Biomedical subjects

G M Rossolini

Publications and source records attributed to G M Rossolini.

At least 73 records · Page 4Linked to original sources

PubMed1995

Development of a yeast retrotransposon-based system useful for screening of potentially active anti-retroviral compounds.

We have developed a new system suitable for easy inexpensive screening of substances potentially active against HIV and other retroviruses. The system evaluates the ability of substances to inhibit the high-frequency induced retrotransposition of the yeast transposable element Ty917, and is based on an engineered version of the retrotransposon which carries a dominant selectable marker useful for scoring transposition events. The system performance was evaluated using two nucleoside analogues of proved anti-HIV activity, 3'-azido-3'deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC). Both substances were able to inhibit Ty917 transposition and could have been detected as potentially active antiretroviral drugs using the new screening system.

Antiviral Agents↗

PubMed1995

Engineering human monoclonal antibody fragments: a recombinant enzyme-linked Fab.

A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed. pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii. Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase. In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity. Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.

Acid Phosphatase↗

PubMed1995

Cloning and characterization of human recombinant antibody Fab fragments specific for types 1 and 2 herpes simplex virus.

Twenty-six bacterial clones producing human recombinant Fab fragments specific for Herpes Simplex virus (HSV) antigens were obtained from an IgG1k human antibody combinatorial library displayed on filamentous phage, following panning against an HSV lysate. All the Fabs reacted against the HSV lysate in enzyme-linked immunosorbent assay and were able to recognize both type 1 and type 2 HSV in an indirect immunofluorescence assay (IFA). DNA sequencing of the heavy chain variable regions showed that these Fabs were different from those already described. One of these Fabs (Fab19) was purified and subjected to further characterization. Purified Fab19 was able to specifically recognize several different HSV-1 and HSV-2 strains (including 2 reference strains and 12 clinical isolates) in IFA. It was also able to neutralize the infectivity of both HSV-1 and HSV-2 strains, although the neutralizing activity was somewhat lower against HSV-2. In fact, 100% neutralization of infectivity was observed at a Fab concentration of 2 micrograms/100 TCD50 for the majority of HSV-1 strains, while a concentration of 8 micrograms/100 TCD50 was needed for 100% neutralization of all the HSV-2 strains tested. Owing to the above properties, Fab19 appears to be useful for diagnostic purposes and might also prove useful for in vivo immunoprophylaxis and therapy of HSV infections.

Animals↗

PubMed1995

A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.

Acid Phosphatase↗

PubMed1994

fimA-folD genes linkage in Salmonella identifies a putative junctional site of chromosomal rearrangement in the enterobacterial genome.

Analysis of the Salmonella chromosomal region located upstream of the fimA gene (coding for the major type 1 fimbrial subunit) showed a close linkage of this gene to the folD gene (coding for the enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase), indicating that the fim gene cluster of Salmonella, unlike that of Escherichia coli, has no regulatory genes located upstream of fimA and apparently terminates with this gene. The respective locations of the fim and folD genes in the E. coli and Salmonella genetic maps suggests that the fimA-folD intergenic region of Salmonella encompasses a junctional site of a genetic rearrangement that probably originated from the different chromosomal location of the fim genes in these species.

Amino Acid Sequence↗

PubMed1994

Identification of an Escherichia coli periplasmic acid phosphatase containing of a 27 kDa-polypeptide component.

An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique. The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p-nitrophenyl phosphate and other phenolic phosphate esters. Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose.

Acid Phosphatase↗

PubMed1994

Detection of the Vibrio cholerae heat-stable enterotoxin gene by polymerase chain reaction.

A polymerase chain reaction assay was developed for detection of the Vibrio cholerae heat-stable enterotoxin gene (sto). The assay is based on two oligonucleotide primers suitable for amplification of the entire sto open reading frame. Reaction conditions were defined to obtain optimal results in terms of specificity and sensitivity. Under these conditions the assay was highly sensitive and enabled good results to be obtained using, as a template, crude DNA preparations from single bacterial colonies. A rapid protocol for direct sequence analysis of the amplification product, which may be used in combination with the PCR assay to confirm the product identity and analyse sequence polymorphisms within the sto gene, was also developed. Twenty-two V. cholerae non-O1 isolates from sporadic cases of V. cholerae non-O1-associated gastroenteritis from Cuba were analysed by this PCR assay. Four strains (18.2%) were found to carry the sto gene. The prevalence of V. cholerae non-O1 strains carrying the sto gene among clinical isolates is higher than that reported for other geographical areas, except in the case of epidemics.

Amino Acid Sequence↗

PubMed1994

Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information.

