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Biomedical subjects

M A Baker

Publications and source records attributed to M A Baker.

At least 19 recordsLinked to original sources
PubMed2004

A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

Animals↗
PubMed2003

Multiple forms of redox activity in populations of human spermatozoa.

In this study we have examined the biochemical attributes of the redox systems that regulate human sperm function using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt (WST-1), lucigenin and luminol-peroxidase as probes. WST-1 was readily reduced by human spermatozoa in the presence of an intermediate electron acceptor (IEA) or NAD(P)H. The IEA-mediated activity resembled a previously described trans-membrane NADH oxidase in being inhibited by capsaicin, superoxide dismutase (SOD) and N-ethyl maleimide, but differed in its sensitivity to p-chloromercuriphenylsulphonic acid (pCMBS). The NAD(P)H-induced WST-1 reduction resembled the superficial oxidase described previously, in its sensitivity to pCMBS, but differed in its suppression by capsaicin. Lucigenin was reduced by human spermatozoa in a manner that could be inhibited by SOD and stimulated by NAD(P)H or 12-myristate, 13-acetate phorbol ester. A23187 also stimulated human spermatozoa via a diphenylene iodonium-sensitive pathway detectable with luminol-peroxidase but not lucigenin. Defective sperm populations recovered from the low-density region of Percoll gradients were characterized by high levels of redox activity that was only discernable with lucigenin. We conclude that human spermatozoa possess multiple plasma membrane redox systems that are involved to varying extents in the physiological control and pathological disruption of sperm function. Their distinct pharmacological profiles should significantly assist attempts to resolve and characterize these systems.

Acridines↗
PubMed2002

Didemnin B induces apoptosis in proliferating but not resting peripheral blood mononuclear cells.

We have previously shown that didemnin B, a branched cyclic depsipeptide composed of seven amino acids and two hydroxy acids, can induce rapid and complete apoptosis in HL-60 cells (Grubb, D.R. et al. (1995) Biochem. Biophys. Res. Commun. 215, 1130-1136). We now report that didemnin B can induce apoptosis in a wide range of transformed cell lines. Resting normal lymphocytes, however, are apparently unaffected by exposure to the drug. To investigate whether cell transformation, and/or cell proliferation is necessary for didemnin B to induce apoptosis, we examined the effect of didemnin B on freshly harvested human lymphocytes before and after stimulation with concanavalin A. Didemnin B induced apoptosis in normal lymphocytes only after mitogenic stimulation and therefore warrants further examination for its potential as a chemotherapeutic agent, especially for treatment of leukemia.

Antineoplastic Agents↗
PubMed2001

Surface and biological evaluation of hydroxyapatite-based coatings on titanium deposited by different techniques.

Hydroxyapatite coatings have been deposited on titanium cp by plasma spray, sol-gel, and sputtering techniques for dental implant applications. The latter two techniques are of current interest, as they allow coatings of micrometer dimensions to be deposited. Coating morphology, composition, and structure have been investigated by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). All coatings were homogeneous and exhibited a rough morphology suitable for implant applications. The sputtered (after annealing), plasma spray, and sol-gel coatings all showed diffraction peaks corresponding to hydroxyapatite. The surface contaminants were observed to be different for the different coating types. The sputtered coatings were found to have a composition most similar to hydroxyapatite; the sol-gel deposits also showed a high concentration of hydroxyl ions. A discrepancy in the Ca/P ratio was observed for the plasma spray coatings, and a small concentration of carbonate ions was found in the sputter-deposited coatings. The in vitro cell-culture studies using MG63 osteoblast-like cells demonstrated the ability of cells to proliferate on the materials tested. The sol-gel coating promotes higher cell growth, greater alkaline phosphatase activity, and greater osteocalcin production compared to the sputtered and plasma-sprayed coatings.

Alkaline Phosphatase↗
PubMed2001

Malignant lymphoma of mucosa-associated lymphoid tissue of the lacrimal gland: case report and review of literature.

