PubMed Health⌕ Search

Biomedical subjects

D Crane

Publications and source records attributed to D Crane.

At least 19 recordsLinked to original sources
PubMed2004

Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1.

The production, purification and characterisation of recombinant gp140 oligomeric envelope glycoproteins derived from six primary isolates of HIV-1 (covering clades A, B, C, D, F and O) are described. Using a Chinese hamster ovary cell expression system, expression levels of between 0.1 and 1 mg/l cell-conditioned culture media were obtained, and purified to >95% by affinity chromatography. A, B, D, F and O clade gp 140s were found to be multimeric, bind to a panel of defined env-specific monoclonal antibodies and interact with CD4 and CXCR4, demonstrating correct folding. Their immunogenicity was confirmed by the generation of high-titre anti-gp140 antibodies in rabbits. The C clade gp140 was incorrectly folded and poorly antigenic. Despite the presence of an unmodified gp120/41 cleavage site, only the B clade gp140 showed significant processing to gp120 and gp41. Each gp140 has a specific pattern of oligomerisation, and varies in its resistance to reducing agents and salt concentration. The binding of gp140 to soluble and cell-surface CD4 and CXCR4 is related to the degree of oligomerisation. The C1 and C5 regions, CD4 binding domain and the epitope defined by the 2G12 monoclonal antibody were well exposed, but the C-terminal region of the extracellular domain of gp41 appears to be occluded by oligomerisation. These reagents have potential as immunogens for use in vaccine development.

Animals↗
PubMed2001

Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity.

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.

Animals↗
PubMed2001

Solution stability studies of the subunit components of meningococcal C oligosaccharide-CRM197 conjugate vaccines.

Spectroscopic methods were used to detect modifications in the structures of CRM197, the mutant diphtheria toxin, and meningococcal C capsular oligosaccharide following their conjugation and incubation at various temperatures. Meningococcal C oligosaccharide-CRM197 conjugate vaccines obtained from two different manufacturers were incubated at -20, 4, 23, 37 or 55 degrees C for 5 weeks or subjected to ten cycles of freeze-thawing. The CRM197 carrier protein and the saccharide components of the treated vaccines were monitored by CD and NMR spectroscopic techniques. CD data indicated incubation temperature-dependent conformational changes in the carrier protein from vaccine A. Modifications appeared in both secondary and tertiary structures of the conjugated CRM(197) when incubated at 23 degrees C or above. This was characteristic of the 'open' conformation previously observed for this protein component. The NMR spectra also indicated modification of the structure of the conjugated CRM197 component of vaccine A when incubated at 23 degrees C or above, but failed to show any modification in the conjugated oligosaccharide. On the other hand, the structure of the oligosaccharide chains in vaccine B appeared to be degraded following incubation at 55 degrees C, even though the thermal effect on the conjugated CRM197 was less apparent. Repeated freeze-thawing did not affect the CD or NMR spectra. In conclusion, the two meningococcal C oligosaccharide-CRM197 conjugate vaccines were stable when stored at their recommended temperatures, but were differently affected by elevated temperatures. The conjugates differ in their conjugation chemistry, attachment positions, oligosaccharide chain length and loading, as well as recommended pH and storage buffer, and their different stability properties can probably be attributed to a combination of these factors.

Circular Dichroism↗
PubMed1999

Differences in the carboxy-terminal (Putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes.

The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.

Amino Acid Sequence↗
PubMed1999

New methods for the characterisation of biopharmaceuticals: conjugate vaccines against Haemophilus influenzae type b.

Modern physicochemical methods allow biological pharmaceuticals, particularly those arising from recombinant DNA technology, to be characterised with a degree of precision not previously possible. These techniques, which tell us what a material is (rather than what it does) provide an approach complementary to traditional bioassays for the control of biological pharmaceuticals. As we come to understand the mechanisms by which structural variation modulates the various biological activities of a product, structure-based assays will be able to replace biological identity and potency assays, although replacement of safety tests to find trace impurities (such as endotoxin) may be more difficult.

Animal Testing Alternatives↗
PubMed1998

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae.

Pneumolysin, a member of the thiol-activated cytolysin family of toxins, is a virulence factor from the Gram-positive bacterium Streptococcus pneumoniae. The toxin forms large oligomeric pores in cholesterol-containing membranes of eukaryotic cells. A plethora of biochemical and mutagenesis data have been published on pneumolysin, since its initial characterization in the 1930s. Here we present an homology model of the monomeric and oligomeric forms of pneumolysin based on the recently determined crystal structure of perfringolysin O and electron microscopy data. A feature of the model is a striking electronegative surface on parts of pneumolysin that may reflect its cytosolic location in the bacterial cell. The models provide a molecular basis for understanding the effects of published mutagenesis and biochemical modifications on the toxic activity of pneumolysin. In addition, spectroscopic data are presented that shed new light on pneumolysin activity and have guided us to hypothesise a detailed model of membrane insertion. These data show that the environment of some tryptophan residues changes on insertion and/or pore formation. In particular, spectroscopic analysis of a tryptophan mutant, W433F, suggests it is the residue mainly responsible for the observed effects. Furthermore, there is no change in the secondary structure content when the toxin inserts into membranes. Finally, the basis of the very low activity shown by a pneumolysin molecule from another strain of S. pneumoniae may be due to the movements of a key domain-domain interface. The molecular basis of pneumolysin-induced complement activation may be related to the structural similarity of one of the domains of pneumolysin to Fc, rather than the presumed homology of the toxin to C-reactive protein as previously suggested.

