Biomedical subjects

R D Smith

Publications and source records attributed to R D Smith.

At least 199 records · Page 11Linked to original sources

PubMed1996

Effects of short-term exercise on appetite responses in unrestrained females.

OBJECTIVE: To examine the short-term effects of exercise and diet composition on appetite in unrestrained females. DESIGN: A 2 x 2 repeated measures design study was conducted, with exercise and lunch type as the repeated factors. SETTING: The Human Appetite Research Unit at Leeds University Psychology department. SUBJECTS: 13 unrestrained, normal weight females were recruited from the student and staff population of Leeds University. INTERVENTIONS: Subjects were assigned to four experimental treatments in a counterbalanced order. These were a control (no-exercise) session and an intense (70% VO2 max) exercise session followed by a free-selection test lunch consisting of high-fat/low-carbohydrate or low-fat/high-carbohydrate foods. The effects of exercise and lunch type were measured by monitoring energy intake and macronutrient intake during the test lunches only. Visual analogue scales were used to monitor profiles of motivation to eat during the treatments. RESULTS: Subjects did not experience the marked suppression of hunger induced by exercise, which is a striking and robust finding in men. Moreover, unlike men, females rated a range of foods to be more palatable after exercise (F(1,12) = 8.0, P < 0.05). Similar to male subjects, exercise in females had no significant short-term effect on energy or macronutrient intake. Energy intake was significantly influenced by the fat:carbohydrate composition of the foods available (F(1,12) = 64.7, P < 0.001), and the short-term energy deficit induced by exercise when followed by low-fat lunch was completely wiped out when exercise was followed by a high-fat lunch (F(1,12) = 66.2, P < 0.001). CONCLUSIONS: These results show that there are similarities and differences between males and females in the appetite response to foods varying in macronutrient composition following vigorous exercise. The differences may help to explain why exercise so often produces a disappointing effect on weight control in females.

Adult↗

PubMed1996

The diversified pharmacology of angiotensin II-receptor blockade.

The discovery of orally active nonpeptide angiotensin II (A II)-receptor antagonists has initiated a growing understanding of the physiologic and pathophysiologic roles of A II. Losartan is the first of the new class of antagonists that block all the well-known effects of A II, including vasoconstriction, aldosterone release, renin release (negative feedback), and the stimulation of thirst. A II-receptor subtypes have been described, with losartan antagonism defining the AT1 subtype and with PD123319 antagonism defining the AT2 subtype. The AT1 receptor is G-protein-coupled, involving PLC, PLA2, PLD, or adenylate cyclase and the release of intracellular calcium. The receptor-response coupling of the AT2 site remains elusive but may involve protein tyrosine phosphatase and subserve an antiproliferative role. Losartan as the prototype of an AT1-selective antagonist: i) inhibits A II binding, ii) antagonizes effects of A II in vivo and in vitro, and iii) lowers blood pressure in models of A II-dependent hypertension A II stimulates growth in vitro (DNA and protein synthesis) and in vivo (cardiac and vascular hypertrophy), and these effects are blocked by losartan. Losartan, like angiotensin-converting enzyme inhibitors, has significant renal, cardiac, and cerebral protective effects in models of renal failure, cardiac failure, and stroke, confirming the pathologic role of A II in these models. The pioneering studies in experimental animals are being confirmed by a growing number of other AT1-selective blockers and provide the basis of use of losartan for hypertension and its clinical trial in other disease states.

Angiotensin II↗

PubMed1995

Identification of atypical (non-AT1, non-AT2) angiotensin binding sites with high affinity for angiotensin I on IEC-18 rat intestinal epithelial cells.

Specific high-affinity (Kd = 3.4 nM) binding sites for 125I-labelled angiotensin I ([125I]Ang I) were identified on an epithelial cell line (IEC-18) derived from the rat small intestine. The sites, which also have high affinity for Ang II, are insensitive to both AT1- and AT2-specific angiotensin receptor antagonists. The rank order of potency with which various angiotensin peptides inhibited [125I]Ang I binding to the cells (Ang I > or = Ang II > Ang(1-7) > [Sar1,Ile8]-Ang II > Ang(3-8) > Ang III) also distinguishes these sites from AT1 and AT2 angiotensin receptors.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1995

Characterization of cytochrome c variants with high-resolution FTICR mass spectrometry: correlation of fragmentation and structure.

