μ-Calpain Activation and Calpain-Mediated Cytoskeletal Proteolysis Following Traumatic Brain Injury

Abstract: Increasing evidence suggests that excessive activation of the calcium-activated neutral protease μ-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of μ-calpain to its 80-kDa precursor was measured by western blotting. This μ-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ-calpain activation on proteolysis of the neuronal cytoskeletal protein α-spectrin. Immunoreactivity for α-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ-calpain autolysis, calpain-mediated α-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.     

The genus Pyrus L. (Rosaceae) in south-west Europe and North Afkica

A multivariate morphometric study of the genus Pyrus in south-west Europe and North Africa shows that five species may be recognized in the area: P. bourgaeana Decne., P. communis L., P. cordata Dew., P. spinosa Forssk, and P. nivalis Jacq. Some valuable characters for identification of these species are proposed. In particular the width of fruit peduncle, petal size, leaf width and petiole length served to discriminate the taxa. Several names such as P. gharbiona Trab., P. cossonii Rehder (|M= P. longipes Balansa ex Coss. & Durieu) and P. boisseriana Buhse, are regarded as synonyms of P. cordata , while P. marnormis Trab. of P. bourgaeana. Consequently a check-list and a key to these species are provided.     

Effects of beta 2-agonist administration and exercise on contractile activation of skeletal muscle fibers

Lynch, Gordon S., Alan Hayes, Siun P. Campbell, and David A. Williams. Effects of₂-agonist administration andexercise on contractile activation of skeletal muscle fibers.J. Appl. Physiol. 81(4):1610-1618, 1996.Clenbuterol, a₂-adrenoceptor agonist, hastherapeutic potential for the treatment of muscle-wasting diseases, yetits effects, especially at the single-fiber level, have not been fullycharacterized. Male C57BL/10 mice were allocated to three groups:Control-Treated mice were administered clenbuterol (2 mg ^· kg^{1 ·} day¹)via their drinking water for 15 wk; Trained-Treated mice underwent low-intensity training (unweighted swimming, 5 days/wk, 1 h/day) inaddition to receiving clenbuterol; and Control mice were sedentary anduntreated. Contractile characteristics were determined on membrane-permeabilized fibers from the extensor digitorum longus (EDL)and soleus muscles. Fast fibers from the EDL and soleus muscles ofTreated mice exhibited decreases inCa²⁺ sensitivity. Enduranceexercise offset clenbuterol's effects, demonstrated by similarCa²⁺ sensitivities in theTrained-Treated and Control groups. Long-term clenbuterol treatment didnot affect the normalized maximal tension of fast or slow fibers butincreased the proportion of fast fibers in the soleus muscle. Trainingincreased the proportion of fibers with high and intermediate succinatedehydrogenase activity in the EDL and soleus muscles, respectively. Ifclenbuterol is to be used for treating muscle-wasting disorders, someform of low-intensity exercise might be encouraged such thatpotentially deleterious slow-to-fast fiber type transformations areminimized. Indeed, in the mouse, low-intensity exercise appears toprevent these effects.

     

N-acetyl-6-hydroxytryptophan a natural substrate of a monophenol oxidase from Aspergillus nidulans

An ivo B mutant of Aspergillus nidulans, deficient in conidiophore pigment has been shown to accumulate N-acetyl-6-hydroxytryptophan. This substrate for a specific monophenol oxidase present in the wild type but absent in the mutant.     

An Improved Method for In Situ Freezing of Cat Brain for Metabolic Studies

This study introduces a new method for rapid freezing of the cat brain. The method employed a Styrofoam box which was fitted around the head of the animal. Liquid nitrogen was poured into the box until the head was submerged. Temperature changes in three brain sites (ventral hypothalamus, the fourth ventricle, and the corpus callosum) and levels of labile carbohydrate metabolites (glycogen, glucose, ATP, P-creatine, and lactate) in five brain regions (cortex, thalamus, midbrain, cerebellum, and pons) frozen by the box method were compared with those frozen by a conventional cup method in which liquid nitrogen was poured into a hollow Styrofoam cup placed on top of the skull. The box method shortened the time of arrival of the freezing front and improved the freezing rate. The time required to bring the tissue to -20 degrees C was shortened, from 20 min at the ventral hypothalamus and 10-12 min at the fourth ventricle with the cup method, to less than 5 min at both sites with the box technique. Continued perfusion of brainstem prior to freezing was demonstrated. Levels of metabolites frozen by either method were similar. Lactate levels in any of the five brain regions studied by either method were not elevated, indicating no ischemic change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measuring leukotrienes of slow reacting substance of anaphylaxis: development of a specific radioimmunoassay

Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.     

