Spatial distribution of disease-associated variants in three-dimensional structures of protein complexes.

Next-generation sequencing enables simultaneous analysis of hundreds of human genomes associated with a particular phenotype, for example, a disease. These genomes naturally contain a lot of sequence variation that ranges from single-nucleotide variants (SNVs) to large-scale structural rearrangements. In order to establish a functional connection between genotype and disease-associated phenotypes, one needs to distinguish disease drivers from neutral passenger variants. Functional annotation based on experimental assays is feasible only for a limited number of candidate mutations. Thus alternative computational tools are needed. A possible approach to annotating mutations functionally is to consider their spatial location relative to functionally relevant sites in three-dimensional (3D) structures of the harboring proteins. This is impeded by the lack of available protein 3D structures. Complementing experimentally resolved structures with reliable computational models is an attractive alternative. We developed a structure-based approach to characterizing comprehensive sets of non-synonymous single-nucleotide variants (nsSNVs): associated with cancer, non-cancer diseases and putatively functionally neutral. We searched experimentally resolved protein 3D structures for potential homology-modeling templates for proteins harboring corresponding mutations. We found such templates for all proteins with disease-associated nsSNVs, and 51 and 66% of proteins carrying common polymorphisms and annotated benign variants. Many mutations caused by nsSNVs can be found in protein-protein, protein-nucleic acid or protein-ligand complexes. Correction for the number of available templates per protein reveals that protein-protein interaction interfaces are not enriched in either cancer nsSNVs, or nsSNVs associated with non-cancer diseases. Whereas cancer-associated mutations are enriched in DNA-binding proteins, they are rarely located directly in DNA-interacting interfaces. In contrast, mutations associated with non-cancer diseases are in general rare in DNA-binding proteins, but enriched in DNA-interacting interfaces in these proteins. All disease-associated nsSNVs are overrepresented in ligand-binding pockets, and nsSNVs associated with non-cancer diseases are additionally enriched in protein core, where they probably affect overall protein stability.

Human skeletal muscle HSP70 response to physical training depends on exercise intensity.

We have previously reported that HSP70 in human skeletal muscle could be induced by training. However, whether HSP70 induction is dependent upon exercise volume or exercise intensity remains unknown. The aim of the present study was to investigate the relationship between HSP70 and training intensity in rowers. Fourteen well-trained male rowers were divided into two groups (group A, n = 6; group B, n = 8). Group A performed higher intensity exercise during 1st phase, whereas group B performed higher intensity exercise during 2nd training phase. Training volume in 2nd phase increased in both groups. Both training intensity and volume were reduced in 3rd phase. Muscle samples were taken from m. vastus lateralis by fine needle biopsy before training, at the end of the 1st, 2nd and 3rd training phases. HSP70 was quantitatively determined using SDS-PAGE with silver stain. In group A, HSP70 increased significantly from 38 +/- 12 etag before training to 59 +/- 16 etag at the end of the lst training phase (loaded total protein 2.5microg), and decreased afterwards. In group B, HSP70 increase (from 36 +/- 11 etag to 50 +/- 13 etag) in the 1st phase was significantly smaller, there was a further increase of HSP70 in the 2nd phase (60 +/- 14 etag). At the end of the training, HSP70 decreased in both groups. Thus, HSP70 response to training seems to be dependent upon exercise intensity.

Expression of myosin heavy chain isoforms in skeletal muscle of patients with peripheral arterial occlusive disease.

PURPOSE: Peripheral arterial occlusive diseases (PAODs) not only compromise blood flow but lead to a series of subsequent metabolic and structural changes in the relevant muscles. Changes in myofibrillar proteins (eg, of myosin heavy chain [MHC] isoforms), one of the determinants of muscle structure as well as of muscular function, have not been reported in patients with PAOD and were therefore the aim of this study. METHODS: Thirteen consecutive patients with PAOD were examined (clinical stage according to Fontaine II, three patients; III, three patients, and IV, seven patients) and compared with five age-matched control patients who had been in traffic accidents. A calf muscle sample (gastrocnemius muscle) in the ischemic region was taken for MHC isoform analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver stain, and the relative content of MHC isoforms was measured. RESULTS: Compared with the control patients, there was no significant change of MHC isoforms in patients with PAOD II. In patients with PAOD III, MHC IIb decreased significantly (P <.05) although MHC IIa remained unchanged; in patients with PAOD IV, both MHC IIa and IIb decreased significantly (P <.05). Accordingly, there was a progressive increase of the relative amount of MHC I with more critical ischemia in PAOD. CONCLUSION: In patients with PAOD, the content of MHC II decreased with a higher grade of ischemia. That seems to be consistent with an increased resistance to ischemia for myosin isoforms in the order of I more than in IIa more than IIb. Whether the decrease of MHC II in patients with PAOD is related to atrophy of muscle fibers or to muscle-fiber transition must be investigated further.

