Effects of electrically induced fatigue on the twitch and tetanus of paralyzed soleus muscle in humans

Shields, Richard K., Laura Frey Law, Brenda Reiling, KellySass, and Jason Wilwert. Effects of electrically induced fatigueon the twitch and tetanus of paralyzed soleus muscle in humans.J. Appl. Physiol. 82(5):1499-1507, 1997.We analyzed the twitch and summated torque(tetanus) during repetitive activation and recovery of the human soleusmuscle in individuals with spinal cord injury. Thirteen individualswith complete paralysis (9 chronic, 4 acute) had the tibial nerveactivated every 1,500 ms with a 20-Hz train (7 stimuli) for 300 ms anda single pulse at 1,100 ms. The stimulation protocol lasted 3 min andincluded 120 twitches and 120 tetani. Minimal changes were found forthe acute group. The chronic group showed a significant reduction inthe torque and a significant slowing of the contractile speeds of boththe twitch and tetanus. The decrease in the peak twitch torque was significantly greater than the decrease in the peak tetanus torque early during the fatigue protocol for the chronic group. The twitch time to peak and half relaxation time were prolonged during fatigue, which was associated with improved fusion of the tetanus torque. At theend of the fatigue protocol, the decrease in the peak twitch torque wasnot significantly different from the decrease in the peak tetanustorque. After 5 min of rest, the contractile speeds recovered causingthe tetanus to become unfused, but the tetanus torque became lessdepressed than the twitch torque. The differential responses for thetwitch and the tetanus suggest an interplay between optimal fusioncreated from contractile speed slowing and excitation contractioncoupling compromise. These issues make the optimal design of functionalelectrical stimulation systems a formidable task.

     

Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Genetic polymorphism of serum complement components in the chimpanzee

Significant polymorphism of serum complement components Bf, C2, C3, C6, and C8 in the chimpanzee has been demonstrated. The data are consistent with the hypothesis thatC2 andBf are closely linked toChLA and argue against close linkage ofChLA toC3 or toC8, as in man. In addition, a blank allele for C6 and C6 deficiency was detected in several chimps.     

The effect of complex-formation with polyanions on the redox properties of cytochrome c.

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN'N'-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.     

Ionic currents across pancreatic acinar cell membranes and their role in fluid secretion

Fluid and enzyme secretion from a number of mammalian exocrine glands is controlled by the action of neurotransmitters and hormones on acinar cell membranes. Sustained stimulation evoking sustained fluid and enzyme secretion also evokes sustained membrane depolarization and increase in conductance. Mouse and rat pancreatic fluid and enzyme secretion, as well as membrane depolarization and conductance increase evoked by sustained stimulation with acetylcholine or cholecystokinin-gastrin peptides, are acutely dependent on extracellular calcium. However, the initial stimulant-evoked conductance increase and secretion appear to be triggered by calcium released from inside the cells. Direct measurement of membrane current during sustained stimulation in voltage-clamp experiments with resolution of the total current into its Na, Cl and K components has allowed calculations of stimulant-evoked Na and Cl uptake into the acinar cells. The NaCl uptake is quantitatively sufficient to account for the stimulant-evoked fluid secretion. The role of the stimulant-evoked transmembrane ionic current appears to be the supply of salt for the fluid secretion. Calcium derived from intracellular sources in the initial phase of secretion, and from the extracellular fluid in the sustained phase, couples fluid and enzyme secretion to hormone-receptor interaction.     

Drilomermis leioderma n. gen., n. sp. (Mermithidae:Nematoda) parasitizing Cybister fimbriolatus (Say) (Dystiscidae-Coleoptera)

The nematode Drilomermis leioderma n. gen., n. sp. (Merrnithidae) is described from larvae of Cybister fimbriolatus (Say) (Dytiscidae: Coleoptera) in Louisiana. Diagnostic characters of the genus Drilomermis are: medium-sized nematodes with the cuticle appearing smooth (lacking cross fibers) under the light microscope, six cephalic papillae, without mouth papillae, six hypodermal cords at midbody, 2 extremely long spicules (longer than 10 times body width at anus) which are separate and parallel (not twisted), an S-shaped vagina, medium-sized amphids located near head papillae, and postparasitic juvenile with a tail appendage. D. leioderma possesses a ventrally displaced mouth, very long vagina, and male genital papillae arranged in 3 double rows in the vicinity of the cloacal opening. Even when containing multiple parasites, about 40% of the hosts sulwived emergence of the memithids and lived several more days. In nature, some of these hosts may be able to continue their development, which is unusual since most mermithid-parasitized hosts die soon after the nematode emerges.     

Subcellular distribution and kinetic parameters of HS mouse liver aldehyde dehydrogenase

1. Aldehyde dehydrogenase subcellular distribution studies were performed in a heterogeneous stock (HS) of male and female mice (Mus musculus) with propionaldehyde (5 mM and 50 microM) and formaldehyde (1 mM) and NAD+ or NADP+. 2. The relative percents of distribution were: cytosolic 55-68%, mitochondrial 12-20%, microsomal 9-18% and lysosomal 3-15% for both propionaldehyde concentrations and NAD+. 3. Kinetic experiments using propionaldehyde and acetaldehyde with NAD+ revealed two separate enzymes, Enzyme I (low Km) and Enzyme II (high Km) in the cytosolic and mitochondrial fractions. 4. The kinetic data also indicated a spectrum of cytosolic low Km values that exhibited a bimodal distribution with one congruent to 40 microM and one congruent to 5 microM. 5. It was concluded that there was no significant difference in aldehyde-metabolizing capability between male and female HS mice, compared on a per gram of liver basis. The cytosolic low Km enzyme plays a major role in aldehyde oxidation at moderate to low aldehyde concentrations.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.