The dissociation of cytochrome c ions (15+ charge state) generated by electrospray ionization has been studied by Fourier transform ion cyclotron resonance mass spectrometry (FTICR) using a sustained off-resonance irradiation/collision-induced dissociation (SORI-CID) technique. Over 95% of the fragment ions can be accurately assigned (to better than 10 ppm), yielding information on the primary sequences of the various proteins. Up to four stages of mass spectrometry (MS4) have been achieved without the need for quadrupole excitation/collisional cooling of the product ions. The subtle structural differences among the cytochrome c variants (from bovine, tuna, rabbit, and horse) are clearly reflected in their fragmentation patterns: replacing 3 out of 104 residues of the cytochrome c is shown to dramatically change the dissociation pattern. Of particular importance are a variety of results indicating that the dissociation of the cytochrome c's is influenced by higher-order structure and charge location, in addition to the primary structure (i.e., sequence). No fragmentation is observed in the region between residues 10-20 and little dissociation between residues 70-90. This is most likely due to the interactions of the heme group with the polypeptide chain, and such a heme "footprinting" pattern is analogous to the protein conformation in solution. These studies demonstrate that electrospray ionization-FTICR using SORI-CID can be a useful tool to probe not only the small differences in the primary sequences of proteins but also suggest the potential for probing their higher-order structures and yielding information not readily available from H/D exchange or circular dichoism studies.

Amino Acid Sequence↗

PubMed1995

Capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for direct analysis of cellular proteins.

The combination of capillary electrophoresis (CE) with electrospray ionization (ESI) mass spectrometry has proven to be broadly applicable to a wide range of biologically important compounds. When combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, the combined method, in addition to high-resolution separations, affords high-resolution precision mass measurements for analytes separated from complex mixtures. Direct chemical analysis of single cells has received considerable attention in recent years; the single cell approach provides a major step toward answering important questions in the field of cellular biochemistry. In this work we present preliminary results which demonstrate the feasibility of using the CE-ESI-FTICR combination as a high-performance detection scheme for the analysis of cellular proteins acquired directly from small populations (i.e., 5-10) of intact living cells. The human erythrocyte was chosen as a model system owing to its availability, relatively homogeneous composition, and thorough documentation of contents by previous researchers. In this work we demonstrate the on-line acquisition of high-resolution mass spectra (average resolution > or = 45,000 fwhm) of both the alpha and the beta chains of hemoglobin acquired from the injection of 10 human erythrocytes (corresponding to 4.5 fmol of hemoglobin). Given the extremely small volume of the human erythrocyte (typically 87 fl/cell), the techniques implemented here should also be adaptable to the study of larger mammalian cell systems.

Adult↗

PubMed1995

Efficiency considerations in the expansion of radiation therapy services.

PURPOSE: An economic option appraisal to determine whether early investment in capital is an efficient means of expanding radiation therapy services. METHODS AND MATERIALS: Costs were based on 1991 data from a center in western Sydney. Two options were costed: Option 1 based on an increase in overtime performed by existing staff, using capital more intensively and possible use of shifts; Option 2 based on an investment in new capital and associated increases in levels of staffing. The health sector costs of both options were determined in one center at workloads of between 70,940 and 98,525 fields per year to assess relative efficiency. RESULTS: There was very little difference in cost between both options, with Option 1 slightly cheaper at workloads up to 98,525 fields per year. CONCLUSIONS: The results suggest that capital investment may be introduced at a fairly early stage without efficiency loss. Sensitivity analysis reinforces these conclusions and the generalizability of the results.

Costs and Cost Analysis↗

PubMed1995

On-line capillary electrophoresis-electrospray ionization mass spectrometry of nucleotides.

Capillary zone electrophoresis (CZE) and reversed anionic capillary isotachophoresis (CITP) conditions have been developed for the separation of mixtures comprised of monophosphate nucleosides, pyridine and flavin dinucleotides, and monophosphate dinucleosides. Results for the on-line coupling of CZE and CITP with electrospray ionization mass spectrometry (MS) are presented. CITP-tandem MS is utilized to provide both molecular weight and structural information of monophosphate dinucleotides. The fragmentation pattern of dinucleotides in the low collision energy range is described. The resulting mass spectra are readily interpreted in terms of dinucleotide structures. These results demonstrate the new capability for applications for the study of DNA and RNA.