The performance of oligonucleotide primers containing deoxyinosine (dl) at all ambiguous positions for polymerase chain reaction, based on ambiguous sequence information derived either from compilations of consensus nucleotide sequences or from amino acid sequences, has been evaluated in two model systems represented respectively by amplification of conserved genomic regions from different types of human papillomavirus and by amplification of a region of the human lysozyme cDNA on the basis of the protein amino acid sequence. In both instances the dl-containing primers obtained the expected amplification products. When using short primers or primers with very high dl contents, however, peculiar reaction conditions had to be adopted to obtain successful amplification and, in the latter case, performance remained suboptimal. Comparison of results with those obtained using corresponding degenerate primers showed that the use of dl-containing primers can be advantageous in terms of both specificity and yield of the amplification product. Sequence analysis of amplification products showed that dG residues are always found at positions corresponding to the dl residues of the primers.

Amino Acid Sequence↗

PubMed1994

In-vitro evaluation of cefpodoxime.

In-vitro antimicrobial activity of cefpodoxime was evaluated against several microbial species by both conventional tests and additional parameters which take into consideration some of the conditions likely to be encountered in infected tissues. MICs for 414 recent clinical isolates, including staphylococci, streptococci, Haemophilus influenzae, Moraxella catarrhalis, several Enterobacteriaceae, Aeromonas hydrophila and Campylobacter jejuni were determined. MIC values overall were similar to those observed for strains from other geographical areas. Inhibition of growth by cefpodoxime was virtually unaffected by the inoculum size, even using bacterial populations as large as 10(9) cfu of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, H. influenzae, beta-lactamase-negative M. catarrhalis, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Growth in the presence of human serum as the only source of nutrients did not significantly affect the inhibition exerted by cefpodoxime, even against large bacterial populations of S. aureus, S. pneumoniae, E. coli, and K. pneumoniae. For K. pneumoniae, E. coli, P. mirabilis and beta-lactamase-negative M. catarrhalis it was also found that sub-MIC concentrations of cefpodoxime were still able to inhibit the majority of cells in microbial populations as large as 10(9) cfu. Evaluation of bactericidal activity demonstrated that cefpodoxime concentrations comparable to those achievable in plasma or in the respiratory tract were able to kill rapidly large bacterial populations of S. pneumoniae and S. pyogenes. The bactericidal activity was apparently lower against M. catarrhalis, H. influenzae, S. aureus, E. coli, and K. pneumoniae.

Bacteria↗

PubMed1994

Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii.

Phosphatase activities were investigated in Morganella morganii, which is one of the few enterobacterial species producing high-level phosphate-irrepressible acid phosphatase activity (HPAP phenotype), and the gene encoding the major phosphate-irrepressible acid phosphatase was cloned, sequenced, and its product characterized. Using p-nitrophenyl phosphate as substrate, Morganella produced a major phosphate-irrepressible acid phosphatase (named PhoC) which is associated with the HPAP phenotype, a minor phosphate-irrepressible acid phosphatase, and a phosphate-repressible alkaline phosphatase. The presence of the PhoC activity prevented induction of alkaline phosphatase when a PhoC-hydrolysable organic phosphate ester, such as glycerol 2-phosphate, was the sole phosphate source. PhoC is a secreted nonspecific acid phosphatase apparently composed of four 25 kDa polypeptide subunits. The enzyme is resistant to EDTA, P(i), fluoride and tartrate. The M. morganii PhoC showed 84.6% amino acid sequence identity to the PhoN nonspecific acid phosphatase of Providencia stuartii, 45.3% to the PhoN nonspecific acid phosphatase of Salmonella typhimurium, and 37.8% to the principal acid phosphatase (PhoC) of Zymomonas mobilis. Comparison of sequence data and of regulation of these enzymes suggested a different phylogeny of members of this gene family within the Enterobacteriaceae.

Acid Phosphatase↗

PubMed1994

Purification and characterization of type 1 fimbriae of Salmonella typhi.

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose-inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS-PAGE analysis of the fimbrial preparation showed the presence of a 20-kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino-terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti-fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.

Amino Acid Sequence↗

PubMed1993

Analysis of the Salmonella fim gene cluster: identification of a new gene (fimI) encoding a fimbrin-like protein and located downstream from the fimA gene.

The fimA gene coding for the major component (fimbrin) of type 1 fimbriae was mapped within the Salmonella typhi fim gene cluster, and its nucleotide sequence determined. The deduced amino acid sequence of S. typhi fimbrin is highly homologous to that of S. typhimurium type 1 fimbrin and showed similarity to that of other enterobacterial type 1 fimbrins. Downstream of fimA, an open reading frame was found, named fimI, able to encode a fimbrin-like protein. The fimI product could represent the counterpart, in type 1 fimbriae, of the PapH protein involved in cell anchoring and length modulation of Escherichia coli Pap pili. This genetic organization was found to be common to other Salmonella serovars, including S. typhimurium and S. choleraesuis.

