Mode and speed specificity of eccentric and concentric exercise training

This research was supported by a Duke University Research Council Grant. The purpose of this study was to examine mode and speed specificity of strength training by comparing concentric and eccentric isokinetic exercise of the quadriceps. Forty-eight healthy men (mean age = 23.9 years) were randomly assigned to one of three groups: concentric training (C), eccentric training (E), or control (K). Average force (in Newtons) of 3 concentric and of 3 eccentric quadriceps contractions on the KIN-COM(R) dynamometer at 60, 120, and 180 degrees /sec was evaluated prior to and following a 6 week period during which only the C and E groups trained. Training sessions (3/week) included 4 submaximal and 1 maximal warm-up followed by 10 maximal effort isokinetic contractions of the quadriceps at 120 degrees /sec for each leg. Group C subjects trained concentrically only while Group E subjects trained eccentrically only. A t-test for independent means showed no significant right/left differences. ANOVA and Scheffe's F-tests were then used to assess the differences in training effects among the 3 groups for the left leg only. Results showed that although Group C increased slightly in both concentric and eccentric force at all speeds, the gains were significant only for concentric force at 180 degrees /sec. Group E showed significant gains (p < 0.05) in eccentric force at all speeds but not in concentric force. The K group had no significant change in concentric or eccentric force at any speed. We conclude that the eccentric mode of isokinetic exercise has highly specific strength training effects while the concentric mode has less specific training effects. In addition, speed of exercise does not appear to have specific training effects. J Orthop Sports Phys Ther 1989;11(2):70-75.     

Characterization of enterotoxin purified from Clostridium perfringens type C.

Enterotoxin produced by a sporulating culture of Clostridium perfringens type C, which had been isolated from a case of severe necrotic enteritis, was purified. The molecular weight was estimated to be 36,000 by gel chromatography on Sephadex G-100 and 33,400 by ultracentrifugation. The sedimentation coefficient S20,W was 2.92. The toxin protein exhibited unusual behavior on sodium dodecyl sulfate gels, and toxin aggregates having molecular weights of 68,000, 85,000, 105,000, and 140,000 were obtained. The purified enterotoxin often separated, apparently due to slight charge differences, into two protein bands on 7% polyacrylamide gels. Electrofocusing of enterotoxin on polyacrylamide gels gave an approximate isoelectric point of 4.3, with the enterotoxin being fractionated into four distinct protein bands. The specific toxicity of the enterotoxin was about 1,900 mouse mean lethal doses per mg of calculated nitrogen. The data obtained indicate that the enterotoxin from C. perfringens type C is identical in most respects to that obtained from type A strains. Whether or not this toxin plays a role in the necrotic enteritis caused by type C strains is unknown at present.     

C3d binding to the circumsporozoite protein carboxy-terminus deviates immunity against malaria

The immunogenicity of recombinant protein or anti-viral DNA vaccines can be significantly improved by the addition of tandem copies of the complement fragment C3d. We sought to determine if the efficacy of a circumsporozoite protein (CSP)-based DNA vaccine delivered to mouse skin by gene gun was improved by using this strategy. Instead, we found that C3d suppressed the protective immunity against Plasmodium berghei malaria infection and deviated immunity, most notably by suppressing the induction of antibodies specific for the CSP C-terminal flanking sequence and by suppressing the induction of CSP-specific IL-4-producing spleen cells. We further showed that C3d bound to the C-terminal flanking sequence of the CSP, which may explain the immune deviation observed in CS/C3d chimeric antigen. We have thus identified C3d-mediated epitope masking and shifting of both the humoral and cellular immune responses as a potential novel escape mechanism, which plasmodia may use to divert the induction of protective immunity.     

Birnavirus VP1 proteins form a distinct subgroup of RNA-dependent RNA polymerases lacking a GDD motif

We have cloned and characterized the Drosophila X virus (DXV) genome segment B and its encoded VP1, the putative RNA-dependent RNA polymerase (RdRp) present in the virion. The 2991-bp open reading frame encodes the largest birnavirus VP1 at 977 aa, with a calculated M(r) of 112.8 kDa. As with the VP1 proteins of the type species of the other two genera in the family Birnaviridae, namely, infectious pancreatic necrosis virus (genus Aquabirnavirus) and infectious bursal disease virus (genus Avibirnavirus), the DXV (genus Entomobirnavirus) VP1 protein contains a consensus GTP-binding site and appears to possess self-guanylylation activity. All of the birnavirus VP1 proteins contain conserved RdRp motifs that reside in the catalytic "palm" domain of all classes of polymerases. However, the birnavirus RdRps lack the highly conserved Gly-Asp-Asp (GDD) sequence, a component of the proposed catalytic site of this enzyme family that exists in the conserved motif VI of the palm domain of other RdRps. All three birnavirus RdRps do contain downstream DD motifs that could function as part of the catalytic triad. These motifs are, however, located in spatially distinct regions of the various birnavirus VP1 proteins. These results suggest that the VP1 proteins of birnaviruses form a defined subgroup of polymerases that either are lacking the conserved RdRp motif VI or have repositioned this motif to different structural regions.     