Capillary Action↗

PubMed1995

Bio-affinity characterization mass spectrometry.

A new approach, bio-affinity characterization mass spectrometry (BACMS), aimed at providing a more rapid, sensitive and potentially more flexible alternative to techniques presently employed for the characterization of noncovalent interactions in mixtures, such as would be encountered in combinatorial chemistry, in presented. BACMS avoids some of the difficulties and potential artifacts associated with affinity chromatography since the noncovalent associations occur in solution; thus, BACMS avoids the requirement of solid support media and the development of non-interfering linker species. This paper describes the conceptual basis for the methodology and its potential use in applications which include the screening of high affinity ligands in support of new drug development. BACMS exploits new Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry technologies which, when coupled to electrospray ionization (ESI), allow the investigation of specific noncovalent complexes formed in solution. BACMS utilizes the well-known attributes of FTICR, such as the high resolution mass analysis and (MS)n (n > or = 2) capabilities; however, it is even more directly a result of recently developed techniques involving quadrupolar excitation, such as selected-ion accumulation. These tools are demonstrated and the results illustrate the extraordinary sensitivity achievable (solution concentration of 1 x 10-9 M without the use of separations prior to ESI). Thus, the new capabilities demonstrated here, in conjunction with ESI, will be useful for the investigation of very low relative concentration noncovalent association directly from solution, and promote a faster alternative for combinatorial mixture screening and analysis.

Animals↗

PubMed1995

Changes of pH affect calcium currents but not outward potassium currents in rat myometrial cells.

Spontaneous contraction of uterine smooth muscle is enhanced by alkalinization and depressed by acidification. We have investigated the ionic currents responsible for this in single myometrial cells. Intracellular acidification (20 mM butyrate) at constant external pH depressed the magnitude of the calcium current to 58+/-6% of control, but had little effect on outward currents. Similar but slower effects were also observed when the extracellular pH was lowered to 6.9 (56+/-9% of control). Correspondingly, when the intracellular or extracellular pH was elevated (20 mM NH4Cl or pH 7.9 respectively) the calcium current magnitude increased (165+/-15% in NH4Cl; 136+/-2% at pH 7.9) and there was, again, no effect on the outward currents. These observations are consistent with the effects of pH on spontaneous contractile activity being due to an effect on the membrane calcium current.

Animals↗

PubMed1995

"Strict" blood pressure control and progression of renal disease in hypertensive nephrosclerosis.

Hypertensive nephrosclerosis is a progressive renal disease and the leading cause of end-stage renal disease (ESRD) in blacks in the United States. It is generally believed that hypertensive renal injury is responsible for progressive renal failure; however, it is not known whether pharmacologic lowering of blood pressure to any level prevents progression of renal disease. Accordingly, we performed a long-term prospective randomized trial to determine whether "strict" [diastolic blood pressure (DBP) 65 to 80 mm Hg] versus "conventional" (DBP 85 to 95 mm Hg) blood pressure control is associated with a slower rate of decline in glomerular filtration rate. Eighty-seven non-diabetic patients (age 25 to 73; 68 black, 58 male) with long-standing hypertension (DBP > or = 95 mm Hg), chronic renal insufficiency (GFR < or = 70 m/min/1.73 m2) and a normal urine sediment were studied. DBP was pharmacologically lowered to < or = 80 mm Hg (3 of 4 consecutive measurements at 1 to 4 weeks intervals) after which patients were randomized. DBP and GFR (renal clearance of 125I-iothalamate) were measured at baseline, at three months and every six months post-randomization. The rate of decline in GFR (GFR slope, in ml/min/1.73 m2/year), estimated by the method of maximum likelihood in a mixed effects model, was the primary outcome variable. In a secondary analysis, 50% reduction in GFR (or a doubling of serum creatinine) from baseline, ESRD and death were combined.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery.

