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Biomedical subjects

D Mead

Publications and source records attributed to D Mead.

At least 19 recordsLinked to original sources
PubMed2001

Chlamydia pneumoniae major outer membrane protein is a surface-exposed antigen that elicits antibodies primarily directed against conformation-dependent determinants.

The major outer membrane protein (MOMP) of Chlamydia trachomatis serovariants is known to be an immunodominant surface antigen. Moreover, it is known that the C. trachomatis MOMP elicits antibodies that recognize both linear and conformational antigenic determinants. In contrast, it has been reported that the MOMP of Chlamydia pneumoniae is not surface exposed and is immunorecessive. We hypothesized that the discrepancies between C. trachomatis and C. pneumoniae MOMP exposure on intact chlamydiae and immunogenic properties might be because the focus of the host's immune response is directed to conformational epitopes of the C. pneumoniae MOMP. We therefore conducted studies aimed at defining the surface exposure of MOMP and the conformational dominance of MOMP antibodies. We present here a description of C. pneumoniae species-specific monoclonal antibody (MAb), GZD1E8, which recognizes a conformational epitope on the surface of C. pneumoniae. This MAb is potent in the neutralization of C. pneumoniae infectivity in vitro. Another previously described C. pneumoniae species-specific monoclonal antibody, RR-402, displayed very similar characteristics. However, the antigenic determinant recognized by RR-402 has yet to be identified. We show by immunoprecipitation of C. pneumoniae with GZD1E8 and RR-402 MAbs and by mass spectrometry analysis of immunoprecipitated proteins that both antibodies GZD1E8 and RR-402 recognize the MOMP of C. pneumoniae and that this protein is localized on the surface of the organism. We also show that human sera from C. pneumoniae-positive donors consistently recognize the MOMP by immunoprecipitation, indicating that the MOMP of C. pneumoniae is an immunogenic protein. These findings have potential implications for both C. pneumoniae vaccine and diagnostic assay development.

Amino Acid Sequence↗
PubMed2000

Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter.

The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simplified fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more flexibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the first 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H(+)-ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter.

Alcohol Oxidoreductases↗
PubMed1998

The biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene) transfer to acyl carrier protein bound meromycolic acid in vitro.

A closely related family of enzymes from Mycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-L-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain.

Acyl Carrier Protein↗
PubMed1998

Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid.

Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1996

Using nursing initiatives to encourage the use of research.

This year the International Council of Nurses has decided to make nursing research the theme for its Nurses' Day. To coincide with the launch of the ICN's pack to help nurses get involved with the theme, this article outlines the development of nursing research and discusses what progress has been made. The author also discusses the difficulties involved in getting research into practice and what can be done to rectify the problem.

Humans↗
PubMed1995

Expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes during cell cycle traverse.

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

SilkTouch: a new technology for skin resurfacing in aesthetic surgery.

We describe layer-by-layer char-free facial skin resurfacing at very low CO2 laser power levels with a miniature "SilkTouch" microprocessor-controlled optomechanical flashscanner. This device provides excellent ablation depth control with minimal thermal damage to the dermis. Indications for the laser in aesthetic surgery include perioral, lips, and periorbital wrinkles, among others.

Animals↗
PubMed1993

Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations.

The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF-alpha leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non-selective growth. The stabilized strain was grown to high cell density by fed-batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0.5 g/1 or 0.65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0.43 g/1 or 0.56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g/l) and produced 445 IU/ml(2.25 g/l or 2.2% dry weight) extracellularly and 76 IU/ml (0.38 g/l or 0.4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain.

Aspergillus niger↗
PubMed1993

A neodymium:YAG fiber delivery system for interstitial photothermal therapy.

We describe here a highly durable 600-mu optical fiber with a 20-mm frosted distal tip protected by a smooth transparent cover that is capable of remaining in contact with tissue for prolonged periods. When used with a neodymium:YAG (Nd:YAG) laser, the active fiber surface diffuses optical radiation in a radial pattern, delivering up to 40 W power, and thus providing consistent and uniform interstitial photothermal therapy. Preliminary animal studies have demonstrated the feasibility of using these fibers to treat a variety of soft-tissue tumors, including benign prostatic hyperplasia.

Animals↗
PubMed1993

Analyzing the red-shift characteristics of azulenic, naphthyl, other ring-fused and retinyl pigment analogs of bacteriorhodopsin.

