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Biomedical subjects

A M Berry

Publications and source records attributed to A M Berry.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sequence variation in the Streptococcus pneumoniae pneumolysin gene affecting haemolytic activity and electrophoretic mobility of the toxin.

When 30 clinical isolates of Streptococcus pneumoniae, representing 16 capsular serotypes, were analysed by Western blot for production of the haemolytic toxin pneumolysin (Ply), all strains produced an immunoreactive band of similar intensity. However, six isolates of serotype 8 and two of type 7F expressed Ply whose mobility on SDS-PAGE was anomalously slow. Culture lysates from these strains also had low haemolytic activities compared with those for clinical isolates of other serotypes, suggesting the possibility of mutations affecting specific activity. Genes encoding Ply from one type 8 isolate and one type 7F isolate were cloned into Escherichia coli and sequenced. Compared with the published sequence for Ply, the deduced amino acid sequence for the type 8 Ply variant contained three amino acid substitutions, and the type 7F variant four amino acid substitutions. Both variants also had Val270 and Lys271 deleted. The variant Ply proteins were purified from recombinant E. coli expressing the cloned genes, and shown to have substantially reduced specific haemolytic activities [6.8 x 10(4) haemolytic units (HU)/mg and 2.3 x 10(4) HU/mg for type 8 Ply and type 7F Ply respectively] compared with Ply itself (1.2 x 10(6) HU/mg). Studies with chimeric toxin gene constructs indicated that both the reduced haemolytic specific activity and the anomalous electrophoretic mobility of the variant Plys were attributable to a single amino acid substitution (Thr172-->Ile).

Amino Acid Sequence↗
PubMed1996

Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae.

psaA encodes a 37-kDa putative pneumococcal surface adhesin. Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously been assessed. In this study, we used a PCR-amplified internal fragment of the psaA gene from S. pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA-(1) and PsaA-(2)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA-(1) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene. pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis. Adherence of PsaA-(1) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin. Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S. pneumoniae R36A. The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.

Adhesins, Bacterial↗
PubMed1996

Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli.

Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.

Amino Acid Sequence↗
PubMed1996

Distinct roles for pneumolysin's cytotoxic and complement activities in the pathogenesis of pneumococcal pneumonia.

Pneumolysin, the major Streptococcus pneumoniae cytotoxin, contributes to the early pathogenesis of invasive pneumococcal pneumonia by facilitating intrapulmonary bacterial growth and invasion into the blood. Pneumolysin is a multifunctional toxin, with distinct cytolytic ("hemolytic") and complement-activation ("complement") activities that have been mapped to several regions of the molecule. To characterize the specific contributions of pneumolysin's hemolytic and complement properties to the pathogenesis of pneumococcal pneumonia, we compared the in vivo effects of type 2 S. pneumoniae mutant strains, which produce pneumolysins deficient in these activities. The absence of either pneumolysin's hemolytic or complement activities rendered mutant strains less virulent than the wild-type strain during pulmonary infection. Pneumolysin's hemolytic activity correlated with acute lung injury and bacterial growth at 3 and 6 h after endotracheal instillation. In contrast, pneumolysin's complement activity correlated with bacterial growth and bacteremia at 24 h after pulmonary infection. Pneumolysin's complement activity was not associated with the degree of alveolar-capillary injury or recruitment of leukocytes during initial pulmonary infection. However, pneumolysin's complement activity inhibited killing of mutant bacteria in an in vitro complement-dependent neutrophil killing assay. Thus, both pneumolysin's hemolytic and complement activities made specific contributions to the early pathogenesis of pneumococcal pneumonia at different stages of infection and by different mechanisms.

Animals↗
PubMed1995

Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae.