Mucosa-associated lymphoid tissue (MALT) lymphomas are increasingly recognized as a distinct clinical-pathologic entity among the non-Hodgkin's lymphomas. It usually presents as a localized disease process in extranodal tissues or organs such as stomach, salivary gland, thyroid gland, and not infrequently in orbital adnexa. Radiotherapy has an important role in the management, although long-term clinical results specifically addressing localized MALT lymphomas are lacking. We report a case of localized MALT lymphoma of the lacrimal gland, with successful treatment by radiation therapy (total dose 25 Gy) with 3 years of clinical follow-up. A review of the published literature was undertaken to assess the role of radiotherapy in the treatment of this disease involving orbital tissues, specifically, the lacrimal gland. Based on previous reports of patients with orbital lymphomas (low grade) and pseudolymphomas, of which many will now be recognized as MALT lymphomas, radiotherapy has an excellent local control rate and would be the treatment of choice. However, long-term results of pathologically confirmed cases of MALT lymphomas need further study because occasional relapses at distant sites can occur.

Eye Neoplasms↗
PubMed2000

Isolation of MFO inducers from tissues of white suckers caged in bleached kraft mill effluent.

White sucker (Catostomus commersoni) caged for 3 days in a bleached kraft mill effluent (BKME) stream had elevated mixed function oxygenase (MFO) activities 15-90 fold those of fish caged in a reference stream. Liver composites of male and female fish were ground and extracted with dichloromethane (DCM), methanol or 50% DCM/methanol, and tested for MFO activity in rat hepatoma cells (H4IIE). There was no difference in the potency of H4IIE EROD induction among the three solvents, so DCM extracts were split into 31 fractions using reverse phase high pressure liquid chromatography (HPLC). H4IIE MFO activity was elevated in several fractions, with three early peaks and several later peaks of induction, indicating several classes or compounds causing MFO induction were present in the fish livers. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and chlorinated diphenylethers (CDPEs) were detected in several late-eluting fractions, but concentrations were not high enough for these compounds to be solely responsible for the observed induction. Induction by liver extracts decreased as cell exposure times increased (24, 48 or 72 h), suggesting that some inducers were more easily metabolized and eliminated from the H4IIE cells. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TCDD) had similar potency over 24, 48 and 72 h, as it was relatively resistant to metabolism.

Animals↗
PubMed2000

Plasma membrane NADH-oxidoreductase system: a critical review of the structural and functional data.

The observation in the early 1970s that ferricyanide can replace transferrin as a growth factor highlighted the major role plasma membrane proteins can play within a mammalian cell. Ferricyanide, being impermeant to the cell, was assumed to act at the level of the plasma membrane. Since that time, several enzymes isolated from the plasma membrane have been described, which, using NADH as the intracellular electron donor, are capable of reducing ferricyanide. However, their exact modes of action, and their physiological substrates and functions have not been solved to date. Numerous hypotheses have been proposed for the role of such redox enzymes within the plasma membrane. Examples include the regulation of cell signaling, cell growth, apoptosis, proton pumping, and ion channels. All of these roles may be a result of the function of these enzymes as cellular redox sensors. The emergence of many diverse roles for ferricyanide utilizing redox enzymes present in the plasma membrane might also, in part, be due to the numerous redox enzymes present within the membrane; the poor molecular characterization of the enzymes may be the reason for some of the diverging results reported in the literature as various researchers may be working on different enzymes. Here we review the diverse proposals given for structure and function to the plasma membrane NADH-oxidoreductase system(s) with a specific focus on those enzyme activities which can couple ferricyanide and NADH. Although they are still ill-defined enzymes, evidence is rising that they are of utmost significance for cellular regulation.

Animals↗
PubMed1999

Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts.

We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5-14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10-30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400-550 nM; Vmax 11 pmol/min/10(6) cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death.