Amino Acid Sequence↗
PubMed1998

On the role of the peroxisome in cell differentiation and carcinogenesis.

This article reviews the currently available data on the role of peroxisomal function in relation to the processes of cell differentiation and carcinogenesis. In regard to tumourigenesis, both genotoxic and non-genotoxic processes have been considered, and the peroxisomal relationships with these phenomena and with differentiation are described at the level of organelle characteristics, enzyme contents, and the involvement of retinoids, steroid hormones, oxygen free radicals, growth factors, apoptosis, omega-3 polyunsaturated fatty acids and the cellular signalling networks. Overall these data serve to illustrate the unique and distinctive role of the peroxisome in differentiation and carcinogenesis, and point to the advantages of considering the peroxisomal involvement in the holistic context of the differentiation dedifferentiation continuum rather than the narrower focus of non-genotoxic carcinogenesis. The review also outlines the potential for medical benefit arising from a fuller understanding of these peroxisomal affiliations.

Animals↗
PubMed1997

Structural and functional characterisation of two proteolytic fragments of the bacterial protein toxin, pneumolysin.

Proteolytic cleavage of the bacterial protein toxin pneumolysin with protease K creates two fragments of 37 and 15 kDa. This paper describes the purification of these two fragments and their subsequent physical and biological characterisation. The larger fragment is directly involved in the cytolytic mechanism of this pore-forming protein, via membrane binding and self-association. The smaller fragment lacks ordered structure or discernible activity.

Bacterial Proteins↗
PubMed1996

Recent developments in peroxisome biology.

Peroxisomes are subcellular organelles that are present in all eukaryotic organisms. These organelles are the focus of much contemporary interest among cellular and medical biologists--an interest which coincides with the realization of their vital role in higher organisms, their unique metabolic and biogenetic characteristics, and their widespread involvement in genetic and degenerative disease. This article reviews some of the major recent developments in peroxisome biology.

Animals↗
PubMed1994

Developmental variations in the interactions of pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase with subcellular structure in cavian tissues.

The activities and interactions with cellular structure of glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase have been studied in the major tissues of the guinea pig during development. The extent of activity variation in these tissues is described along with the putative physiological determinants of these alterations in activity. As to binding, overall the present data provide a firm indication that the extent of enzyme-structure interactions is appreciable at all ontogenic stages, and when viewed in conjunction with other parallel studies on other enzymes and other animals, serve to confirm the broad biological significance of enzyme-structure associations in the compartmentation of glycolysis. The existence and significance of genetic and epigenetic modifications of these enzymes during development is also discussed.

Aging↗
PubMed1994

Radiosensitization with carotid intra-arterial bromodeoxyuridine +/- 5-fluorouracil biomodulation for malignant gliomas.

Bromodeoxyuridine (BUdR), a nonhypoxic radiosensitizing drug, is a halogenated pyrimidine analog that is incorporated into the DNA of dividing cells in a competitive process with thymidine. BUdR sensitizes cells to radiation therapy. 5-Fluorouracil (5-FU) inhibits the endogenous synthesis of thymidine, resulting in increased incorporation of the BUdR. Neurons and glial cells have a very low mitotic rate; they will not incorporate BUdR and will not be sensitized. BUdR and 5-FU are best delivered intra-arterially (IA) because of their regional advantage. We infused BUdR +/- 5-FU over 8 1/2 weeks, before and during 59.4-Gy focal conformal external beam radiation therapy, through a permanently implanted pump with a catheter placed retrograde through the external carotid artery to the carotid bifurcation. Sixty-two patients with grades III or IV glioma were entered into one of two trials, with 23 patients receiving BUdR alone and 39 patients receiving BUdR + 5-FU. The maximum tolerated dose (MTD) of BUdR alone was 400 mg/m2/d for 8 1/2 weeks. The Kaplan-Meier median survival (KMS) was 20 months. In the BUdR + 5-FU trial, the MTD of BUdR was also 400 mg/m2/d and 5-FU was 5 mg/m2/d with a KMS of 17 months. The KMS of all 62 patients in both trials 1 and 2 was 18 months. Pathologic grading used both the original World Health Organization (WHO) and 1993 modified WHO systems. The KMS of grade IV patients was 13.8 months (48 patients) with the original system and 17 months (58 patients) with the modified system.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Ontogenic activities and interactions of the lactate dehydrogenase isozymes with cellular structure in the guinea pig.

The LDH activities and isozyme distributions associated with soluble and particulate fractions of five major tissues have been followed during the development of the guinea pig. Evidence has been provided for an appreciable degree of interaction between LDH and cellular structure in all these tissues (liver, kidney, skeletal muscle, brain and heart) at all stages of development, but particularly in the early foetal stages. These data have been discussed in relation to the nature and extent of this binding and the correlations with the metabolic emphases in these tissue situations during development.

Animals↗