Prompted by the near infrared-absorbing properties of some of the azulenic bacteriorhodopsin (bR) analogs, we have analyzed their absorption characteristics along with 11 new related ring-fused analogs and the corresponding Schiff bases (SB) and protonated Schiff bases (PSB). The following three factors are believed to contribute to the total red shift of each of the pigment analogs (sigma RS): perturbation of the basic chromophore (SB shift, delta SB), protonation of the SB (PSB shift, PSBS) and protein perturbation (the opsin shift, OS). For each factor, effects of structural modifications were examined. For the red-shifted pigments, percent OS has been suggested as an alternate way of measuring protein perturbation. Computer-simulated chromophores provided evidence against any explanation involving altered shapes of the binding pocket as a major cause for absorption differences. Implications of the current bR results on preparation of further red-shifted bR and possible application to visual pigment analogs are discussed.

Absorption↗
PubMed1992

The integration of a database and a statistical program in the analysis of a large scale survey in nursing.

Large scale surveys have conventionally been analysed using a statistical package. This saves considerable time and permits many more analyses to be undertaken than with more traditional methods. It does, however, have certain drawbacks. First, it does not help with the problem of data management. SPSS and Epfino, for example, have some facilities for data entry restrictions and cross-checking. They are excellent for the static purpose for which they are designed, but rather crude in comparison with those provided by a dedicated database tool. Second, it requires considerable experience to achieve maximum benefit. Third, it does not help at all in the major undertaking of data exploration. The authors have analysed a large scale survey using a statistical package and a database in collaboration. Their experience is reported here.

Databases, Bibliographic↗
PubMed1991

NMR studies of fluorinated visual pigment analogs.

The 19F-nmr chemical shift data of isomeric pigments (11-cis and 9-cis) of four vinyl fluororhodopsins and two trifluororhodopsins have been recorded. When compared with model protonated Schiff bases, a set of F-nmr opsin shift parameter (FOS) was obtained. The data revealed regiospecific protein perturbations on the F-resonances. They can be interpreted in terms of specific protein interactions such as the postulated second point charge and other polar interactions as well as the common hydrophobic protein perturbation.

Fluorine↗
PubMed1990

14-Fluorobacteriorhodopsin and other fluorinated and 14-substituted analogues. An extra, unusually red-shifted pigment formed during dark adaptation.

Five vinyl-substituted fluororetinal analogues (8-F, 10-F, 12-F, 14-F, and 13,14-F2) were found to give bacteriorhodopsin analogues with properties similar to those of the parent system. Of these, only 14-fluororetinal was found to give an extra red-shifted BR analogue (lambda max less than or equal to 680 nm) in equilibrium with the normal 587-nm pigment. The 680-nm pigment was enriched upon irradiation. It rearranged to the 587-nm pigment at room temperature (delta E [symbol: see text] = 20.8 kcal/mol). Chromophore extraction experiments revealed the all-trans geometry for the 680-nm pigment. 14-Chlororetinal gave a similarly red-shifted pigment while 14-methylretinal did not. A scheme for dark adaptation of the 14-halogenated bacteriorhodopsins has been proposed in which the new red-shifted pigment was assigned the all-trans, 15-syn geometry.

Bacteriorhodopsins↗
PubMed1990

Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues.

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide.

The NH2-terminal sequences of cytochromes P-450 resemble signal peptides, but these sequences are not cleaved during the insertion of these integral membrane proteins into the microsomes. To examine whether these putative signal peptides are functionally equivalent to signal peptides of secretory proteins, cDNA coding for a fusion protein was produced, in which the signal peptide for preproparathyroid hormone was replaced with the putative signal peptide of cytochrome P450IIC2. The translational product of RNA synthesized in vitro from the cDNA was neither processed nor translocated by chicken oviduct microsomal membranes in a reticulocyte cell-free system but was resistant to extraction from the membranes by alkaline solutions. In addition, the translation of the hybrid RNA was arrested by signal recognition particle. Unlike most signal peptides, the cytochrome P450IIC2 NH2-terminal sequence does not contain basic amino acids preceding the hydrophobic core. Introduction by oligonucleotide-directed mutagenesis of lysine and arginine at the NH2 terminus resulted in a fusion protein that was partially processed by the microsomal membranes, with translocation across the membrane of both the processed and unprocessed proteins. The positive charges convert the cytochrome P450IIC2 NH2 terminus from a combination membrane insertion-halt transfer signal to a more classical secretory membrane-insertion signal, possibly by altering the orientation of the signal peptide in the membrane.

Amino Acid Sequence↗