The conditioning role of saliva in streptococcal attachment to hydroxyapatite surfaces

The solution properties of saliva and its role in conditioning both the substrate and the bacterial surface have been investigated with regard to the attachment of oral streptococci to hydroxyapatite surfaces. Saliva from eight subjects was used and the attachment of three organisms, Streptococcus mutans strains FA-1 (serotype b) and KPSK2 (serotype c) and S. sanguis T175-1, was studied. An adsorbed salivary layer on a hydroxyapatite surface substantially reduced the affinity of the organisms for the surface. Adsorbed saliva on the bacterial surfaces, however, tended to increase the organisms' affinity for saliva-coated apatite. The source of saliva was important in determining the extent of inhibition of attachment. The data indicated that the negatively charged and hydrophilic nature of salivary conditioning films was important in controlling bacterial adsorption to hydroxyapatite. The results also suggested that hydrophobic salivas could promote binding of the more hydrophobic bacteria known to be early colonizers of the teeth.     

Effect of extraction time on ability of calmodulin to activate 30S and 14S dynein ATPases

Cilia from the protozoan Tetrahymena pyriformis were demembranated and then extracted for 5 min with a buffer containing 0.5 M NaCl. The briefly extracted axonemal pellet was then reextracted for about 20 hr. The soluble material obtained from each extraction was resolved into 14S and 30S dynein ATPases by sedimentation on sucrose density gradients and tested for sensitivity to added calmodulin. The 14S dynein obtained by a 5-min extraction was generally insensitive to added calmodulin, whereas that obtained by 20-hr extraction of the 5-min extracted axonemes was activated by calmodulin, the activation being much larger in the “light” 14S fractions than in the “heavy” fractions. The 30S dynein ATPase obtained by a 5-min extraction was generally activated over 1.6-fold by added calmodulin, whereas that obtained by the subsequent long extraction was usually activated only 1.3-fold. After further purification of the 5-min extracted 30S dynein and of the 5-min to 20-hr-extracted 14S dynein on DEAE-Sephacel, these dyneins retained much of their calmodulin activatability. The ATPase activity of both 14S and 30S dyneins was inhibited more strongly by erythro-9-[3-(2-hydroxynonyl)] adenine and by vanadate in the presence of added calmodulin than in its absence. These data suggest that the only ATPase activity present in the fractions studied is that of the dyneins and demonstrate that both the 14S and 30S dynein ATPases may be obtained in forms mat are activated by added calmodulin as well as in forms that are insensitive to added calmodulin.     

The distribution and nutrient status of phytoplankton in the Southern Ocean between 20° and 70° W

Summary The distribution of phytoplankton along transects amounting to about 10,000 nautical miles in the sector of the Southern Ocean between 20° and 70°W was determined during the austral summer of 1978/79. Chlorophyll a concentration was monitored by the continuous measurement of in vivo fluorescence (IVF). Surface samples were collected for the determination of temperature, salinity, chlorophyll a concentration, carbon fixation rate and species of the phytoplankton. Phytoplankton distribution was found to be extremely patchy both locally and regionally. High phytoplankton concentrations were often associated with either hydrographic features, such as upwelling or the presence of sea-ice, or with bathymetric features, such as shelf breaks, submarine mountain ranges or islands. Enrichment experiments, in which the effects of various nutrient additions on the rate of ¹⁴C fixation by the natural phytoplankton were compared, and bioassay experiments, in which the growth of Thalassiosira pseudonana (Hustedt) Hasle and Heimdal in enriched water samples was measured, were carried out using water samples collected at various stations throughout the study area. Although these techniques were effective in demonstrating nutrient limitation elsewhere, the results suggest that availability of nitrate, phosphate, silicate, trace metals or vitamins exerts no primary control over phytoplankton abundance south of the Polar Front.