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Biomedical subjects

G E Martin

Publications and source records attributed to G E Martin.

At least 19 recordsLinked to original sources
PubMed2001

Thalibealine, a novel tetrahydroprotoberberine--aporphine dimeric alkaloid from Thalictrum wangii.

A novel tetrahydroprotoberberine-aporphine dimeric alkaloid, (-)-thalibealine (1), was isolated from the roots of Thalictrum wangiii, and its structure established via spectroscopic analysis. Three other alkaloids were isolated, including the benzyltetrahydroisoquinoline-aporphine dimer (+)-thalmelatidine, the aporphine (+)-magnoflorine, and the protoberberine berberine. This is the first reported isolation of a tetrahydroprotoberberine-aporphine dimer from nature, as well as the first reported isolation of constituents from Thalictrum wangii.

Berberine Alkaloids↗
PubMed2001

Synthesis of thalprzewalskiinone, a revision of structure.

A direct comparison of the spectral data for synthetic 2-methyl-6,7-dimethoxy-3'-methoxy-4'-hydroxyoxobenzylisoquinoline iodide (1) and its positional isomer 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2) with the data obtained for the oxobenzylisoquinoline alkaloid thalprzewalskiinone revealed that the original structural assignment of the alkaloid as 1 was in error. These results mandate the revision of structure of thalprzewalskiinone to 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2).

Drugs, Chinese Herbal↗
PubMed2000

NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus. This enzyme (His(6)-POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented. Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide. Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis.

Chlorophyllides↗
PubMed2000

Long-range (1)H-(15)N heteronuclear shift correlation at natural abundance.

Despite the inherently low sensitivity of (15)N NMR because of its low gyromagnetic ratio (gamma(N)) and its relatively low natural abundance (0.37%), this important nuclide still has useful potential as a structural probe even at natural abundance. Inverse-detected NMR methods coupled with major advances in NMR probe designs have made it possible to acquire long-range (1)H-(15)N heteronuclear shift correlation data on samples as small as a micromole overnight. Chemical shift referencing schemes for (15)N and the range of (15)N shifts are discussed, followed by a discussion of the currently available pulse sequences, pulse calibration, parametrization and processing of long-range (1)H-(15)N data, and the implications of probe selection. These topics are followed by a review of the applications contained in the literature that have utilized (1)H-(15)N heteronuclear shift correlation experiments at natural abundance, with emphasis placed on the observed long-range coupling pathways.

Calibration↗
PubMed1999

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A(1) receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [3H]cyclohexyladenosine ([3H]CHA) to A(1) receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81, 723 (0.3-100 microM) produced a concentration-dependent enhancement of [3H]CHA binding to rat brain A(1) receptors. These effects were also species-dependent with larger enhancements (150-200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [3H]CHA binding to A(1) receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 microM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A(1) receptor selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 microM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [3H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 microM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A(1) receptor in a high-affinity state and that the relative proportions of high-affinity A(1) receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A(1) receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A(1) receptor.

Adenosine↗
PubMed1999

Improved performance accordion heteronuclear multiple-bond correlation spectroscopy-IMPEACH-MBC.

A modification of the recently reported ACCORD-HMBC long-range heteronuclear shift correlation experiment is described. The new experiment, IMPEACH-MBC (improved performance accordion heteronuclear multiple-bond correlation), introduces a new pulse sequence element, a constant time variable delay. The incorporation of the constant time variable delay into the IMPEACH-MBC sequence suppresses (1)H-(1)H coupling modulation inherent to the utilization of the accordion principle to sample a broad range of potential long-range heteronuclear couplings. (1)H-(1)H coupling modulation, which introduces an F(1) modulation or a "skew" of responses in the second frequency domain of the ACCORD-HMBC experiment, is suppressed in the IMPEACH-MBC experiment. Results of identically optimized IMPEACH-MBC and ACCORD-HMBC experiments performed on a sample of strychnine are compared.

Hydrogen Bonding↗
PubMed1999

The relationship between clinical assessments of nutritional status and adverse outcomes in older hospitalized medical patients.

