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Biomedical subjects

A Martin

Publications and source records attributed to A Martin.

At least 307 records · Page 17Linked to original sources
PubMed1997

Characterization of a recombinant Neisseria meningitidis alpha-2,3-sialyltransferase and its acceptor specificity.

The structure and specificity of the recombinant alpha-2,3-sialyltransferase from Neisseria meninigitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use alpha-terminal and beta-terminal Gal residues as acceptors. In addition (beta1-->4)-linked and (beta1-->3)-linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane-associated protein in Escherichia coli at a level of 750 U/l (approximately 250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X-100 and purified to homogeneity using immobilized-metal-affinity chromatography. Electrospray-ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95% of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP-Neu5Ac synthetase, the alpha-2,3-sialyltransferase could sialylate fluorescent derivatives of N-acetyllactosamine with N-acetylneuraminic acid, N-propionylneuraminic acid and N-glycoloylneuraminic acid.

Amino Acid Sequence↗
PubMed1997

A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix.

The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition.

Alanine-tRNA Ligase↗
PubMed1997

Diacylglycerol and phosphatidate generated by phospholipases C and D, respectively, have distinct fatty acid compositions and functions. Phospholipase D-derived diacylglycerol does not activate protein kinase C in porcine aortic endothelial cells.

Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.

3T3 Cells↗
PubMed1997

The genes for gonadotropin-releasing hormone and its receptor are expressed in human breast with fibrocystic disease and cancer.

While gonadotropin-releasing hormone (GnRH) or GnRH receptor (GnRHR) have been reported to exist in tissues other than brain and pituitary, there is no report concerning co-expression of GnRH and GnRHR in human breast tissues. To address this question, we have examined whether mRNA for GnRH as well as GnRHR was present in different human breast samples, by employing the reverse transcription-polymerase chain reaction (RT-PCR) protocol followed by Southern blotting of the PCR products. Coexpression of GnRH and GnRHR genes was further confirmed by dot blot hybridization using appropriate [32P]-labeled probes. We thus tested fibrocystic breast (4 cases), invasive ductal carcinomas (6 cases) and 1 adjacent non-neoplastic tissue. GnRHR and GnRH mRNAs were found in all actin-positive samples including malignant as well as nonmalignant tissues. Quantitative determinations of mRNA did not reveal significant differences between malignant and non-malignant breast samples for either GnRH or GnRHR gene expression. Our data show that neither gene was overexpressed in the breast cancer samples compared with normal breast tissue. Since GnRH agonists inhibit breast epithelial cell growth, the presence of GnRHR mRNA suggests that GnRH may specifically affect breast cell growth. Our data thus raise the possibility of an autocrine/paracrine role for GnRH in human mammary gland.

Breast↗
PubMed1997

Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts.

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras- and tyrosine kinase (fps)-transformed fibroblasts but not in control fibroblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3- and 5.5-fold higher respectively in the ras-transformed compared to control fibroblasts. These concentrations in fps-transformed fibroblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed fibroblasts. Ceramide concentrations were about 2.5- and threefold higher in the fps- and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control fibroblasts from Days 1 to 3 with phosphatidylcholine-specific phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg2+-independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic effect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

Animals↗
PubMed1997

Hypercalcemia may indicate Richter's syndrome: report of four cases and review.

