gene fusions. M6 is a streptococcal surface protein already proven useful as a fusion partner for the delivery of foreign antigens to the surface of Sg [Pozzi et al., Infect. Immun. 60 (1992) 1902-1907]. Insertion vectors carry a drug-resistance marker, different portions of emm6 and a multiple cloning site to allow construction of a variety of emm6-based fusions. Upon transformation of a recipient host with an insertion vector, 100% of transformants acquire both the drug-resistance marker and the capacity of displaying the M6 molecule on the cell surface. Chromosomal integration occurred at high frequency in recipient host GP1221. Transformation with 1 microgram of insertion vector DNA yielded 8.1 X 10(5) transformants per ml of competent cells.

Base Sequence↗
PubMed1996

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae.

Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252, to form the composite element Tn5253 of Streptococcus pneumoniae. We show that Tn5251 is identical in structure and size to Tn916. DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the two CT. Essentially all differences (66/73) were clustered in a 688-bp segment of tet(M), which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae. DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site (attB) of Tn5251 into Tn5252. Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10(6) chromosomes.

Base Sequence↗
PubMed1996

The tetracycline resistance gene tet(M) exhibits mosaic structure.

Tetracycline resistance genes of the M class, tet(M), are typically found on mobile genetic elements as the conjugative transposons of gram-positive bacteria. By comparing the sequences of eight different tet(M) genes (from Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Ureaplasma urealyticum, and Neisseria), a mosaic structure was detected which could be traced to two distinct alleles. The two alleles displayed a divergence of 8% and a different G/C content. The block structure of these genes provides evidence for the contribution of homologous recombination to the evolution and the heterogeneity of the tet(M) locus. Unlike described cases of chromosomally located mosaic loci, tet(M) is a relatively recently acquired determinant in the species examined and it would appear that mosaic structure within tet(M) has evolved after acquisition of the gene by the mobile genetic elements upon which it is located.

Bacterial Proteins↗
PubMed1996

Gram-positive commensal bacteria for mucosal vaccine delivery.

To avoid the use of engineered pathogens for vaccine delivery, systems have been developed that allow the expression of heterologous antigens in commensal Gram-positive bacteria. In some cases, both a serum IgG and secretory IgA response are induced to the recombinant protein after vaccination, verifying the validity of the approach. These live recombinant bacteria may be used in the future to introduce a protective immune response to pathogenic microorganisms after mucosal colonization.

Adjuvants, Immunologic↗
PubMed1996

Conjugative transposon Tn916: evidence for excision with formation of 5'-protruding termini.

Conjugative transposons are genetic elements able to promote their own intracellular transposition and intercellular conjugal transfer. They move by an excision-integration system related to that of lambdoid phages, in which the first step is the excision of the transposon from the donor replicon to form a covalently closed circular intermediate which contains a heteroduplex joint. In this work, sequencing both strands of the circular intermediate heteroduplex joint, it was found that, as during lambda phage excision, Tn916 excises from the host DNA by 5'-protruding staggered endonucleolytic cleavages.

Bacteriophage lambda↗
PubMed1996

Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone.

The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Havarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.

Alleles↗
PubMed1996

Exercise-induced hypoxaemia in emphysematous type chronic obstructive pulmonary disease.

To investigate gas exchange response to exercise, we studied 16 male patients with moderate-to-serve airflow obstruction (forced expiratory volume in one second (FEV1) 39 +/- 10% of predicted value), mild-modest arterial hypoxaemia (arterial oxygen tension (Pa,O2) 9.6 +/- 0.87 kPa) and no arterial hypercapnia (arterial carbon dioxide tension (Pa,CO2) 5.04 +/- 0.45 kPa), referred to as emphysematous-type chronic obstructive pulmonary disease (COPD) clinical pattern. During maximal exercise tests, Pa,O2 increased by more than 0.3 kPa in eight patients (Group A) and fell by more than 0.3 kPa in the other eight patients (Group B). Pulmonary function tests, maximal inspiratory pressure at the mouth, values at maximum cycle incremental exercise and baseline arterial blood gases did not differ significantly between the two groups. We, therefore, showed that common pulmonary function measurements at rest and during exercise were not useful in identifying patients who underwent exercise-induced hypoxaemia. Furthermore, we suggest that patients with the same clinical pattern of chronic obstructive pulmonary disease and the same degree of airflow obstruction and gas exchange impairment could develop a different adaptation to a maximal exercise test, and that the presence of exercise-induced hypoxaemia might be related to pathological features of emphysema more than to different respiratory functional measurements.

Aged↗