BACKGROUND: Malnutrition is common in hospitalized older people and may predict adverse outcomes. Previous studies of the relationship between nutritional status and hospital outcomes are limited by inadequate accounting for other potential predictors of adverse outcomes, the failure to consider functional outcomes, and the omission of clinical assessments of nutritional status. OBJECTIVE: To measure the relationship between a clinical assessment of nutritional status on hospital admission and subsequent mortality, functional dependence, and nursing home use. DESIGN: Prospective cohort study SETTING: A tertiary care hospital PATIENTS: A total of 369 patients at least 70 years old (mean age 80.3, 62% women) admitted to a general medical service MEASUREMENTS: Nutritional status was measured with the Subjective Global Assessment, a validated measure of nutritional status based on historical and physical exam findings. Patients were classified as severely malnourished (generally at least a 10% weight loss over the previous 6 months and marked physical signs of malnutrition), moderately malnourished (generally a 5 to 10% weight loss and moderate physical signs), or well nourished. Vital status, independence in activities of daily living, and nursing home use were determined through patient or surrogate interview at admission and 90 days and 1 year after discharge. Indices of comorbidity and illness severity were determined from chart review. RESULTS: 219 patients (59.3%) were well nourished, 90 (24.4%) were moderately malnourished, and 60 (16.3%) were severely malnourished. Severely malnourished patients were more likely than moderately malnourished or well nourished patients to die by 90 days (31.7%, 23.3%, and 12.3%, respectively, P < .001) and 1 year (55.0%, 35.6%, and 27.9%, P < .001) after discharge. In logistic regression models controlling for acute illness severity, comorbidity, and functional status on admission, severely malnourished patients were more likely than well nourished patients to die within 1 year of discharge (OR = 2.83, 95% CI, 1.47-5.45), to be dependent in activities of daily living 3 months after discharge (OR = 2.81, 1.06-7.46), and to spend time in a nursing home during the year after discharge (OR = 3.22, 1.05-9.87). CONCLUSION: Malnutrition was common in hospitalized patients with medical illness and was associated with greater mortality, delayed functional recovery, and higher rates of nursing home use. These adverse outcomes were not explained by greater acute illness severity, comorbidity, or functional dependence in malnourished patients on hospital admission.

APACHE↗
PubMed1997

Purification and kinetic analysis of pea (Pisum sativum L.) NADPH:protochlorophyllide oxidoreductase expressed as a fusion with maltose-binding protein in Escherichia coli.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key reaction in the chlorophyll biosynthetic pathway. To facilitate structure-function studies, POR from pea (Pisum sativum L.) has been overexpressed in Escherichia coli as a fusion with maltose-binding protein (MBP) at 5-10% of the total soluble cell protein. The fusion protein (MBP-POR) has been purified to greater than 90% homogeneity by a two-step affinity-purification procedure. This represents the first successful overexpression and purification of a plant POR. MBP-POR was found to be active, and the kinetic properties were determined using a continuous assay in which the rate of chlorophyllide formation was measured. The Vmax was 20.6+/-0.9 nmol.min-1.mg-1 and the Km values for NADPH and protochlorophyllide were 8.7+/-1.9 microM and 0.27+/-0.04 microM respectively. These results represent the first determination of the kinetic properties of a pure POR and the first report on the kinetics of POR from a dicotyledenous plant. The experiments described here demonstrate that the enzyme is not a 'suicide' enzyme, and the only components required for catalysis are NADPH, protochlorophyllide and light. Size-exclusion chromatography on a Superose 6 HR column indicated that MBP-POR has a molecular mass of 155 kDa (compared with the molecular mass of 80 kDa estimated by SDS/PAGE), indicating that it behaves as a dimer in solution. This is the first direct determination of the oligomerization state of POR.

ATP-Binding Cassette Transporters↗
PubMed1997

Granulosin, a new chromone from Galipea granulosa.

A novel chromone, granulosin (1), has been isolated from the bark of Galipea granulosa. The extract of the bark, as well as granulosin (1), exhibited lethality in the brine shrimp test. The structure of granulosin (1) as 2-propyl-7,8-(methylenedioxy)chromone was established via spectroscopic analysis.

Animals↗
PubMed1995

The role of tryptophans 371 and 395 in the binding of antibiotics and the transport of sugars by the D-galactose-H+ symport protein (GalP) from Escherichia coli.

