Biomedical subjects
Publications and source records attributed to F Iannelli.
UNLABELLED: GeneSyn is a software tool that allows automatic detection of conserved gene order from annotated genomes. AVAILABILITY: Available free of charge for Unix/Linux/Cygwin platforms at ftp://159.149.110.11/pub/GeneSyn_1.0/ SUPPLEMENTARY INFORMATION: ftp://159.149.110.11/pub/GeneSyn_1.0/
Susceptibilities to macrolides were evaluated in 267 Streptococcus pneumoniae isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried mef. Strains that carried both mef(A) and mef(E) were found: 17 strains carried mef(A) and 3 carried mef(E). The characteristics of the strains carrying the mef genes and the properties of the mef-containing elements were studied. Strains carrying mef(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three mef(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the mef-containing element, which encompassed mef and the msr(A) homolog, were identical among the three mef(E)-positive strains and among the three mef(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all mef(A)-positive strains, the mef element was inserted in celB, which led to impairment of the competence of the strains. In line with insertion of the mef(E) element at a different site, the competence of the mef(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three mef(A) strains but from none of three mef(E) strains. Due to the important different characteristics of the strains carrying mef(A) or mef(E), we suggest that the distinction between the two genes be maintained.
Choline-binding proteins (CBPs) from Streptococcus pneumoniae are involved in several important processes. Inactivation of zmpB, a gene that encodes a surface-located putative zinc metalloprotease, in a S. pneumoniae serotype 4 strain was recently reported to reveal a composite phenotype, including extensive chain formation, lysis defect and transformation deficiency. This phenotype was associated with the lack of surface expression of several CBPs, including the major autolysin LytA. LytA, normally 36 kDa in size, was reported to form an SDS-resistant 80 kDa complex with CinA. ZmpB was therefore proposed to control translocation of CBPs to the surface, possibly through the proteolytic release of CBPs (and RecA) from CinA. Based on the use of 12 independent mariner insertions in the zmpB gene of the well-characterized R6 laboratory strain, we could not confirm several of these observations. Our zmpB mutants: (i) did not form chains; (ii) lysed normally in the presence of deoxycholate, which indicates the presence of a functional autolysin; (iii) transformed at normal frequency; and (iv) contained bona fide CinA and LytA species. Polymorphism of ZmpB between R6 and the serotype 4 isolate could not account for the discrepancy, as inactivation of zmpB (through replacement by transposon-inactivated zmpB R6 alleles) in the latter strain did not affect separation of daughter cells and autolysis. The conflicting observations could be explained by our finding that the reportedly serotype 4 zmpB 'mutant' differed from its S. pneumoniae parent in lacking capsule and in exhibiting characteristic traits of the Streptococcus viridans group, including resistance to optochin.
The important human pathogen Streptococcus pneumoniae was found to absorb factor H, an inhibitor of complement, from human plasma. We identified the gene encoding a novel surface protein, factor H-binding inhibitor of complement (Hic), in the pspC locus of type 3 pneumococci. Unlike PspC proteins in other serotypes, Hic is anchored to the cell wall by means of an LPXTG motif, and the overall sequence homology to various PspC proteins is low. However, the NH(2)-terminal region showed significant homology to the NH(2)-terminal region of several PspC proteins. A fragment of Hic, covering this homologous region, was expressed as a glutathione S-transferase (GST) fusion protein. GST:Hic(39-261) bound radiolabeled factor H and inhibited binding of factor H to pneumococci of different serotypes. Interaction kinetics between GST:Hic(39-261) and factor H were studied with surface plasmon resonance and showed a high affinity binding (K(A) = 5 x 10(7), K(D) = 2.3 x 10(-)(8)). Mutant pneumococci lacking Hic showed no absorption of factor H in human plasma and no binding of radiolabeled factor H, suggesting that Hic is responsible for factor H-binding in type 3 pneumococci. Factor H-dependent inhibition of the alternative pathway was not diminished by the presence of GST:Hic(39-261). In addition, an intrinsic inhibitory effect of Hic is suggested.
The mef(A) gene from a clinical isolate of Streptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames. orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance gene msr(SA).
Cryptic plasmids pDP1 and pSMB1 from clinical strains of Streptococcus pneumoniae isolated 74 years apart were found to be essentially identical in their nucleotide sequence. pDP1, 3161 bp, contains five codirectional ORFs and presents all the general features of plasmids replicating by the rolling circle mechanism. The rep gene, 963 bp, is highly homologous to the rep gene of other streptococcal plasmids of the pC194 family.
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.
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).
Tumor Proliferative Fraction (TPF) has been shown to correlate with prognosis in some malignancies. A reliable, accurate method for application in a clinical practice is still being sought. The aim of this study is to compare TPF as determined by Proliferating Cell Nuclear Antigen (PCNA) and Flow Cytometry (FC) in 36 consecutive patients affected by Renal Cell Carcinoma (RCC). Proliferating cells were identified in paraffined sections using a anti-PCNA monoclonal antibody (PC 10 Dako). Cell suspension for FC were prepared from fresh/frozen samples DNA index and S phase were evaluated using a computerized program (Multicycle, Phoenix). 16 samples (47.1%) were found to be aneuploid by FC (DI range 0.72-2.40). Aneuploid vs diploid tumors had significantly higher mean FC-S phase (p = 0.049) and PCNA LI (p = 0.034). Weak correlation (r-Spearman 0.416 p = 0.01) was found between PCNA LI and grading and near to significativity between PCNA LI and tumor size (r = 0.335 p = 0.0061). When patients are classified according to nuclear grading, is evident that all PCNA G4 are aneuploid and that 62.5% of PCNA G1 are diploid. A week correlation near to significativity is found between PCNA LI and S phase only in the aneuploid tumors. A more reliable measurement of TPF in RCC could be provided by combining the two methods. Further research on larger series is needed.
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.
A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1 pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3' end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selective PCR.
HBsAg, HBeAg, anti-HBc, anti-HBs, IgM anti-HAV, total and IgM anti-delta (RIA) and IgM anti-HBc (ELISA) have been determined on sera of 72 male patients with acute hepatitis (68 HBsAg positive and 4 anti-HBc-positive) at the onset of the disease and at 2, 4 and 6 months thereafter, and of 77 patients with HBsAg-positive chronic active hepatitis. The results indicate that testing for IgM anti-HBc in acute hepatitis is necessary to identify cases of hepatitis B even in the absence of positivity for HBsAg and/or HBeAg. HBsAg-positive cases, negative for IgM anti-HBc, were due to delta superinfection of chronic carriers of HBsAg. Recovery was always associated with negativity of IgM anti-HBc; IgM anti-HBc persistence was not found in all cases evolved to chronicity. In chronic hepatitis cases positivity for IgM anti-HBc was found only in 39% of the anti-delta-negative and only in 5.5% of the anti-delta-positive cases.