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetically-engineered streptavidin variants designed for enhanced bioseparation and drug delivery performance. Subunit mixing is accomplished by guanidine thiocyanateinduced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by renaturation and reassociation of mixed tetramers. In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutant. The WT/n49C tetramers can be used for site-specific and stoichiometric attachment of therapeutics/imaging agents or targeting proteins through the genetically-engineered thiol while retaining unhindered access to biotin-binding at the WT subunits. Second, we demonstrate that the His127Cys mutation (H127C) results in a streptavidin mutant that forms a disulfide-linked dimer under non-reducing conditions. Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is controlled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-directed mutant, Trp120Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 10(4) compared to WT streptavidin. The W120 biotin-binding affinity is sufficiently high (Ka approximately equal to 10(7) M-1) to immobilize the mutant on a biotinagarose affinity chromatography column, but the engineered off-rate allows for facile elution with excess biotin at physiological pH, whereas WT streptavidin is irreversibly immobilized on the column. We demonstrate that the purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated targets at the WT subunit, while retaining the reversible separation capabilities of the W120A subunits via biotin-agarose affinity chromatography.

Dimerization↗

PubMed1995

Pharmacology of XR510, a potent orally active nonpeptide angiotensin II AT1 receptor antagonist with high affinity for the AT2 receptor subtype.

The angiotensin II (Ang II) type 1 receptor (AT1) mediates all known physiological effects of ANG II, whereas functions of the type 2 (AT2) receptor are not clear. Should undesirable AT2 effects be identified, it may be advantageous to combine antagonism of AT1 and AT2 receptors. XR510 was shown to inhibit the specific binding of [125I]Sar1,Ile8-Ang II for AT1 and AT2 subtype binding sites in rat adrenal membranes with IC50 of 0.26 and 0.28 nM, respectively, and in human tissues with subnanomolar binding affinity. In isolated rabbit aorta, XR510 exerted insurmountable Ang II antagonism with a Kb value of 4 nM. In conscious renal hypertensive rats, XR510 decreased blood pressure (BP) with intravenous (i.v.) and oral (p.o.) ED30 of 0.08 and 0.27 mg/kg, respectively. In spontaneously hypertensive rats (SHR), repeated daily oral dosing of XR510, losartan, and enalapril at 30 mg/kg/day decreased BP similarly. In conscious furosemide-treated dogs, XR510, given either intravenously or orally, decreased BP. These results suggest that XR510 is an orally active and selective Ang II receptor antagonist with equal binding affinities for AT1 and AT2 receptor binding sites.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1995

Karyotype polymorphism in the slender loris (Loris tardigradus).

Three wild-caught Sri Lankan and two captive-bred slender lorises (Loris tardigradus) in Adelaide Zoo, South Australia, were karyotyped from leucocyte cultures. The founder male and one of the founder females were of the grey subspecies L. tardigradus grandis, of which L. tardigradus nordicus is probably a synonym. The other founder female was a small red-brown L. tardigradus tardigradus. All three wild-caught individuals were karyotypically distinct from each other and their offspring showed the expected heterozygosity. The L. tardigradus tardigradus karyotype was no more distinct from the grey subspecies karyotypes than they were from each other. On present evidence, Indian and Sri Lanka lorises appear to be chromosomally distinct.

Animals↗

PubMed1995

Evidence for autocrine growth inhibition of rat intestinal epithelial (RIE-1) cells by transforming growth factor type-beta.

The proliferation of sparse cultures of RIE-1 rat intestinal epithelial cells was potently inhibited by transforming growth factor type beta 1 (TGF-beta 1), with a half-maximal effect at 10-30 pg/ml. As judged by [3H]thymidine incorporation assays, this growth inhibitory action became apparent 4-6 h after addition of TGF-beta 1 to the cells. RIE-1 cells express high-affinity (KD approximately 5.6 pM) receptors for 125I-TGF-beta 1. Affinity cross linking experiments labelled two major species of putative TGF-beta 1 receptors with M(r) values of 235,000 and 65,000. Medium conditioned by confluent cultures of RIE-1 cells contained latent TGF-beta-like activity that was activated at low pH. These findings support a model of autocrine growth inhibition of intestinal crypt cells by TGF-beta.

Animals↗

PubMed1995

The myristoylated alanine-rich C-kinase substrate (MARCKS) is not required for mitogenic signalling via protein kinase C in cultured rat intestinal epithelial (RIE-1) cells.

Activation of protein kinase C (PKC) by angiotensin II or 12-O-tetradecanoylphorbol-13-acetate (TPA) was associated with a mitogenic response in RIE-1 rat intestinal epithelial cells. However, whereas in control experiments using Swiss 3T3 cells TPA stimulated phosphorylation of the major PKC substrate, MARCKS, the agent did not induce the phosphorylation of any protein with the electrophoretic mobility pattern of MARCKS in RIE-1 cells. However, TPA was able to activate PKC in RIE-1 cells since the agent reduced ('transmodulated') 125I-EGF binding to the cells. The failure of TPA to induce phosphorylation of MARCKS in RIE-1 cells was due to the lack of expression of MARCKS protein and mRNA by these cells. MARCKS is not therefore required for mitogenic signalling via PKC in RIE-1 cells.