BACKGROUND: Hypercalcemia has rarely been reported in patients with Richter's syndrome (RS). In this article, the authors report four cases with complementary pathophysiologic examinations. METHODS: Four patients with hypercalcemia that indicated RS were admitted to the study unit between 1980 and 1994 with the following diagnoses: transformation of mixed follicular lymphoma, Waldenstrom's macroglobulinemia, and chronic lymphocytic leukemia, respectively, in 3 men ages 60 to 73 years, and chronic lymphocytic leukemia in a woman 73 years of age. Radiologic and histologic features were reported. Circulating levels of parathormone-related peptide (PTHrP), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were measured. RESULTS: Radiologic skeletal abnormalities were found in three of three cases: osteopenia twice and multifocal osteolysis once. All four patients had large cell lymphoma (LCL). Bone resorption assessed histomorphometrically was elevated in two of two cases. Serum TNF-alpha and IL-6 levels were high in three of three and one of three cases, respectively. Elevated values were also found in four patients with LCL but without hypercalcemia. The serum PTHrP level was increased in the only hypercalcemic patient tested and values were normal in the three control patients. CONCLUSIONS: Hypercalcemia arising in a patient with a low grade lymphoproliferative disorder may indicate RS. Hypercalcemia is due to increased bone resorption, which may be caused by the secretion of osteoclast-stimulating factors by the LCL invading the bone marrow. Independently, TNF-alpha and/or IL-6 were unlikely causative factors of hypercalcemia in the patients in this study; however, synergism with PTHrP was suspected in one case.

Aged↗
PubMed1997

Purification and characterization of sn-1-stearoyl-2-arachidonoylglycerol kinase from pig testes.

1-Stearoyl-2-arachidonoylglycerol (SAG) kinase was identified in the particulate fraction of pig testes. This activity was enriched by hydroxyapatite and blue dye chromatography. The enzyme was selective for polyunsaturated diradylglycerol species and activity was not modulated by other diradylglycerol species or sphingomyelin metabolites. Further purification resulted in the isolation of 55 and 50 kDa proteins that corresponded with SAG kinase activity. These results support the view that the phosphorylation of polyunsaturated diradylglycerol is regulated by structural determinants in the molecule.

Animals↗
PubMed1997

Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b strain Eagan.

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is capable of expressing a heterogeneous population of LPS which exhibits extensive antigenic diversity among multiple oligosaccharide (OS) epitopes. Structural elucidation of variable and conserved OS epitopes of H. influenzae serotype b strain Eagan was determined by the application of high-field NMR techniques and MS-based methods on oligosaccharides obtained from LPS samples by a deacylation strategy. LPS extracted by the hot aqueous phenol method gave complex electrophoretic patterns consisting of at least six low-molecular mass bands. Electrospray ionization-mass spectrometry of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues as well as subpopulations of glycoforms containing additional phosphoethanolamine (PEA) groups. It was demonstrated that the LPS contains a conserved PEA-substituted, heptose-containing trisaccharide inner core moiety attached via a KDO 4-phosphate unit to a lipid A component. Tandem MS experiments unambiguously established the presence of a KDO 4-pyrophosphoethanolamine unit in the subpopulation of LPS containing additional PEA groups. The occurrence of LPS containing this structural feature was found to be dependant on the isolation procedure used. Each heptose of the common inner core element L-alpha-D-Hepp(1-->2)-L-alpha-D-Hepp(1-->3)-L-alpha-D-Hep p(1-->5)-alpha-KDO is substituted by a hexose residue with further chain elongation from the central unit. The structures of the major glycoforms containing four (three Glcs and one Gal), five (three Glcs and two Gals), and six (three Glcs and three Gals) hexoses were determined in detail. The Hex6 glycoform contains the terminal structure, alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glc, providing, for the first time, definitive structural evidence for the expression of the Pk-blood group antigen in H. influenzae LPS. Moreover, an analogue of the Hex4 glycoform was identified in which the third heptose residue carries phosphate at 0-4.

Epitope Mapping↗
PubMed1997

Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide.

By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the IgtA-E genes.

Acetylglucosamine↗
PubMed1997

VBH head holder to improve frameless stereotactic brachytherapy of cranial tumors.