Anemia, Megaloblastic↗
PubMed1997

Estrogen replacement in ovariectomized rats results in physiologically significant levels of circulating progesterone, and co-administration of progesterone markedly reduces the circulating estrogen.

Estrogen and progesterone replacement in ovariectomized rats in an often-used experimental system for determination of the specific effects of these hormones. In this study, two different delivery systems and two different dosage levels of estrogen, progesterone or a combination of the two have been used. Estrogen and progesterone in the circulation have been measured in response to each treatment. It is reported that estrogen treatment (237.2 +/- 49.2 pg/mL) results in physiologically significant levels of circulating progesterone (11.1 +/- 1.3 ng/mL). Also, co-administration of progesterone (23.7 +/- 2.0 ng/mL) with estrogen decreases the level of estrogen over that seen with estrogen alone (96.7 +/- 19.2 pg/mL with progesterone vs 237.2 +/- 49.2 pg/mL without progesterone). Thus, contrary to expectations, estrogen replacement therapy is not specific to estrogen and some of the antagonistic effects of progesterone are the result of a decrease in circulating estrogen, and not a specific effect on a target tissue. Whereas the mechanism of these effects has not been determined, obvious artifactitous phenomena have been excluded as being their cause. These results could have a major impact on the interpretation of past and future experiments of this kind.

Animals↗
PubMed1997

Thermoregulation during exercise in women who are taking oral contraceptives.

Thermoregulation during exercise was studied in seven women who were taking oral contraceptive pills for 3 weeks of each month. The subjects were studied once in the 3rd week of taking the pill (P) and once during the following week when they took no pil (N). Rectal temperature (Tre), heart rate (fc) and evaporative water loss (EWL, ventilated capsule technique) were measured while they walked on a treadmill for 60 min at 4.8 km.h-1 at a 10% gradient. Ambient temperature was 22 degrees C. A venous blood sample was drawn 30 min before each experiment for measurement of hematocrit (packed cell volume, PCV), plasma osmolality (Osmpl), and plasma levels of the endogenous pyrogens interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6). Resting Tre was 0.31 degree C higher in P than in N (P < 0.01) and Tre remained higher in P throughout the entire exercise period (P < 0.01). Threshold Tre for the onset of EWL was 0.32 degree C higher in P than in N (P < 0.01). Exercise fc was 6.5 beats.min-1 higher in P than in N (P < 0.01). There was no significant difference in PCV, Osmpl, IL-1 beta or IL-6 between P and N. It was concluded that the administration of synthetic progestins in oral contraceptives causes an upward shift in the threshold for heat loss responses, resulting in higher body core temperatures both at rest and during exercise. There was no evidence that these alterations in thermoregulation were mediated by changes in body fluid balance or in plasma levels of IL-1 beta or IL-6.

Adult↗
PubMed1997

Oral immunization with attenuated vaccine strains of Vibrio cholerae expressing a dodecapeptide repeat of the serine-rich Entamoeba histolytica protein fused to the cholera toxin B subunit induces systemic and mucosal antiamebic and anti-V. cholerae antibody responses in mice.

Entamoeba histolytica is a significant cause of morbidity and mortality worldwide. The serine-rich E. histolytica protein (SREHP) is a surface-expressed trophozoite protein that includes multiple hydrophilic tandem repeats. A purified fusion protein between the dodecapeptide repeat of SREHP and cholera toxin B subunit (CTB) has previously been shown to be immunogenic in mice after oral inoculation when cholera toxin is coadministered as an immunoadjuvant. We engineered a live attenuated El Tor Vibrio cholerae vaccine strain, Peru2, to express the SREHP-12-CTB fusion protein to the supernatant from either a plasmid [Peru2 (pETR5.1)] or from a chromosomal insertion (ETR3). Vector strains were administered orally to germfree mice that were subsequently housed under nongermfree conditions; mice received one (day 0) or two (days 0 and 14) inoculations. No immunoadjuvant or cholera holotoxin was administered. Mice that received two inoculations of Peru2(pETR5.1) had the most pronounced antiamebic systemic and mucosal immunologic responses. Less marked, but significant, anti-SREHP serum immunoglobulin G antibody responses were also induced in mice that received either one or two oral inoculations of strain ETR3. Anti-V. cholerae responses were also induced, as measured by the induction of serum vibriocidal antibodies and by serum and mucosal anti-CTB antibody responses. These results suggest that V. cholerae vector strains can be successful delivery vehicles for the SREHP-12-CTB fusion protein, to induce mucosal and systemic antiamebic and anti-V. cholerae immune responses. The magnitude of these responses is proportional to the amount of SREHP-12-CTB produced by the vector strain.