Training of junior rowers before world championships. Effects on performance, mood state and selected hormonal and metabolic responses.

BACKGROUND: Few data have been published on training of competitive athletes and about metabolic, hormonal and psychological reactions to overreaching (transient over-training) and tapering in successful athletes. METHODS: Training was recorded and effects on mood state and metabolic and hormonal responses were examined in 10 rowers and spares of the coxed eight during preparation for the World Championships 1995. Mood state was determined using the Recovery-Stress-Questionnaire for Athletes. Resting morning blood parameters as well as performance were measured every week over a period of five weeks. RESULTS: Very high training loads of approximately 3.2 hours per day were sustained for 18 days. Maximum performance (Pmax) and maximum lactate (Lamax) were decreased during high-load training phases (overreaching), Pmax, Lamax and endurance increased after the tapering period. There were decreases in gonadal and hypothalamic steroid hormones (fsh, 1h, prolactin, testosterone) during overreaching and increases in these hormones in tapering. Both performance and hormonal indices of training load were reflected by deterioration of recovery in the Recovery-Stress-Questionnaire for Athletes. CONCLUSIONS: Clear signs of overreaching were found after 18 days of intense training of about 3 h.d(-1) in these highly-trained athletes, i.e. decreases in performance, gonadal and hypothalamic steroid hormones and deterioration of recovery in the psychological questionnaire. After tapering values returned to baseline values before the World Championship. The findings indicate that overreaching is an integral part of successful training regimens and can be analyzed by a multi-factorial approach involving biological and psychometric data.

Human skeletal muscle HSP70 response to training in highly trained rowers.

Previous studies have demonstrated exercise-induced heat shock protein 70 (HSP70) in animals. The purpose of this study was to investigate human skeletal muscle HSP70 response to rowing training. Ten male rowers trained for 4 wk with different forms, durations, and intensities of exercise. Biopsy was performed in the right musculus vastus lateralis before training and at the end of each week. HSP70 in 5 microg of total protein from the muscle sample was determined by using Western blot and immunodetection with chemiluminescence technique, by means of laser densitometer referring to a series of known standard HSP70. Compared with pretraining (100%), HSP70 increased during training (181, 405, 456, and 363% from the first to fourth training week, respectively) with the maximum HSP70 production at the end of second training week. Thus HSP70 is induced in highly trained human muscle by long-term training.

Molecular cloning of a lobster G alpha(q) protein expressed in neurons of olfactory organ and brain.

We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, hG alpha(q), with >80% identity to mammalian and arthropod G alpha(q) sequences. In brain and olfactory organ, hG alpha(q) mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. G alpha(q) protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hG alpha(q) plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hG alpha(q) mRNA species (6 kb) in the olfactory organ, and the localization of hG alpha(q) mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hG alpha(q) is to mediate olfactory transduction.

Training and overtraining: an overview and experimental results in endurance sports.

Overtraining can be defined as "training-competition > > recovery imbalance", that is assumed to result in glycogen deficit, catabolic > anabolic imbalance, neuroendocrine imbalance, amino acid imbalance, and autonomic imbalance. Additional non-training stress factors and monotony of training exacerbate the risk of a resulting overtraining syndrome. Short-term overtraining called overreaching which can be seen as a normal part of athletic training, must be distinguished from long-term overtraining that can lead to a state described as burnout, staleness or overtraining syndrome. Persistent performance incompetence, persistent high fatigue ratings, altered mood state, increased rate of infections, and suppressed reproductive function have been described as key findings in overtraining syndrome. An increased risk of overtraining syndrome may be expected around 3 weeks of intensified/prolonged endurance training at a high training load level. Heavy training loads may apparently be tolerated for extensive periods of time if athletes take a rest day every week and use alternating hard and easy days of training. Persistent performance incompetence and high fatigue ratings may depend on impaired or inhibited transmission of ergotropic (catabolic) signals to target organs, such as: (I) decreased neuromuscular excitability, (II) inhibition of alpha-motoneuron activity (hypothetic), (III) decreased adrenal sensitivity to ACTH (cortisol release) and increased pituitary sensitivity to GHRH (GH release) resulting in a counter-regulatory shift to a more anabolic endocrine responsibility, (IV) decreased beta-adrenoreceptor density (sensitivity to catecholamines), (V) decreased intrinsic sympathetic activity, and (VI) intracellular protective mechanisms such as increased synthesis of heat-shock proteins (HSP 70) represent a complex strategy against an overload-dependent cellular damage.

