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Biomedical subjects

David J Smith

Publications and source records attributed to David J Smith.

At least 37 records · Page 2Linked to original sources

Training principles and issues for ultra-endurance athletes.

Ultra-endurance competition is defined as events that exceed than 6 hours in duration. The longer events rely on long-term preparation, sufficient nutrition, accommodation of environmental stressors, and psychologic toughness. Successful ultra-endurance performance is characterized by the ability to sustain a higher absolute speed for a given distance than other competitors. This can be achieved through a periodized training plan and by following key principles of training. Periodization is an organization of training into large, medium and small training blocks which are referred to as macro-, meso-, and microcycles, respectively. When the sequencing of training is correctly applied, athletes can achieve a high state of competition readiness and during the months of hard training, avoid the overtraining syndrome. A plan is executed in accordance with the following principles of training: all-around development, overload, specificity, individualization, consistent training, and structural tolerance. Training relies heavily on the athlete's tolerance to repetitive strain. Today's ultra-endurance athlete must also follow appropriate nutritional practices in order to recover and prepare for daily training and remain injury free and healthy. Rehydration after exercise, together with the timing and method of increased food intake to cope with heavy training, are essential for optimal performance. Furthermore, the treatment of soft tissue after training or racing is necessary to control inflammation.

Adaptation, Physiological↗

Crystal structure of the human vascular adhesion protein-1: unique structural features with functional implications.

The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role.

Amine Oxidase (Copper-Containing)↗

Vibration training: an overview of the area, training consequences, and future considerations.

The effects of vibration on the human body have been documented for many years. Recently, the use of vibration for improving the training regimes of athletes has been investigated. Vibration has been used during strength-training movements such as elbow flexion, and vibration has also been applied to the entire body by having subjects stand on vibration platforms. Exposure to whole-body vibration has also resulted in a significant improvement in power output in the postvibratory period and has been demonstrated to induce significant changes in the resting hormonal profiles of men. In addition to the potential training effects of vibration, the improvement in power output that is observed in the postvibratory period may also lead to better warm-up protocols for athletes competing in sporting events that require high amounts of power output. These observations provide the possibility of new and improved methods of augmenting the training and performance of athletes through the use vibration training. Despite the potential benefits of vibration training, there is substantial evidence regarding the negative effects of vibration on the human body. In conclusion, the potential of vibration treatment to enhance the training regimes of athletes appears quite promising. It is essential though that a thorough understanding of the implications of this type of treatment be acquired prior to its use in athletic situations. Future research should be done with the aim of understanding the biological effects of vibration on muscle performance and also the effects of different vibration protocols on muscle performance.

Clinical Protocols↗

Endotaxial silicide nanowires.

We demonstrate the growth of self-assembled nanowires of cobalt silicide on Si(111), (100), and (110) substrates during deposition of Co onto a heated Si substrate. Silicide islands form via an endotaxial mechanism, growing into the substrate along inclined Si{111} planes, which breaks the symmetry of the surface and leads to a long, thin nanowire shape. During growth, both the length and width of the islands increase with time in a fixed proportion that varies strongly with growth temperature, which shows that the nanowire shape is kinetically determined. It is expected that nanowires could form in many other overlayer/substrate systems via this mechanism.

Journal Article↗

Molecular basis of slow activation of the human ether-a-go-go related gene potassium channel.

The human ether-á-go-go related gene (HERG) encodes the pore forming alpha-subunit of the rapid delayed rectifier K(+) channel which is central to the repolarization phase of the cardiac action potential. HERG K(+) channels have unusual kinetics characterized by slow activation and deactivation, yet rapid inactivation. The fourth transmembrane domain (S4) of HERG, like other voltage-gated K(+) channels, contains multiple positive charges and is the voltage sensor for activation. In this study, we mutated each of the positively charged residues in this region to glutamine (Q), expressed the mutant and wild-type (WT) channels in Xenopus laevis oocytes and studied them using two-electrode voltage clamp methods. K525Q channels activated at more hyperpolarized potentials than WT, whereas all the other mutant channels activated at more depolarized potentials. All mutants except for R531Q also had a reduction in apparent gating charge associated with activation. Mutation of K525 to cysteine (C) resulted in a less dramatic phenotype than K525Q. The addition of the positively charged MTSET to K525C altered the phenotype to one more similar to K525Q than to WT. Therefore it is not charge per se, but the specific lysine side chain at position 525, that is crucial for stabilizing the closed state. When rates of activation and deactivation for WT and mutant channels were compared at equivalent total (chemical + electrostatic) driving forces, K525Q and R528Q accelerated activation but had no effect on deactivation, R531Q slowed activation and deactivation, R534Q accelerated activation but slowed deactivation and R537Q accelerated deactivation but had no effect on activation. The main conclusions we can draw from these data are that in WT channels K525 stabilizes the closed state, R531 stabilizes the open state and R534 participates in interactions that stabilize pre-open closed states.