Precise target localization is essential for brachytherapy. We have adapted the VBH (Vogele-Bale-Hohner) head holder (Wellhoefer Dosimetry, Schwarzenbruck, Germany), originally developed at the University of Innsbruck, for frameless stereotactic surgery, for use in brachytherapy of cranial tumors. The VBH head holder allows for rigid, noninvasive head fixation by means of an individualized upper dental cast. Registration rods, rigidly attached to the dental cast, provide stable external points of reference. The dental cast is sucked against the upper palate by vacuum, and then the fixated patient is scanned. During simulation, the targeting device can be positioned with respect to the virtual patient using the ISG Viewing Wand. Following simulation, the real patient is repositioned under vacuum control, the targeting device repositioned as well, and the actual brachytherapy initiated. The VBH head holder is well tolerated by patients and simple to use, and various studies have confirmed submillimeter accuracy. The modified head holder in combination with a new targeting device allows for precise and well-planned insertion of hollow needles into a tumor using frameless stereotactic systems as well as being compatible for uses in other fields.

Brachytherapy↗
PubMed1997

Modulation of human medial temporal lobe activity by form, meaning, and experience.

Clinically, the hallmark of the human amnesic syndrome is an impaired ability to consciously recollect or remember daily events. If the medial region of the temporal lobes, including the hippocampus and related structures, is critical for establishing these new memories, then this brain region should be active whenever events are experienced, regardless of whether subjects are asked explicitly to learn and remember. Here we show that the medial temporal region is active during encoding and that the hemisphere activated and the amount of activation depend on the type of stimulus presented (objects or words), whether the stimulus can be encoded for meaning (real objects and words versus nonsense objects and words), and task experience (first versus the second time a task is performed). These findings demonstrate that the medial temporal lobe memory system is engaged automatically when we attend to a perceptual event and that the location and amount of activation depend on stimulus characteristics (physical form, meaning) and experience.

Adult↗
PubMed1997

Growth factor control of growth and epithelial differentiation in embryonic lungs.

embryonic lung cultures were exposed to either EGF (10 ng/ml) or TGF beta 1 (2 ng/ml) for 72 h, and branching morphogenesis, cell proliferation, and epithelial differentiation (the expression of DSPC synthesis and of surfactant protein C (SP-C) mRNA) were studied. EGF treatment stimulated branching morphogenesis (measured as the number of terminal left lung buds), epithelial differentiation, and cell proliferation. Branching morphogenesis was increased compared to controls after 48 h of culture by 47% and after 72 h by 34% (P < 0.0005). Choline incorporation into DSPC was stimulated by 343% (P = 0.05). SP-C expression was increased sixfold. Thymidine incorporation was stimulated by 49% (P < 0.05). The effects of EGF on thymidine labeling were distributed among epithelial cells of the airway walls and of the branching tips, and also the mesenchyme (P < 0.01 for each area compared to controls). In contrast, TGF beta 1 did not alter the number of terminal left lung buds, inhibited choline incorporation into DSPC by 35% (P < 0.05), and had no effect on thymidine incorporation (87% of control). There was increased thymidine labeling at the branching tips (P < 0.01), while other areas were not different from controls. We conclude that both EGF and TGF beta 1 affect the development of branching morphogenesis and of epithelial differentiation in the embryonic lung.

Animals↗
PubMed1997

Phospholipase D.

Phospholipase D catalyses the hydrolysis of phosphatidylcholine to generate phosphatidate. The regulation of PLD activity is complex involving a number of small GTP binding proteins, but in particular Arf and Rho, phosphatidylinositol 4,5-bisphosphate and protein kinase C. The cDNA for PLD1 has recently been cloned and shows homology to the yeast and plant genes but only within four domains. Domains I and IV each contain a putative catalytic triad. PLD activity has been detected in plasma membranes, Golgi membranes and in nuclear membranes; it is unclear if different isoenzymes are responsible for this variation, or if the PLDs are differently regulated. The product of PLD activity, PA, appears to be a messenger molecule regulating the actin cytoskeleton and maybe playing a role in the control of membrane traffic and secretion.

Journal Article↗
PubMed1997

The reproducibility of cardiac baroreceptor activity assessed non-invasively by spectral sequence techniques.