3T3 Cells↗

PubMed1995

Discovery of losartan, the first angiotensin II receptor antagonist.

The 'discovery' of losartan represents three separate discoveries: (1) losartan as the unique biphenyltetrazole molecule and the first of a new chemical class; (2) losartan as a tool to identify AT1-subtype receptors; and (3) losartan as a specific probe for exploring the multiple roles of angiotensin II (Ang II) in normal physiology and pathologic states. Losartan is the first nonpeptide orally active Ang II receptor antagonist to reach clinical trials. Losartan was selected for its affinity for Ang II receptors, functional antagonism of Ang II, lack of agonist properties, and oral anti-hypertensive effects. Losartan has been widely used to define the distribution and function of AT receptor subtypes. Although possible roles of the AT2 subtype have been reported, virtually all of the known effects of Ang II are blocked by losartan. Specific AT1 receptor blockade has been broadly compared with ACE inhibition. Possible differences on the basis of AT1 selectivity, bradykinin potentiating effects and Ang II formed by non-ACE pathways are discussed. Losartan blocks the vascular constrictor effect of Ang II, the Ang II-induced aldosterone synthesis and/or release, and the Ang II-induced cardiovascular 'growth' in vitro and in vivo. In various models of experimental hypertension, losartan prevents or reverses the elevated blood pressure and the associated cardiovascular hypertrophy similar to ACE inhibitors. Likewise, in models of renal failure (for example reduced renal mass, puromycin, ochratoxin), losartan, like ACE inhibition, markedly reduced the elevation in blood pressure, proteinuria or sclerosis. In aortocaval shunt, coronary ligation and ventricular pacing models of heart failure, losartan demonstrated a pathological role for Ang II by reversing the associated haemodynamic findings. In SHR-stroke prone, losartan dramatically increased survival while having a limited effect on blood pressure, suggesting a non-pressure dependent effect of Ang II. These collective data show that Ang II exerts complex pathological effects in experimental models of vascular, cardiac, renal and cerebral disease. The effectiveness of losartan in experimental models of heart failure supports its evaluation in clinical trials with patients with heart failure.

Angiotensin II↗

PubMed1994

Interaction of a protein phosphatase with an Arabidopsis serine-threonine receptor kinase.

A protein phosphatase was cloned that interacts with a serine-threonine receptor-like kinase, RLK5, from Arabidopsis thaliana. The phosphatase, designated KAPP (kinase-associated protein phosphatase), is composed of three domains: an amino-terminal signal anchor, a kinase interaction (KI) domain, and a type 2C protein phosphatase catalytic region. Association of RLK5 with the KI domain is dependent on phosphorylation of RLK5 and can be abolished by dephosphorylation. KAPP may function as a signaling component in a pathway involving RLK5.

Amino Acid Sequence↗

PubMed1994

Activation of AT1 angiotensin receptors induces DNA synthesis in a rat intestinal epithelial (RIE-1) cell line.

Proliferation of the rat intestinal epithelial cell-line, RIE-1, has previously been shown to be stimulated by certain polypeptide growth factors acting via receptors that possess intrinsic tyrosine kinase activity. In this study, we show that the octapeptide hormone angiotensin II (AII), apparently acting through the AT1 G-protein-coupled receptor, is also a mitogen for RIE-1 cells. Maximal stimulation of DNA synthesis and cellular proliferation occurred at an AII concentration of 10-100 nM, with half-maximal stimulation at 1 nM. The mitogenic response to AII was completely inhibited by the AT1 angiotensin-receptor antagonist, DuP753, but not by the AT2-receptor antagonist, PD123319. The early signalling responses activated by AII in RIE-1 cells include increased production of inositol phosphates, a transient increase in the intracellular concentration of free calcium, an activation of protein kinase C, and a rapid change in the pattern of cellular protein-tyrosine phosphorylation. These results implicate an activation of the inositol lipid signalling pathway via the AT1 receptor subtype in the AII-stimulated mitogenic response of this normal epithelial cell line.

Angiotensin II↗