Induced luteolysis in the primate: rapid loss of luteinizing hormone receptors

The molecular mechanisms involved in luteolysis are still unclear in the primate. This study aimed to investigate the effect of induced luteolysis on the ovarian luteinizing hormone (LH) receptor and the steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the marmoset monkey. Luteolysis was induced in the mid-luteal phase either directly by systemic prostaglandin F2alpha (PGF2alpha), or indirectly by LH withdrawal using systemic gonadotrophin releasing hormone antagonist (GnRHant) treatment. The LH receptor was studied by isotopic mRNA in-situ hybridization and in-situ ligand binding and 3beta-HSD expression was studied using isotopic mRNA in-situ hybridization and immunohistochemistry. Induced luteolysis was associated with a reduction in the expression of LH receptor (P < 0.0001) and 3beta-HSD mRNA, closely followed by a reduction in the LH receptor (P < 0.05) and 3beta-HSD protein concentrations within 24 h. There were no differences in the findings whether luteolysis was induced with PGF2alpha or GnRHant. This study shows that disparate mechanisms to induce luteolysis in the primate result in an identical rapid loss of the LH receptor and 3beta-HSD. In conclusion, induced luteolysis leads to rapid loss of the steroidogenic pathway in luteal cells.      

Absorption of histamine from the gastrointestinal tract of dogs in vivo

1. In anaesthetized dogs, various amounts of [(14)C]histamine were introduced into the lumen of a ligated intestinal loop or of the ligated stomach and the absorption of this histamine was studied by determining the radioactive histamine in the venous blood coming from the ligated part.2. After the introduction of 5-5000 mug [(14)C]histamine, into a loop of jejunum, or of 50 mug into a loop of duodenum, ileum or colon, radioactive histamine was detected in all eight successive 15 min samples of venous blood collected during 2 hr. The percentage recovery of the [(14)C]histamine in the blood during this period varied between 0.04 and 3.7.3. After the introduction of 10 mg [(14)C]histamine into the stomach, radioactive histamine was detected in all samples of gastric venous blood collected at various times during the following 4 hr.4. After the introduction of 50-5000 mug [(14)C]histamine into a loop of jejunum, radioactive histamine was also detected in the general arterial blood.5. When a jejunal loop was perfused through its artery with a dextransaline solution, the absorption of [(14)C]histamine from the lumen into the venous effluent was much greater than when the blood supply was kept intact.6. A large part of the [(14)C]histamine introduced into an intestinal loop was inactivated or destroyed either in the lumen or the wall since only a part was recovered in the venous blood, contents and wall of the loop at the end of 2 hr. When different amounts of [(14)C]histamine were introduced into a jejunal loop the recovery was shown to be dependent on the dose. With 5 mug it amounted to about 1% whereas with 5000 mug to 29-42%. The recovery of the [(14)C]histamine introduced into a perfused jejunal loop was greater.7. In dogs and cats large amounts of free histamine were found in the contents of the stomach 2 hr after a meat meal, and much smaller amounts in the contents of loops from the small intestine. The amounts found in the contents of loops from the colon varied greatly.8. In eighteen commercial dog and cat foods the free histamine contents were found to vary over fiftyfold, from 0.06 to 3.5 mg/100 g.9. The significance of the results is discussed in relation to the role of histamine as a humoral agent in the ;gastric' and ;intestinal phases' of gastric secretion.     

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.     

Interaction of pre-programmed control and natural stretch reflexes in human landing movements

Pre-programmed mechanisms of motor control are known to influence the gain of artificially evoked stretch reflexes. However, their interaction with stretch reflexes evoked in the context of unimpeded natural movement is not understood. We used a landing movement, for which a stretch reflex is an integral part of the natural action, to test the hypothesis that unpredicted motor events increase stretch reflex gain. The unpredicted event occurred when a false floor, perceived to be solid, collapsed easily on impact, allowing the subjects to descend for a further 85 ms to a solid floor below. Spinal stretch reflexes were measured following solid floor contact. When subjects passed through the false floor en route to the solid floor, the amplitude of the EMG reflex activity was double that found in direct falls. This was not due to differences in joint rotations between these conditions. Descending pathways can modify H- and stretch-reflex gain in man. We therefore manipulated the time between the false and real floor contacts and hence the time available for transmission along these pathways. With 30 ms between floors, the enhancement of the reflex was extinguished, whereas with 50 ms between floors it reappeared. This excluded several mechanisms from being responsible for the doubling of the reflex EMG amplitude. It is argued that the enhanced response is due to the modulation of reflex gain at the spinal level by signals in descending pathways triggered by the false platform. The results suggest the future hypothesis that this trigger could be the absence of afferent signals expected at the time of false floor impact and that salient error signals produced from a comparison of expected and actual sensory events may be used to reset reflex gains.     

Adaptation of the plaque assay methodology for dengue virus infected HepG2 cells

The HepG2 cell line is a useful tool for studying dengue virus-cell interactions but as it grows in clumps rather than monolayers, it does not readily adapt itself to the standard plaque assay technique. We therefore sought to develop an indirect plaque assay methodology. Initially HepG2 cells were infected with dengue virus serotype 2 and post-infection incubated for between 0 and 16 h before being treated with trypsin to separate the cells, followed by dilution and plating onto pre-grown monolayers of Vero cells in six well plates. After 7 days incubation and crystal violet staining, plaques were observed at all time points, although there was a relationship between number of plaques and post-infection incubation time, with the longest post-infection incubation time giving the highest number of plaques. To validate the assay with respect to virus input, the experiment was repeated at both the 0 and 16 h post-infection incubation times with different virus: cell levels. At both post-infection incubation times the response of input virus to plaque number was linear. This is a useful adaptation of the plaque assay methodology and one that may be applicable to other virus/cell line combinations.