The interactions between the D-galactose-H+ symporter (GalP) from Escherichia coli and the inhibitory antibiotics, cytochalasin B and forskolin, and the substrates, D-galactose and H+, have been investigated for the wild-type protein and the mutants Trp-371-->Phe and Trp-395-->Phe, so that the roles of these residues in the structure-activity relationship could be assessed. Neither mutation prevented photolabeling by either [4-3H]cytochalasin B or by 3-[125I]iodo-4-azidophenethyl-amido-7-O-succinyldesacetylforskolin ([125I]APS-forskolin). However, measurements of protein fluorescence show that both residues are in structural domains, the conformations of which are perturbed by the binding of cytochalasin B or forskolin. Moreover, both mutations cause a substantial decrease in the affinity of the inward-facing site of the GalP protein for cytochalasin B, 10- and 43-fold, respectively, but have little effect upon the affinity of this site for forskolin, 0.8- and 2.6-fold reductions, respectively. Both these mutations change the equilibrium between the putative outward- (T1) and inward-facing (T2) conformations, so that the inward-facing form is more favored. They also stabilize a different conformational state, "T3-antibiotic," in which the initial interactions between the protein and antibiotics are tightened. Overall, this has the effect of compensating for the reduction in affinity for cytochalasin B, so that the respective overall Kd values are 0.74- and 3.5-fold that of the wild type, while causing a slight increase, 1.5- and 3.2-fold, respectively, in affinity of the mutants for forskolin. The Trp-371-->Phe mutation causes a 15-fold reduction in the affinity of the inward-facing site for D-galactose, suggesting that this residue forms part of the sugar binding site. In contrast, the Trp-395-->Phe mutation has no effect upon the affinity of the inward-facing site for D-galactose. These effects may be related to the reduction in galactose-H+ symport activity only in the Trp-371-->Phe mutant, although it still effects active transport to the same extent as the Trp395-->Phe mutant. However, there is a 10-20-fold increase in the Km values for energized transport of D-galactose for both mutants.

Anti-Bacterial Agents↗
PubMed1995

Kinetics and thermodynamics of the binding of forskolin to the galactose-H+ transport protein, GalP, of Escherichia coli.

The binding of the transport inhibitor, forskolin, to the galactose-H+ symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 microM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.9-1.4 microM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (kon) and dissociation (koff) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 microM-1.s-1 and 5.1-11.5 s-1 respectively, and the Kd was calculated to be 1.5 microM. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by sugar provided an additional method to calculate the dissociation rate constant for forskolin (koff = 12.4-13.0 s-1). The rate of the slow change in protein fluorescence (3-5 s-1) was independent of the forskolin concentration, indicating an isomerization of the transporter between different conformations, possibly outward- and inward-facing forms. These kinetic parameters were determined at a series of temperatures, so that the thermodynamics of forskolin binding and transporter re-orientation could be analysed. The binding process was entropically driven (delta S = 83.7 J.K-1.mol-1; delta H = 8.25 kJ.mol-1), similar to that for cytochalasin B, which is also an inhibitor of GalP. Measurements of the binding of [3H]forskolin by equilibrium dialysis revealed competitive displacement of bound forskolin by cytochalasin B, possibly suggesting that the sugar, forskolin and cytochalasin B binding sites are overlapping; the Kds for forskolin and cytochalasin B were calculated to be 0.85 microM and 4.77 microM respectively, and the concentration of binding sites was 10.2 nmol.mg-1.

Bacterial Proteins↗
PubMed1995

Identification of an N-acetyl keto derivative of fumonisin B1 in corn cultures of Fusarium proliferatum.

A method is presented for the separation and identification of a new N-acetyl keto derivative of fumonisin B1 (FB1) produced in solid corn culture. Cultures of Fusarium proliferatum (M-1597) were purified using preparative hplc, and the new fumonisin was detected by negative-ion esms. Structures were confirmed by 1H- and 13C-nmr spectroscopy. The new fumonisin differs from FB1 in that the tricarballylic acid functionality at the C-15 position of the eicosane backbone is replaced by a ketone and the amino group is acetylated. Direct analysis of the culture material by negative-ion electrospray lc/ms confirmed that the new fumonisin is produced naturally by the fungus.

Acetylation↗
PubMed1995

RP 73870, a gastrin/cholecystokinin-B receptor antagonist with potent anti-ulcer activity in the rat.

RP 73870, the racemic potassium salt of (([N-(methoxy-3-phenyl)-N-(N-methyl-N-phenyl-carbamoylmethyl)- carbamoylmethyl]-3-ureido)-3-phenyl)-2-ethylsulfonate-(RS) is a potent, reversible antagonist of both gastrin and cholecystokinin-B receptors in guinea pig and rat tissues. This compound is a potent inhibitor of pentagastrin-stimulated gastric acid secretion in the perfused rat stomach. RP 73870 also inhibits basal gastric acid secretion in the rat, although at doses higher than that required for inhibition of pentagastrin-stimulated gastric acid secretion. RP 73870 is a potent inhibitor of aspirin-induced gastric damage in the rat. In the prevention of aspirin-induced gastric damage, RP 73870, given p.o., was 10-fold less potent than when given i.v. RP 73870 was as potent as a H2 receptor antagonist or proton pump inhibitor in the prevention of cysteamine-induced duodenal ulcers in the rat. Relative to other gastrin/cholecystokinin-B antagonists, RP 73870 demonstrates greater affinity to gastrin binding sites, and possesses a unique spectrum of in vivo biological activities appropriate for an anti-ulcer indication.

Animals↗