Administration, Oral↗
PubMed1996

Use of cleaved amplified polymorphic sequences to distinguish strains of Staphylococcus epidermidis.

We examined the utility of a PCR-based method termed cleaved amplified polymorphic sequences (CAPS) to type 35 well-characterized isolates of Staphylococcus epidermidis. The results were compared with detailed epidemiologic information and typing obtained by using pulsed-field gel electrophoresis (PFGE). To identify CAPS markers for this study, eight pairs of oligonucleotide primers corresponding to five previously sequenced S. epidermidis genes were synthesized and then used to amplify DNA sequences from the S. epidermidis strains by using PCR. Amplified products were reproducibly obtained for seven of eight primer pairs from chromosomal DNA of 33 of the 35 isolates. Seven restriction site polymorphisms were found in five of the amplified products when they were subjected to digestion with a panel of restriction endonucleases. Each fragment-enzyme combination that was polymorphic demonstrated only two alleles in the 33 S. epidermidis isolates analyzed, corresponding to the presence or absence of a single restriction site. Overall, five distinct combinations of alleles were detected and were designated CAPS types A through E. There was a close correlation between the CAPS grouping, the epidemiologic information for the strains, and grouping by PFGE following SmaI digestion of chromosomal DNA. Although PFGE analysis was more discriminatory than typing based on the limited number of CAPS markers used in this study (isolates from the same CAPS group were sometimes distributed into more than one PFGE group), no isolates from the same PFGE group were found in more than one CAPS group. The CAPS procedure was highly reproducible, in contrast to published experience with arbitrarily primed PCR. These preliminary data suggest that CAPS represents a PCR-based technique for strain typing that is highly reproducible, rapid, utilizes widely available technologies, and provides results that are relatively easy to interpret and express.

Bacterial Typing Techniques↗
PubMed1995

Expression of selenium-dependent glutathione peroxidase in human breast tumor cell lines.

In estrogen receptor (ER)-positive breast cancer cell lines, very low expression of glutathione peroxidase-1 (GPX-1) activity and hgpx1 mRNA has been observed. Such cell lines have been used as models in studies of resistance to redox cycling anticancer drugs. In particular, large increases in GPX-1 activity levels by expression of transfected GPX-1 cDNA have been shown to confer some resistance to such drugs. It has never been determined that such low GPX-1 expression is a common feature of breast cancer. Based on previous limited surveys of breast cancer cell lines, it has been suggested that there may be an inverse correlation between ER status and GPX-1 production. Here we report the results from a larger survey of breast cancer cell lines, including six recently isolated cell lines. A near absence of hgpx1 mRNA expression was observed in 3 of 13 ER-negative cell lines; 1 of 4 ER-positive cell lines had high production of GPX-1. Both observations weaken the proposed inverse correlation between ER status and GPX-1 production. We have evidence to suggest that one cell line, COH-BR-5 (ER-negative), lacked hgpx1 gene expression prior to culture. This is based on the finding of stable hgpx1 gene expression during serial culture of ER-negative breast cancer cell lines newly isolated from malignant effusion and absence of hgpx1 mRNA expression in COH-BR-5. Expression of hgpx2 mRNA (producing GPXGI, the GI tract GPX) was detected in several long and newly established, ER-negative breast cancer cell lines. Cell lines, COH-BR-5 and MDA-MB-175, expressed only hgpx2 mRNA. The hgpx2 mRNA was detected in COH-BR-5 and COH-BR-7 at low passage number, suggesting that hgpx2 gene expression occurs in breast cancer malignant effusion. Thus, studies of the role of GPX in redox drug resistance may account for changes in hgpx2 gene expression. Phospholipid hydroperoxide GPX activity was not found to be generally elevated above normal tissue levels in newly established breast cancer-derived cell lines.