Amino Acid Sequence↗

Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol.

Zilpaterol is an beta-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growth promoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the development of an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in 0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLH resulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigen for ELISA development. The average IC(50) derived from the developed zilpaterol immunoassay was 3.94 +/- 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (dobutamine, fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists. The assay was tolerant of up to 10% (v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrations ranging from 0.05 to 1.0 M minimally affected B(0) or IC(50) values. When buffer pH was <7 or >8.8, the IC(50) values increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specific zilpaterol ELISA has been developed that can serve as a rapid screening assay.

Adrenergic beta-Agonists↗

Granulocyte transmigration through the endothelium is regulated by the oxidase activity of vascular adhesion protein-1 (VAP-1).

Polymorphonuclear leukocytes (PMNs) migrate from the blood into areas of inflammation by binding to the endothelial cells of blood vessels via adhesion molecules. Vascular adhesion protein-1 (VAP-1) is one of the molecules mediating leukocyte-endothelial cell interactions. It is also an endothelial cell-surface enzyme (amine oxidase) that produces reactive oxygen species during the catalytic reaction. To study the role of the enzymatic activity of VAP-1 in PMN extravasation, we used an enzymatically inactive VAP-1 mutant, specific amine oxidase inhibitors (including a novel small molecule compound), and anti-VAP-1 antibodies in several flow-dependent models. The enzyme inhibitors diminished PMN rolling on and transmigration through human endothelial cells under conditions of laminar shear stress in vitro. Notably, the enzyme inactivating point mutation abolished the capacity of VAP-1 to mediate transmigration. Moreover, the new VAP-1 inhibitor effectively prevented the extravasation of PMNs in an animal model of inflammation. These data show that the oxidase activity of VAP-1 controls PMN exit from the blood during the relatively poorly understood transmigration step.

Amine Oxidase (Copper-Containing)↗

Giant folliculosebaceous cystic hamartoma of the upper extremity.

BACKGROUND: Folliculosebaceous cystic hamartoma (FSCH) is a rare cutaneous hamartoma consisting of dilated folliculosebaceous units invested in mesenchymal elements. These lesions have a striking predilection for the central face and scalp of adults. The vast majority of lesions present as 0.5-1.5-cm papules or exophytic nodules. A single case of giant FSCH has been reported on the upper back. METHODS: A 32-year-old woman presented with a (15 cm in greatest dimension) plaque-like, multinodular lesion on her left upper arm for several years. The lesion was clinically suspected to be a nevus sebaceus. RESULTS: The skin excision showed numerous dermal and subcutaneous dilated follicular structures with peripherally radiating sebaceous lobules, hair follicles, and surrounding mesenchymal elements consistent with FSCH. CONCLUSION: To our knowledge, this is the second case of giant FSCH. Our case is unique for its larger size, more plaque-like growth, and location on an extremity when compared to the seminal case of giant FSCH.

Adult↗

New magnetic order in buried native iron oxide layers.

The properties of a magnetically ordered buried Fe oxide layer are presented. This oxide has a room-temperature magnetization exceeding that of Fe3O4 by 42% and of gamma-Fe2O3 by 89%. The oxide consists of a component (70%) with a net moment of 2.0 micro(B)/Fe ion, while the remaining spins yield no net moment. The oxide magnetization is stabilized in part by the proximate Fe metal.

Journal Article↗

Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer.

Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR). Protein kinase Cmu (PKCmu) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC. Studying PKCmu interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCmu kinase domain (KD) in PC, which was quantified by beta-gal assay, confirmed in PC cells by immunoprecipitation, and PKCmu-MT 2A co-localization in vivo by immunofluorescence studies. PKCmu domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains. Peptide library and in silico analysis strongly suggest that MT 2A is a novel substrate of PKCmu and our data indicate that the PKCmu-MT 2A interaction depends on PKCmu kinase activity. Because metallothioneins are associated with cell proliferation and CR, the PKCmu-MT 2A interaction may contribute to CR and/or AI in PC.

