The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Neurogenic peptides and the cardiomyopathy of magnesium-deficiency: Effects of substance P-receptor inhibition

Dietary deficiency of magnesium (Mg) in rodents results in cardiomyopathic lesion formation. In our rat model, these lesions develop after 3 weeks on the Mg-deficient diet; significant elevation of several cytokines, IL-1, IL-6 and TNF also occurs. In probing the mechanisms of lesion formation, we obtained data supporting the participation of free radicals (Freedman AMet al.: Bioch Biophys Res Commun 1990; 170: 1102). Recently, we identified an early elevation of circulating substance P and proposed a role of neurogenic peptides during Mg-deficiency (Weglicki WB, Phillips TM: Am J Phys 1992; 262: R734). The present study was designed to evaluate the contribution of neurogenic peptides to the pathogenesis of Mg-deficiency. In the blood, substance-P and calcitonin gene related peptide (CGRP) are elevated during the first week on the diet. During the second week, circulating histamine, PGE₂ and TBAR-materials were elevated and red cell glutathione was reduced, all prior to the elevation of the inflammatory cytokines during the third week. When the rats were treated with the substance P-receptor blocker [CP-96,345], the levels of substance P and CGRP remained elevated; however, increases in histamine, PGE₂, TBAR-materials, and the decrease in red cell glutathione were inhibited; also, the development of cardiac lesions was inhibited significantly. These data support a central role for neurogenic peptides, especially substance P, in the development of cardiomyopathic lesions during Mg-deficiency.     

Simple electromyographic biofeedback treatment for chronic pediatric constipation/encopresis: Preliminary report

Pediatric constipation/encopresis is thought to be due, in part, to paradoxical constriction of the external anal sphincter (EAS) muscle during attempted defecation. This inappropriate contraction can lead to delayed, impacted, painful, and infrequent bowel movements. Standard Medical Care (SMC) involves disimpaction with enemas, followed by laxative therapy and diet modification, to maintain frequent soft stools. Using the case control method, the efficacy of SMC alone was compared with SMC plus EAS electromyographic biofeedback aimed at eliminating paradoxical contraction. Thirteen consecutive chronically constipated children received SMC plus biofeedback, and were compared with 13 age- and sex-matched children who received only SMC. Biofeedback subjects demonstrated post-treatment elimination of EAS paradoxical constriction. At 16 months follow-up parents of biofeedback children reported significantly greater improvement in constipation, encopresis, laxative use, and painful bowel movements compared to SMC.This research report was supported by the NIH under grant No. RO1 HD 28160.     

Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

The Receptor-Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine beta-hydroxylation is not increased. Preincubation experiments suggest that the carbachol-induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.     

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral‐derived macroporous constructs

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca⁺⁺ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca⁺⁺ activating stem cell differentiation and the induction of bone formation.