Breast Neoplasms↗
PubMed1995

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R beta 1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues.

UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R (GlcNAc to Man) beta 1,6- N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc beta 1-6 branch to bi- and triantennary N-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc beta 1-2Man alpha 1-6Glc/Man beta-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man alpha 1-3 residue and the 4-hydroxyl of the Man beta- residue of the Man alpha 1-6(Man alpha 1-3)Man beta-R N-glycan core are not essential for catalysis but influence substrate binding. GlcNAc beta 1-2(4,6-di-O-methyl-)Man alpha 1-6Glc beta-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Animals↗
PubMed1995

Characterization of a novel mucin sulphotransferase activity synthesizing sulphated O-glycan core 1,3-sulphate-Gal beta 1-3GalNAc alpha-R.

A novel sulphotransferase (sulpho-T) activity from rat colonic mucosa was characterized using O-glycan core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl. Derivatives of Gal beta 1-3GalNAc- were used to demonstrate that the 3- and 4-hydroxyl of Gal and the 2-acetamido group of the GalNAc residue of Gal beta 1-3GalNAc alpha-benzyl substrates were important for activity. Sulphated product using Gal beta 1-3GalNAc alpha-benzyl as substrate was analysed by ion spray mass spectrometry, methylation analysis, high-pH anion-exchange chromatography and beta-galactosidase digestion. The results suggested that sulphate was added to the 3-position of the Gal residue. The synthesis of core 2 from core 1 by UDP-GlcNAc: Gal beta 1-3GalNAc beta 6-GlcNAc-transferase was inhibited by sulphation of the Gal residue, indicating that GlcNAc beta 1-6 branching has to precede sulphation in the O-glycan core 1 processing pathway. These data demonstrate several novel pathways in the synthesis of sulphated mucin-type oligosaccharides.

Animals↗
PubMed1995

Inhibitors of carbohydrate processing: A new class of anticancer agents.

There is a need for anticancer agents with novel mechanisms of action. Recently identified molecular targets for new anticancer agents include inducers of cell differentiation, cell cycle arrest, and apoptosis, as well as signaling pathways for growth factors and cytokines. Another unexplored opportunity is presented by the ubiquitous intracellular glycoprotein glycosylation pathway. This complex process, concerned with the addition of sugars onto newly synthesized proteins, occurs in the lumen of the rough endoplasmic reticulum and in the Golgi. There are estimates of over 200 glycosyltransferase enzymes in this pathway, which results in considerable structural diversity of carbohydrates found on secreted and transmembrane glycoproteins. The specificity of glycosyltransferases for acceptors and sugar-nucleotide donors dictates linkage positions between sugars, anomeric configuration of linkages, and monosaccharide composition. Specific carbohydrate structures participate in cell-cell and cell-substratum interactions affecting processes such as lymphocyte trafficking, immune cell stimulation, embryogenesis, and cancer metastasis. Of the carbohydrate-processing inhibitors presently available, the alkaloid swainsonine, a Golgi alpha-mannosidase II inhibitor, is the first to have been selected for clinical testing based on its anticancer activity, p.o. availability, and low toxicity in mice. Herein, we review the rationale for targeting Golgi carbohydrate processing pathways in the treatment of cancer, and summarize the preclinical and clinical results with swainsonine. Prospects for the development of second generation inhibitors with improved specificity for Golgi-processing enzymes are discussed. Potential clinical applications of this new class of anticancer agents are emphasized.

Animals↗