Androgens↗

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation.

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.

Amino Acid Sequence↗

Protein kinase C mu is down-regulated in androgen-independent prostate cancer.

Progression to androgen independence (AI) is the main cause of death in prostate cancer. Our prior differential gene expression studies by microarray analysis in progressive prostate cancer cell line model identified dysregulation of protein kinase C mu (PKCmu) expression in prostate cancer. In this study, quantitative ribonuclease protection assay and immunoblot analysis demonstrate down regulation of PKCmu at transcription and translational level, respectively, in AI C4-2 cells compared to its parental androgen dependent (AD) LNCaP prostate cancer cells. Significantly lower PKCmu kinase activity was confirmed in C4-2 cells by in vitro kinase assay. Immunohistochemical studies of prostate cancer tissue from patient progressing to AI prostate cancer demonstrated that PKCmu expression is decreased in 100% of AI human prostate cancers. The consistent down regulation of PKCmu in cell line models and human prostate cancer tissues suggests a possible functionally significant role for PKCmu in progression to AI in prostate cancer.

Androgens↗

Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis: comparative study with HPLC and ELISA.

A biosensor method, using the surface plasmon resonance (SPR) principle, was developed for the determination of ractopamine in cattle and sheep urine. A monoclonal antibody was used to compete with ractopamine in the sample and ractopamine immobilized on the sensor chip. Addition of bovine serum albumin (BSA, 1 mg/mL) as an antibody stabilizer to the incubation buffer was required to achieve a stable biosensor response throughout each sample set. The calibration curve gave a mean IC(50) of 4.7 +/- 0.21 ng/mL (n = 7). Over sample concentrations from 2.5 to 10 ng/mL recoveries were typically approximately 100-110%, whereas inter- and intra-assay reproducibilities (% CV) were usually less than 10 and 6%, respectively. Comparison of biosensor results with results obtained from high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assays (ELISA) using enzyme-hydrolyzed urine (to convert ractopamine conjugates to free ractopamine) gave correlation coefficients of 0.94 for sheep and 0.86 for cattle. Slopes of the lines, with zero intercepts, equaled 0.80 for sheep and 0.74 for cattle. For untreated (nonhydrolyzed) urine samples, the correlations between biosensor and HPLC results were 0.95 for sheep and 0.72 for cattle with slopes of 1.18 (sheep) and 1.69 (cattle). The slopes greater than unity indicate that the biosensor responded to ractopamine metabolites in addition to free ractopamine. The biosensor assay is an excellent analytical tool to screen ractopamine residues in sheep or cattle urine, and the results should be extendible to other species with suitable validation.

Adrenergic beta-Agonists↗

A framework for understanding the training process leading to elite performance.

The development of performance in competition is achieved through a training process that is designed to induce automation of motor skills and enhance structural and metabolic functions. Training also promotes self-confidence and a tolerance for higher training levels and competition. In general, there are two broad categories of athletes that perform at the highest level: (i) the genetically talented (the thoroughbred); and (ii) those with a highly developed work ethic (the workhorse) with a system of training guiding their effort. The dynamics of training involve the manipulation of the training load through the variables: intensity, duration and frequency. In addition, sport activities are a combination of strength, speed and endurance executed in a coordinated and efficient manner with the development of sport-specific characteristics. Short- and long-term planning (periodisation) requires alternating periods of training load with recovery for avoiding excessive fatigue that may lead to overtraining. Overtraining is long-lasting performance incompetence due to an imbalance of training load, competition, non-training stressors and recovery. Furthermore, annual plans are normally constructed in macro-, meso- and microcycles around the competitive phases with the objective of improving performance for a peak at a predetermined time. Finally, at competition time, optimal performance requires a healthy body, and integration of not only the physiological elements but also the psychological, technical and tactical components.

Body Composition↗

Mapping of electrostatic potential in deep submicron CMOS devices by electron holography.

Quantitative two-dimensional maps of electrostatic potential in device structures are obtained using off-axis electron holography with a spatial resolution of 6 nm and a sensitivity of 0.17 V. Estimates of junction depth and variation in electrostatic potential obtained by electron holography, process simulation, and secondary ion mass spectroscopy show close agreement. Measurement artifacts due to sample charging and surface "dead layers" do not need to be considered provided that proper care is taken with sample preparation. The results demonstrate that electron holography could become an effective method for quantitative 2D analysis of dopant diffusion in deep-submicron devices.