Effect of felodipine on regional blood supply and collateral vascular resistance in patients with peripheral arterial occlusive disease.

This double-blinded, randomized, placebo-controlled study was designed to investigate the acute effect of felodipine on regional blood supply and collateral vascular resistance in patients with peripheral arterial occlusive disease (PAOD). Thirty men with PAOD were treated with a single dose of 5 mg felodipine or placebo. Systolic blood pressure (SBP), Doppler ankle pressure (DAP), calf blood flow (CBF) by venous occlusion plethysmography and calf transcutaneous oxygen tension (tcpO2) were measured during a cycle ergometry. Felodipine reduced SBP significantly (from 149 to 136 mmHg, p < 0.05), while placebo did not. DAP increased slightly but not significantly in both groups. The pressure gradient between SBP and DAP fell significantly in the felodipine group (60 vs 39 mmHg, p < 0.01) but not in the placebo group (59 vs 56 mmHg). There was a trend for lower velocity in tcpO2 decrease during the stress test and higher velocity of tcpO2 increase during recovery from exercise in the felodipine group although the differences between both groups were not significant. In the felodipine group, CBF increased by 35.6% (p < 0.05) whereas it did not change in the placebo group. In conclusion, while lowering SBP, felodipine increased slightly, or at least maintained, the blood supply to the calves in PAOD patients, which probably results from reducing collateral vascular resistance.

Molecular cloning and characterization of a lobster G alphaS protein expressed in neurons of olfactory organ and brain.

We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, lobG alphaS, with >70% identity to mammalian and arthropod G alphaS sequences. In genomic Southern blots, a fragment of lobG alphaS detected only one band, suggesting the lobsters have a single G alphaS gene. In brain and olfactory organ, lobG alphaS mRNA was expressed predominantly in neurons, including many of the neuronal cell body clusters of the brain. G alphaS protein was also expressed broadly, appearing on western blots as a band of 51.8 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that lobG alphaS plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, and the expression of lobG alphaS mRNA in the olfactory receptor neurons of the olfactory organ indicate that lobG alphaS may mediate olfactory transduction. That virtually all ORNs express lobG alphaS mRNA equally predicts that hyperpolarizing odor responses mediated by cyclic AMP are a property of all lobster olfactory receptor neurons.

Comparison of whole-body thallium imaging with transcutaneous PO2 in studying regional blood supply in patients with peripheral arterial occlusive disease.

Quantitatively estimating functional reserve of blood supply to the legs in patients with peripheral arterial occlusive disease (PAOD) remains a clinical issue. This study was designed to investigate the regional blood supply to the legs in PAOD patients during exercise by use of thallium 201 (201Tl) whole-body imaging in comparison with transcutaneous PO2 (tcPO2) measurement. Thirty-three patients with PAOD and 10 subjects without PAOD (control) performed an incremental cycle ergometry (CE), while tcPO2 was continuously registered on the involved calf. In the last minute of exercise, 2 mCi of 201Tl was injected intravenously and the 201Tl whole-body images were taken immediately (stress) and four hours (redistribution) following stress with a dual-head camera system. Regional blood supply (RBS) (%) was calculated from the geometric mean counts of the region of interest divided by the total counts of the whole body. The performance of PAOD patients was reduced in doing CE, and tcPO2 fell distinctly in PAOD patients (from 51 to 19 mmHg) whereas it increased in controls (from 57 to 67 mmHg). The RBS in PAOD patients was obviously reduced in comparison with that of controls. While in controls the RBS of the calf (3.1%) at stress did not differ from that at redistribution (3.4%), in PAOD patients the redistribution RBS (2.8%) increased as compared with that of stress (1.5%). There was a hyperbolic relationship between stress RBS of the calf and the velocity of tcPO2 fall in PAOD patients during exercise test (velocity of tcPO2 fall = -0.032 + 0.39/RBS, r2 = 0.54, P < 0.05). In conclusion, the RBS determined by 201Tl whole-body imaging is comparable to the tcPO2 measurement in differentiating patients with PAOD from subjects without PAOD during exercise. Regional 201Tl uptake reflects regional blood supply in PAOD patients. There is a hyperbolic relationship between the RBS derived from 201Tl whole-body imaging and tcPO2 in PAOD patients during exercise, implying that in a critical ischemia the lower the RBS is, the more steeply the tcPO2 decreases.