The thiol-activated toxin pneumolysin is a known pneumococcal virulence factor, with both cytotoxic (hemolytic) and complement activation properties. Copies of the pneumolysin gene carrying defined point mutations affecting either or both of these properties were introduced into the chromosome of Streptococcus pneumoniae D39 by insertion-duplication mutagenesis. The virulences of these otherwise isogenic strains were then compared. There was no significant difference in either the median survival time or overall survival rate between mice challenged with D39 derivatives producing the wild-type toxin and those expressing a pneumolysin gene with an Asp-385-->Asn mutation, which abolishes the complement activation property. However, mice challenged with strains carrying either His-367-->Arg or Trp-433-->Phe plus Cys-428-->Gly mutations, which reduce hemolytic activity to approximately 0.02 and 0.0001% of the wild-type level, respectively, had significantly greater median survival times and overall survival rates than mice challenged with D39 derivatives expressing a wild-type pneumolysin gene. No additional reduction in virulence was observed when mice were challenged with a D39 derivative carrying Trp-433-->Phe, Cys-428-->Gly, and Asp-385-->Asn, rather than Trp-433-->Phe and Cys-428-->Gly, mutations in the pneumolysin gene. Thus, it appears that in the intraperitoneal challenge model, the contribution of pneumolysin to virulence is largely attributable to its hemolytic (cytotoxic) properties rather than to its capacity to activate complement. Interestingly, however, the amount of pneumolysin required for full virulence may be very small, as D39 derivatives carrying the Trp-433-->Phe mutation (which reduces hemolytic activity to 0.1% of the wild-type level) had intermediate virulence.

Animals↗
PubMed1995

Use of the laryngeal mask airway in otolaryngology.

The laryngeal mask airway (LMA) provides a third type of airway for consideration in otolaryngology that offers some of the benefits of intubation and avoids many of the associated hazards. It can be inserted without the use of a laryngoscope or muscle relaxant, and is designed to produce an airtight seal around the laryngeal inlet. The LMA provides a secure airway suitable for spontaneous or controlled ventilation, acts as an airtight throat pack, and is well tolerated during recovery. The reinforced LMA (RLMA), specifically designed for otolaryngology anaesthesia, has recently become available. The purpose of this article to is provide a brief overview of the LMA with special emphasis on its use in otolaryngology--head and neck surgery.

Equipment Design↗
PubMed1994

Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli.

A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.

Amino Acid Sequence↗
PubMed1993

Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase.

Biological nitrogen fixation in aerobic organisms requires a mechanism for excluding oxygen from the site of nitrogenase activity. Oxygen exclusion in Frankia spp., members of an actinomycetal genus that forms nitrogen-fixing root-nodule symbioses in a wide range of woody Angiosperms, is accomplished within specialized structures termed vesicles, where nitrogen fixation is localized. The lipidic vesicle envelope is apparently a functional analogue of the cyanobacterial heterocyst envelope, forming an external gas-diffusion barrier around the nitrogen-fixing cells. We report here that purified vesicle envelopes consist primarily of two hopanoid lipids, rather than of glycolipids, as is the case in cyanobacteria. One envelope hopanoid, bacteriohopanetetrol phenylacetate monoester, is vesicle-specific. The Frankia vesicle envelope thus represents a layer specific to the locus of nitrogen fixation that is biosynthetically uniquely derived.

Journal Article↗
PubMed1992

Effect of insertional inactivation of the genes encoding pneumolysin and autolysin on the virulence of Streptococcus pneumoniae type 3.

Derivatives of Streptococcus pneumoniae type 3 deficient in production of either pneumolysin or autolysin were constructed. This was achieved by transformation of type 3 pneumococci with DNA from derivatives of a rough strain (Rx1), in which the respective genes had been interrupted by insertion-duplication mutagenesis using internal fragments of the cloned genes in the vector pVA891. Southern blot analysis confirmed that the pneumolysin or autolysin genes in the respective transformants had been interrupted by insertion of the plasmid-derived sequences. Both the pneumolysin-negative and the autolysin-negative strains had significantly reduced (P less than 0.0001) virulence in mice, as judged by survival time after intraperitoneal challenge. The median survival time of mice challenged with type 3 pneumococci in which either pneumolysin or autolysin production had been reconstituted by back-transformation of the mutants with an intact copy of the respective cloned gene (with concomitant elimination of plasmid-derived sequences), was indistinguishable from that of mice challenged with the wild-type strain. These results establish the importance of both pneumolysin and autolysin to the virulence of type 3 pneumococci.