Baroreceptor sensitivity (BRS) is increasingly used as a prognostic indicator in cardiovascular disease. Traditionally it has been measured using invasive techniques with pharmacological manipulation of blood pressure (BP). With the advent of newer methods to measure pulse interval and beat-to-beat changes in BP it is now possible, using sophisticated mathematical modelling techniques, to calculate cardiac BRS non-invasively. However, there are virtually no data on the reproducibility of these newer techniques and what factors may affect the repeatability of these measurements. We studied 39 subjects, aged 22-82 years, with a supine systolic BP range 97-160 mmHg and a diastolic BP range 57-94 mmHg on two occasions between 1 week and 6 months apart. Cardiac BRS was measured by power spectral analysis using Fast Fourier Transformation (FFT), sequence analysis (using up, down and combined sequences) and from phase IV of the Valsalva manoeuvre. There was no significant difference between visits for any of the methods for measuring cardiac BRS. Mean BRS values were similar for FFT (16.7 +/- 11.2 ms/mmHg) and sequence analysis (15.8 +/- 11.4 ms/mmHg); however, results using phase IV of the Valsalva manoeuvre were significantly lower (8.1 +/- 2.9 ms/mmHg, p < 0.0001). The coefficient of variation for the five measures of cardiac BRS varied from 16.8% for Valsalva-derived values to 26.1% for 'down' sequence analysis. However, in ten subjects BRS could not be calculated from the Valsalva manoeuvre. None of the independent variables tested (including age, BP levels and time between testing) significantly influenced the degree of repeatability. In summary, there appears to be little difference between these non-invasive methods in their degree of reproducibility. These techniques would seem suitable for longitudinal studies of changes in cardiac BRS and overcome many of the problems associated with the invasive pharmacological methods.

Adult↗
PubMed1997

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate.

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.

Animals↗
PubMed1997

Evaluation of a gel-immunization technique used with two different Immucox vaccine formulations in battery and floor-pen trials with broiler chickens.

The use of a gel-immunization technique with Immucox vaccination was compared and evaluated against other immunization methods in battery and floor-pen immunization trials. Gel immunization was found to be superior to immunization by gavage, by spray cabinet, or by the conventional delivery method of Immucox in a battery trial. Significantly enhanced protection as measured by weight gain, coupled with the establishment of a more uniform primary immunizing infection as evidenced by greater intestinal lesions and increased oocyst shedding, was seen in gel-immunized birds. In addition, cross-protective battery trials determined that the strain of Eimeria maxima found in the Immucox vaccine failed to elicit protection against a recent field isolate of E. maxima as measured by average weight gain and lesion scores. A reformulation of the Immucox vaccine that included the field isolate of E. maxima was required to elicit a protective immune response against challenge by the field strain. A floor-pen experiment demonstrated that gel immunization of 1-day-old roaster chickens resulted in performance parameters of average weight gain, average bird weight, and feed conversion that did not differ significantly from those recorded for medicated nonimmunized birds.

Animals↗
PubMed1997

Automatic semantic priming of nouns and verbs in patients with Alzheimer's disease.

The status of semantic representations of nouns (concrete and abstract) and verbs (motion and non-motion) was investigated in patients with Alzheimer's disease (AD). Nouns and verbs have been argued to activate different neural substrates, given the selective difficulties reported for one or the other grammatical class in patients with focal lesions. Additionally, category-specific deficits have been reported for either abstract or concrete words, often in patients with bilateral neuropathology. We looked for these types of dissociations in patients with AD in a semantic priming experiment using a pronunciation task and a short stimulus onset asynchrony. The normal control subjects demonstrated automatic activation for both concrete nouns and motion verbs. The AD patients, however, demonstrated priming effects for concrete nouns, but not for motion verbs. This dissociation between concrete nouns and motion verbs found for the AD patients is discussed in terms of differences in the nature of semantic representations involving multiple physical and functional attributes in the case of concrete nouns, but only motion attributes in the case of motion verbs. Moreover, the typical distribution of neuropathology associated with AD may put motion verbs at risk, particularly given the neuroanatomical considerations suggested by recent positron emission tomography (PET) studies.

Aged↗