A magnetic evaluation of peripheral nerve regeneration: I. The discrepancy between magnetic and histologic data from the proximal segment

Histologic techniques can quantify the number of axons in a nerve, but give no information about electrical conductibility. The number of functional myelinated neuronal units in a nerve can be quantified based on a magnetic recording technique. When studying reconstructed peripheral nerves a significant difference between the results found with these two techniques can be observed. A comparison was made between the long-term changes in the number of histologically and magnetoneurophysiologically measured neuronal units proximal to a nerve reconstruction. This study was performed on 6 New Zealand White rabbits, 20 weeks after the peroneal nerve had been reconstructed. The contralateral nerves were used as a control. Histologic examination demonstrates a statistically significant decrease of approximately 5% in the number of myelinated fibers. The magnetoneurophysiological results demonstrate a decrease which is estimated to be caused by the loss of approximately 50% of the functional myelinated neuronal units in the nerve. Therefore we conclude that of the initially available myelinated neuronal units, 5% degenerate completely, 45% are vital but lose their signal conducting capability, and the remaining 50% are vital and continue to conduct signals. Apparently, only this latter group of 50% of the initially available functional neuronal units appears to remain available for functional recovery.     

Intracellular calcium regulates agrin-induced acetylcholine receptor clustering

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR beta-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrin-induced AChR clustering was unaffected in cells loaded with EGTA, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.     

Effects of ammonium dinitramide on preimplantation embryos in Sprague-Dawley rats

Ammonium dinitramide (ADN) is a class 1.1 oxidizer that may be used in rocket propellants and explosives. Previous studies have shown that ADN is a female reproductive toxicant, causing implantation failure in Sprague-Dawley rats when it is administered during the preimplantation period of gestation. The purpose of this follow-up study was to identify the mechanism(s) associated with implantation failure following exposure to ADN. Mated female rats were treated with 2.0 grams per liter (g l-1) ADN in their drinking water for 24, 48, 72, or 96 h before preimplantation embryos were harvested from the oviducts or uterine horns. On gestation day 1 (GD-1), comparable numbers of morphologically normal two-cell embryos were harvested from the oviducts of the treatment and control groups. On GD-2, the development of the embryos harvested from the treated animals was either slowed or halted when compared to the control embryos. By GD-4, 98% of the embryos harvested from the control group had developed to the morula or blastocyst stage; these were collected from the uterine horns. On GD-4 in the treated group, 41% of the harvested embryos remained at the two- to six-cell stage and 59% were degenerate; 82% of these embryos were collected from the oviducts. These data suggest that the implantation failure seen in animals treated with ADN is due to embryolethality.     

Banking: financing trends in an acquisitive health care market--focus on long-term care

This article reviews the long-term care sector of the health care industry, particularly the factors driving sector consolidation and, through the use of four transactions as a platform, discusses key credit issues and risks facing long-term care companies.     

TECHNICAL NOTE: measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide

BACKGROUND: Traditional protein kinase assays include the use of [32P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. METHODS: Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. RESULTS: The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 +/- 0.8% and 14.3 +/- 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20-inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. CONCLUSIONS: These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.     

Ethanol, stroke, brain damage, and excitotoxicity

The N-methyl-d-aspartate (NMDA)-glutamate receptor could contribute to stroke, trauma, and alcohol-induced brain damage through activation of nitric oxide formation and excitotoxicity. In rat primary cortical cultures NMDA was more potent at activating nitric oxide formation than triggering excitotoxicity. Ethanol dose dependently inhibited both responses. In contrast, treatment of neuronal cultures with ethanol (100 mM) for 4 days significantly increased NMDA stimulated nitric oxide formation and excitotoxicity. These findings suggest that ethanol acutely inhibits but chronically causes supersensitivity to NMDA-induced excitotoxicity in neuronal cultures. To investigate ethanol's interaction with stroke induced damage models of global cerebral ischemia were studied. Transient global ischemia resulted in a loss of hippocampal CA1 pyramidal neurons over a 3- to 5-day period. Determinations of the NMDA receptor ligand binding stoichiometry or postischemic receptor binding changes did not show differences between neurons that undergo delayed neuronal death following ischemia and those that show no toxicity, for example, CA1 and dentate gyrus, respectively. Acute ethanol (3 g/kg) was found to protect against ischemia-induced CA1 hippocampal damage by lowering body temperature, but not under temperature controled conditions. These studies indicate that the factors contributing to stroke-induced brain damage are complex, although they are consistent with chronic ethanol increasing stroke-induced brain damage by increasing NMDA excitotoxicity.     

Estrus detection using radiotelemetry or visual observation and tail painting for dairy cows on pasture

The efficiency and accuracy of estrus detection using HeatWatch (DDx Inc., Denver, CO) or visual observation were compared in an autumn-calving Friesian herd (n = 48 per group) and a spring-calving Jersey herd (n = 50 per group) grazing on pasture. Cows in the group monitored by the HeatWatch system were fitted with a pressure-sensitive transmitter that signaled mounting activities associated with estrus. Visual observation was carried out for about 20 min before the morning and afternoon milkings and was aided by a strip of paint applied over the tailhead. Ovarian cyclicity was monitored with progesterone concentrations in milk samples collected twice a week. The efficiency and accuracy of estrus detection were, respectively, 98.4 and 97.6% for visual observation and 91.7 and 100% for HeatWatch detection. Autumn-calving herds differed from spring-calving herds in duration of estrus (9.7 vs. 7.3 h), number of mounts (13.6 vs. 8.5), total duration of mounts (36.8 vs. 19.9 s), and mean duration of a mount (2.6 vs. 2.3 s). There was no significant variation in the distribution of the time of onset of estrus or mounting activities at different hours of the day. Conception rate was similar for AI after estrus detection with HeatWatch (65.8%) or after visual observation (65.0%). The highest conception rate was obtained when AI was carried out between 12 and 18 h after the first mount. Both the HeatWatch system and visual observation plus tail painting can be used for estrus detection of dairy cows on pasture.