Animals↗
PubMed1992

Topical anaesthesia for extracorporeal shock wave lithotripsy.

We have investigated 60 patients in a prospective double-blind, placebo-controlled study to assess the efficacy of EMLA (Eutectic Mixture of Local Anaesthetics) cream to provide analgesia during extracorporeal shock wave lithotripsy (ESWL) with a second generation lithotriptor. Before operation, EMLA or placebo cream was applied to the patient's back at the anticipated shock head-skin interface. During the procedure increments of fentanyl 0.5 micrograms kg-1 were given i.v. on patient demand. There was no significant difference (P = 0.83) in the dose of fentanyl given to each group. We cannot recommend, therefore, the use of EMLA cream as an analgesic during ESWL with a second generation lithotriptor.

Anesthesia, Local↗
PubMed1991

Bacteriohopanetetrol: abundant lipid in frankia cells and in nitrogen-fixing nodule tissue.

An unusual class of lipid with amphiphilic properties has been detected in nodule tissue of Alnus and Ceanothus. High levels of the same lipid (20-50% of total cell lipids) were detected in solvent extracts of Frankia spp. cells. However, the lipid was absent in host roots. The lipid was purified and quantified by high-performance liquid chromatography/flame ionization detector. Phenol-sulfuric acid determinations and proton nuclear magnetic resonance indicated that the purified lipid is not a glycolipid. Mass spectra of the predominant species are consistent with published spectra for bacteriohopanetetrol (C(35)H(62)O(4)), a pentacyclic triterpenoid, or hopanoid.

Journal Article↗
PubMed1991

Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide.

As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

Amino Acid Sequence↗
PubMed1989

Presence of a small plasmid in clinical isolates of Streptococcus pneumoniae.

We have detected the presence of a small (2.95 kb) plasmid in a clinical isolate of Streptococcus pneumoniae. A restriction map was constructed for this plasmid and for pDP1 (the only previously reported pneumococcal plasmid); no apparent differences were observed and the two plasmids hybridized strongly to each other. Portions of pDP1 were then cloned into Escherichia coli K-12, using the vector pUC19, and the pneumococcal DNA insert was used as a probe to screen 500 clinical isolates of S. pneumoniae for pDP1 sequences. The plasmid was detected in a total of 8 isolates. These were of various serotypes and no correlation could be found between the presence of the plasmid and the geographical location from which it came, the type of infection, or with resistance to antibacterial drugs. Although no function has yet been assigned to pDP1, it may form the basis of a useful vector for cloning in S. pneumoniae, as it contains at least seven unique restriction sites.

Blotting, Southern↗
PubMed1989

Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.

Animals↗
PubMed1989

Contribution of autolysin to virulence of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct an autolysin-negative derivative of Streptococcus pneumoniae. This derivative was obtained by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 375-base-pair TaqI DNA fragment from the middle of the autolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, autolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these transformants produced autolysin. Southern blot analysis confirmed that the autolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The autolysin-negative mutants showed markedly reduced virulence for mice compared with that of strain D39; intranasal and intraperitoneal 50% lethal doses were increased 10(2)- and 10(5)-fold, respectively. Autolysin production was reinstated in one of the mutants by back-transformation with the cloned autolysin gene, with the concomitant loss of erythromycin resistance; the virulence of this isolate for mice was indistinguishable from that of D39. The importance of autolysin in pathogenesis was confirmed by immunization-challenge studies. Mice immunized with purified autolysin survived significantly longer than did control mice after intranasal challenge with strain D39. This study provides direct evidence that the pneumococcal autolysin contributes to virulence and identifies it as a potential vaccine antigen.

Amidohydrolases↗