Evidence that the redox state has a role in muscular contraction and relaxation

The present work reports that simple oxidizing agents are capable of inducing isotonic contraction of rat aorta in vitro, and that the concentration of agent required depends on its oxidizing potential. Conversely a reducing agent will reverse a muscular contraction induced by oxidizing agents.     

The effect of higher ATP cost of contraction on the metabolic response to graded exercise in patients with chronic obstructive pulmonary disease

To better understand the metabolic implications of a higher ATP cost of contraction in chronic obstructive pulmonary disease (COPD), we used (31)P-magnetic resonance spectroscopy ((31)P-MRS) to examine muscle energetics and pH in response to graded exercise. Specifically, in six patients and six well-matched healthy controls, we determined the intracellular threshold for pH (T(pH)) and inorganic phosphate-to-phosphocreatine ratio (T(Pi/PCr)) during progressive dynamic plantar flexion exercise with work rate expressed as both absolute and relative intensity. Patients with COPD displayed a lower peak power output (WRmax) compared with controls (controls 25 ± 4 W, COPD 15 ± 5 W, P = 0.01) while end-exercise pH (controls 6.79 ± 0.15, COPD 6.76 ± 0.21, P = 0.87) and PCr consumption (controls 82 ± 10%, COPD 70 ± 18%, P = 0.26) were similar between groups. Both T(pH) and T(Pi/PCr) occurred at a significantly lower absolute work rate in patients with COPD compared with controls (controls: 14.7 ± 2.4 W for T(pH) and 15.3 ± 2.4 W for T(Pi/PCr); COPD: 9.7 ± 4.5 W for T(pH) and 10.0 ± 4.6 W for T(Pi/PCr), P < 0.05), but these thresholds occurred at the same percentage of WRmax (controls: 63 ± 11% WRmax for T(pH) and 67 ± 18% WRmax for T(Pi/PCr); COPD: 59 ± 9% WRmax for T(pH) and 61 ± 12% WRmax for T(Pi/PCr), P > 0.05). Indexes of mitochondrial function, the PCr recovery time constant (controls 42 ± 7 s, COPD 45 ± 11 s, P = 0.66) and the PCr resynthesis rate (controls 105 ± 21%/min, COPD 91 ± 31%/min, P = 0.43) were similar between groups. In combination, these results reveal that when energy demand is normalized to WRmax, as a consequence of higher ATP cost of contraction, patients with COPD display the same metabolic pattern as healthy subjects, suggesting that skeletal muscle energy production is well preserved in these patients.     

Cognitive cost of motor reorganizations associated with muscular fatigue during a repetitive pointing task

Muscular fatigue is known to impair motor performance and to catalyse the development of upper limb musculoskeletal disorders. In order to delay the deleterious effects of muscular fatigue, the central nervous system (CNS) employs compensatory strategies. The cognitive cost of such compensatory strategies was assessed in 10 male subjects who alternatively performed two dual-task protocols before and immediately after an exhaustion procedure specific to upper arm abductor musculature. The main motor tasks were an isometric force-matching and a rapid multi-joint pointing. A secondary probe reaction time (RT) task was performed during both protocols and served as an indicator of attentional demands. Overall motor task performance was maintained despite fatigue. Kinematic and electromyographic data revealed that subjects used motor reorganization during the pointing task when fatigued. The RT increased with fatigue in both dual-task protocols, but this increase was significantly higher during the pointing task than during the force-matching task.The results highlight that the motor reorganization used by the CNS under muscular fatigue states require higher attentional demands than the initial motor organization. Finally, the capacity to delay the deleterious effects of muscular fatigue seems to depend on the proportion of cognitive resources available to plan the compensatory motor strategy.     

Evidence that inhibition of cathepsin-B contributes to the neuroprotective properties of caspase inhibitor Tyr-Val-Ala-Asp-chloromethyl ketone.

During the use of tetrapeptide and other proprietary caspase inhibitors in the study of neurodegeneration, we had concluded that mechanisms other than the inhibition of caspases contributed to the protective effects of certain caspase inhibitors. Here we report our studies to identify a target for and hence a mechanism by which the tetrapeptide inhibitor tyrosine-valine-alanine-aspartate-chloromethyl ketone (Ac-YVAD-cmk) is able to rescue neuronal cell cultures from cell death. Ac-YVAD-cmk rescued neuronal cells from cell death in response to oxidative stress and oxygen/glucose deprivation. Affinity labeling with biotinylated YVAD-cmk demonstrated distinct binding proteins for the inhibitor in cells from the central nervous system versus Jurkat cells. Binding to the novel target protein was displaced by class-specific protease inhibitors and suggested that the target is a cysteine protease. Affinity purification and sequencing identified the target as cathepsin-B. Cathepsin-B inhibitors competed with biotinylated YVAD-cmk for the target protein. The availability of the target for binding was reduced in cells that had been rescued by unlabeled inhibitor. Cathepsin-B inhibitors rescue hippocampal slices from cell death induced by oxygen/glucose deprivation. These data provide evidence to support a role for cathepsin-B in neuronal cell death, particularly that following ischemia.     

Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase

Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.     

Evidence that the corpus luteum of pregnancy contributes to the control of tonic secretion of LH in the ewe

Concentrations of LH and FSH were measured in blood samples collected from the jugular vein at 20-min intervals for 7 h (09:00-16:00 h) on Days 60, 80, 100 and 120 of pregnancy in 5 intact ewes and 5 from which the CL had been excised on Day 70. In the 5 intact ewes, plasma LH concentrations remained low and unchanged between Days 60 and 120. During this period, pulsatile release of LH occurred irregularly and infrequently. Removal of the CL resulted in an increase in the basal values of LH and in the frequency and amplitude of LH pulses. Concentrations of FSH were relatively constant in all stages of pregnancy examined and were similar in both groups of ewes. These results show that (1) LH concentrations are low during the second half of pregnancy; and (2) LH, but not FSH, increases after CL excision, presumably by removing some luteal factor inhibitor of LH secretion.     

Evidence that the anterior hypothalamus contributes to the control of tonic secretion of follicle-stimulating hormone in the female rat

A horizontal knife cut was placed between the dorsal anterior hypothalamic area (DAHA) and the medial basal hypothalamus to examine the role of the DAHA in the selective secretion of follicle-stimulating hormone (FSH) following unilateral ovariectomy (ULO) and bilateral ovariectomy of the rat. Complete cuts markedly attenuated the increase in FSH observed 8 h after ULO, whereas incomplete or sham cuts did not. Concentration of luteinizing hormone (LH) did not increase in any group. These cuts also blocked the prolongation of estrous FSH secretion observed in long-term hemicastrated rats, since FSH levels on estrus were significantly lower in rats with complete cuts than in those rats given sham or incomplete cuts. In contrast, neural surgery had no effect on proestrous FSH concentrations. Finally, when FSH levels were monitored 2 days after bilateral ovariectomy, the postcastration rise in FSH was not altered by any neurosurgical procedures. These results support the hypothesis that a neural system originating in, or passing through, the DAHA is necessary for the selective increase in FSH following ULO.     

Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein.

Avian sarcoma virus (ASV) induces sarcomas in animals and transforms fibroblasts to a neoplastic state in cell culture. A single viral gene (src) is responsible for both the induction and maintenance of neoplastic transformation. Recent work has identified a protein with a molecular weight of 60,000 daltons that is apparently encoded in src and may be the effector molecule for the gene (Brugge and Erikson, 1977; Purchio et al, 1978). The putative product of src can be immunoprecipitated by antisera obtained from rabbits bearing tumors induced by ASV. We have used this approach to isolate the protein to characterize further its genetic origins and possible function. Our rabbit tumor antisera precipitated a protein with a molecular weight of 60,000 daltons; according to serological, biochemical and genetic criteria, this protein is encoded in src. We found that this protein is phosphorylated and therefore denoted it pp60. Phosphorylation of pp60 could be accomplished in vitro with extracts of ASV-infected cells. A temperature-sensitive conditional mutation in src had no demonstrable effect on either the production or stability of pp60 in the infected cell, but phosphorylation of the protein was temperature-sensitive. Since the mutant src is not expressed at the restrictive temperature, our findings raise the possibility that phosphorylation of pp60 is required for its function as the putative effector of src. Immunoprecipitates prepared with extracts of ASV-infected cells and the rabbit tumor antisera contained a protein kinase activity that catalyzed phosphorylation of the heavy chains of immunoglobulin molecules, using either ATP or GTP as phosphate donor. The kinase activity immunoprecipitated in parallel with pp60 was obtained only from cells that contained a functioning product of src and could not be precipitated with antisera directed against structural proteins of ASV. A temperature-sensitive conditional mutation in src caused the kinase activity to be thermally inactivated in vitro far more rapidly than the activity from cells infected with wild-type virus. We conclude that both the protein kinase and pp60 are encoded in src, and that the enzymatic activity may be an intrinsic property of pp60. Phosphorylation of pp60 in cellular extracts was inhibited by calcium ion, whereas the immunoprecipitable kinase activity was not, suggesting that the kinase responsible for pp60 phosphorylation may be distinct from that encoded in src. Collett and Erikson (1978) have also identified a protein kinase activity associated with pp60. These findings raise the possibility that phosphorylation of specific cellular targets might account for transformation of the host cell by src.     

Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins.

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.     

Eugenic sterilization: a preliminary comparison of the Scandinavian experience to that of Germany

The paper argues that historical analysis and explanation of eugenics in Germany can benefit much from systematic comparison to Scandinavia. Common cultural background and quite similar development up to 1933 provides a background for isolating salient causes in Nazi population policies. This comparison will also help a more precise understanding of the mutual dependence between science and politics in the case of eugenics. The author holds that many of the geneticists who participated in the eugenics debates of the 1930's and 1940's had a clearer grasp of the distinction between science and politics than most present day historiographers of eugenics.     

Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules

The keratinous capsules surrounding rat sperm mitochondria were isolated 24 days after intratesticular injections of [⁷⁵Se] selenite or [³⁵S] cysteine. Dodecyl sulfate-polyacrylamide gel electrophoresis of purified, doubly labeled mitochondrial capsules revealed only a single ⁷⁵Se-labeled component, whose molecular weight was 17,000, in agreement with previously reported observations obtained with cruder sperm fractions. Most of the ³⁵S label and the major zone of stained protein on the gels coincided with the position of ⁷⁵Se, suggesting that selenium is associated with a cysteine-rich structural protein. The level of selenium in rat sperm, 195 ± 3.2 ng/10⁸ sperm (approximately 30 ppm), determined by hydride generation and atomic absorption spectrophotometry, is consistent with a structural function for this trace element in the sperm.     

Exposure to chemotherapeutic agents and the risk of a second breast cancer: preliminary findings

Chemotherapeutic treatment for cancer has been successful in prolonging survival but may also lead to the development of second cancers. Two case-control studies presented here suggest, however, that breast cancer patients who receive chemotherapy are at significantly lower risk of a contralateral breast cancer than those who do not. Approximately 300 incident cases of contralateral breast cancer and 300 randomly chosen surviving controls with unilateral breast cancer were identified through the Connecticut Tumor Registry for inclusion in each study. The initial study was based on review of medical records at eight hospitals and indicated that the overall association with chemotherapy was modified by body build. The second study obtained information from in-person interviews, hospital records, and outpatient chemotherapy records from across the state. The preliminary results of this second study confirm the previous findings. Both cytotoxic and hormonal drugs were associated with a reduction in the risk of second breast cancers (OR = 0.5, 95 percent CI: 0.3-1.0; OR = 0.5, 95 percent CI: 0.2-1.2, respectively). Significant interaction with body build was observed for hormonal treatment (ratio of ORs = 5.8, 95 percent CI: 1.0-34.3 for a five-unit change in Quetelet's index), with a nonsignificant but detrimental effect suggested for overweight women (OR = 2.3, 95 percent CI: 0.4-13.9 for a Quetelet's score of 35).     

Assembly of microtubules with ATP: evidence that only a fraction of the protein is assembly-competent

Chick brain microtubule protein can be assembled in vitro with ATP, although the extent of assembly is less than that with GTP. The ATP-induced assembly is not the result of generation of GTP by the co-purifying nucleoside diphosphate kinase. Neither an observed increase in the critical concentration nor the phosphorylation of MAP2 can account for the decreased extent of assembly. However, whereas microtubules are formed with both ATP and GTP, incubation with ATP yields additional filaments and polymorphic aggregates. The results demonstrate that of the total protein which can be assembled into microtubules by GTP, about 25-35% is assembled into other structural forms in the presence of ATP.     

Circadian fluctuations in the muscular efficiency of athletes: with sleep versus sleep deprivation

The influence of time of day on muscular performance was studied. From part of the results of two different studies (EAS et EPS), the effects of sleep deprivation were appreciated. Seven times over the 24-h period, developed torque and myoelectric activity were estimated during maximal isometric voluntary contractions using an isokinetic dynamometer: elbow flexion for EAS in standardised sleep, and knee extension for EPS in complete sleep deprivation. The results showed nycthemeral changes in torque in both conditions (p < 0.005), with maximal values recorded at the beginning of night. Although during sleep deprivation (EPS) the rhythm followed neurophysiological factors, during EAS, this rhythm was accounted for by the variations in the contractile state of muscle.     

Evidence that a dodecamer duplication in the gene HOPA in Xq13 is not associated with mental retardation

A recent study suggested that a dodecamer duplication in exon 42 of the HOPA gene in Xq13 may be a significant factor in the etiology of X-linked mental retardation. In an effort to investigate this possibility, we determined the incidence of the dodecamer duplication in cohorts of non-fragile X males with mental retardation from three countries, cohorts of fragile X males from two countries, 43 probands from families with X-linked mental retardation and control cohorts from three countries. The duplication was found in 3.6-4.0% of male patients from two non-fragile X groups (Italy and South Carolina), in 1.2% from another non-fragile X group (South Africa), but in no male patients from families with X-linked mental retardation (South Carolina). The dodecamer duplication was also found in several white males with fragile X syndrome from France (5%) and South Africa (22.2%). Additionally, the duplication was found in 1.5% of South Carolinian newborn males, 2.5% South Carolinian male college students, 5% Italian male controls and 4.5% of the white South African controls. None of the black South African non-fragile X individuals with mental retardation, the fragile X or the control samples tested carried the duplication, suggesting that the duplication is rare in the black South African population. The incidence of the duplication was not significantly different between any of the groups in the study. Therefore, results of our studies in four different populations do not corroborate the findings of the previous study, and indicate that the HOPA dodecamer duplication does not convey an increased susceptibility to mental retardation.