Amino Acid Sequence↗

PubMed1993

Chromosome Polymorphisms among Strains of Hansenula polymorpha (syn. Pichia angusta).

Contour-clamped homogeneous electrophoresis and an embedded-agarose method of sample preparation were combined to carry out an analysis of the chromosome sets of nine strains of Hansenula polymorpha (syn. Pichia angusta). Chromosomal DNA molecules could be separated into a series of bands ranging, approximately, from 650 up to 2,200 kb in size. Polymorphism of the electrophoretic pattern was demonstrated among the strains investigated in this study. Cross-hybridization between H. polymorpha and Saccharomyces cerevisiae ribosomal DNA was also observed.

Journal Article↗

PubMed1993

Unusual clustering of Alu repeats within the 5'-flanking region of the human lysozyme gene.

We report the nucleotide sequence of the 2.2-kb 5'-flanking region of the human lysozyme gene. Four Alu repeats are located within this upstream region. Classification and dating of these four Alu elements, as well as of the four Alu elements present within the human lysozyme structural gene, were performed. Transposition of the eight Alu repeats found in the human lysozyme locus has apparently occurred at four different times during the primate genome evolution. Considering that Alu repeats are interspersed throughout human DNA with an average spacing of 4 kb, the presence of eight such repeats within the 8-kb lysozyme gene region and, in particular, of four of them in the 2.2-kb region upstream of the structural gene, appears quite unusual.

Animals↗

PubMed1993

Rapid access to pharmacokinetics data and correlation between antimicrobial susceptibility results and drug tissue distribution using a personal computer.

MYMIC is a computer-aided system capable of integrating antibiotic susceptibility data with the concentrations the drugs reach in various body tissues and fluids by calculating site concentration/minimal inhibitory concentration quotients. The program can be run on any low-cost personal computer operating under MS-DOS, provided it is equipped with a hard-disk drive and with a minimum of 512 kilobytes of random access memory. The use of the program does not require any knowledge of computer languages. The antibiotic susceptibility data can be entered either as minimal inhibitory concentrations or as inhibitory zone diameters; in the latter case, minimal inhibitory concentrations are automatically calculated via regression formulas. The concentrations obtained by 90 antibiotics in 51 different human tissues and fluids are recorded in a data base of over 1,000 records, obtained from roughly 700 original papers. A MYMIC sample session was simulated by mimicking infections of three different body districts (namely bone, prostate, and sputum) caused by Pseudomonas aeruginosa, Escherichia coli or Providencia stuartii.

Anti-Bacterial Agents↗

PubMed1992

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

Blotting, Northern↗

PubMed1992

Molecular epidemiology of nasopharyngeal corynebacteria in healthy adults from an area where diphtheria vaccination has been extensively practiced.

In addition to conventional biochemical tests, a DNA probe specific for Corynebacterium diphtheriae was used to characterize 53 cystinase-positive and urease-negative corynebacteria strains isolated from pharyngeal and nasal swabs obtained from 515 healthy adults living in an urban area of central Italy. No Corynebacterium diphtheriae strain was found. Six "atypical" strains were isolated, which could not be classified in any of the species so far defined in the Corynebacterium genus. These strains appeared to be biochemically close to Corynebacterium pseudodiphtheriticum and genetically close to Corynebacterium diphtheriae, since their DNAs strongly hybridized, under relatively low stringency conditions, with a Corynebacterium diphtheriae-specific probe and since insertion sequences which are usually found in Corynebacterium diphtheriae genomes were also found to be present in their genomes. No one of these six strains was either toxigenic or susceptible to lysogenization by beta-corynephage carrying the tox gene. Therefore, they do not seem to have any epidemiological relevance as possible hosts for beta-phages.

Adult↗

PubMed1992

A species-specific DNA probe for Providencia stuartii identification.

A DNA probe is described that can be used for identification of Providencia stuartii by means of filter hybridization assays. The probe, which is a fragment of the P. stuartii phoN gene coding for an acid phosphatase, appeared to be able to recognize only P. stuartii strains in slot-blot hybridization experiments performed with total DNA extracted from 545 strains of 64 different Gram-negative bacterial species, including all the major representatives of the family Enterobacteriaceae. Owing to the problems that may be often encountered for correct identification of P. stuartii at the species level when using commercial identification systems, this probe may result useful for fast and reliable identification of P. stuartii strains for taxonomical, epidemiological and diagnostic studies.

Acid Phosphatase↗