Journal Article↗

Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals.

A monoclonal antibody-based ractopamine immunoassay has been applied to incurred samples from sheep and cattle. Results obtained by immunoassay were compared with those from high-performance liquid chromatography (HPLC). Three sets of sample extracts containing primarily unmetabolized ractopamine were analyzed. Correlation of HPLC with enzyme-linked immunosorbent assay (ELISA) for beef liver samples gave an r(2) = 0.98 despite rather low ractopamine concentrations (range 1.1-13.4 ng/mL, n = 6). Ractopamine concentrations in cow urine samples treated by solid phase extraction, to remove ractopamine metabolites, also showed a high correlation between the HPLC and the ELISA results (r(2) = 0.95, range 1.0-275 ng/mL, n = 61). In contrast, HPLC and ELISA analyses of ractopamine in sheep urine were not well-correlated (r(2) = 0.58, range 0.85-51 ng/mL, n = 34). When ractopamine conjugates in urine samples were hydrolyzed with hydrolytic enzymes, ELISA and HPLC methods were highly correlated [r(2) = 0.94 for sheep (range 123-10 554 ppb, n = 60) and an r(2) = 0.98 for cattle (range 14-8159 ppb, n = 62)]. Tissues contained only minute amounts of ractopamine, and after 7-day withdrawal periods, less than 1 ppb of free ractopamine was detected. Ractopamine was rapidly metabolized in both cattle and sheep. The difference in ractopamine concentration of urine samples before and after hydrolysis indicated that only 1-5% of ractopamine was excreted unmetabolized. Results from this study indicate that the monoclonal antibody-based ELISA could be useful for a sensitive, quantitative, or qualitative ractopamine screening assay.

Animal Feed↗

Primary hyperparathyroidism in an elderly woman: surgical reversibility of profound mental state problems due to mild hypercalcaemia.

BACKGROUND: Clinical case of primary hyperparathyroidism in an 89 year old woman causing profound neuropsychiatric symptoms associated with relatively mild increases in serum calcium levels. OBJECTIVES: To demonstrate that lowering of serum calcium levels by medical treatment (biphosphonate), provided a scientific basis from which to treat a very elderly woman with neuropsychiatric symptoms with definitive surgery. METHOD: Case report. RESULTS: The use of biphosphonate therapy led to marked but temporary improvements in her mental state. CONCLUSION: A trial of reducing serum calcium levels with biphosphonate appears to be indicated in elderly patients with mild primary hyperparathyroidism and neuropsychiatric disorders.

Aged↗

Liquid chromatography/electrospray tandem mass spectrometric analysis of incurred ractopamine residues in livestock tissues.

Ractopamine HCl is a beta-adrenergic agonist (beta-agonist) recently approved by the U.S. Food and Drug Administration, but not other governmental agencies, for use in finishing swine. For these reasons, it was important to develop and validate mass spectrometric methods for the detection and confirmation of ractopamine residues in livestock marker tissues. Incurred tissues in cattle, sheep, turkeys, and ducks were generated during 7-day ractopamine feeding (20 ppm in diets) periods. Disposition of ractopamine residues in liver and pigmented retinal epithelium was determined in animals slaughtered with withdrawal periods of 0, 3, and 7 days. Ractopamine residues, purified using solid-phase extraction, were measured using liquid chromatography (LC) and electrospray with detection by tandem mass spectrometry (MS/MS) in the multiple reaction-monitoring (MRM) mode. Total ractopamine residues (parent ractopamine + hydrolyzed conjugates) in liver were detected in all species on withdrawal day 0 (2-97 ppb) and were greatly diminished in all species by withdrawal day 7 (<1 ppb). Bovine and ovine retina had lower levels of ractopamine (0.5-3 ppb) than liver, and occular residues increased with withdrawal time, suggesting redistribution into this tissue. Lower limits of quantification were found to be approximately 0.1 ppb in liver and retina. Incurred ractopamine residues were confirmed by the precise and accurate agreement of MRM intensity ratios of diagnostic fragment ions (m/z 284, 164, and 121) from the protonated molecule between ractopamine residues in incurred samples and an authentic ractopamine standard. The limits of confirmation in liver and retina using recognized acceptance criteria were below 1 ppb. The high sensitivity and specificity for measurement and confirmation of ractopamine residues suggests this method will be applicable for regulatory residue surveillance programs.

Animals↗