Expression of INK4 inhibitors of cyclin D-dependent kinases during mouse brain development

In situ hybridization of mouse embryo sections demonstrated expression of mRNAs encoding two polypeptide inhibitors (p18INK4c and p19INK4d) of cyclin D-dependent kinase (CDK) 4 and CDK6 in the central nervous system. No expression of two other INK4 members, p16INK4a and p15INK4b, was observed. The p19INK4d and p18INK4c proteins formed complexes with either CDK4 or CDK6 in a temporal pattern consistent with the results of in situ hybridization. Expression of INK4c was observed at embryonic day 13.5 in neuroepithelial zones of the developing brain, being restricted to dividing neuroblasts but absent from differentiating postmitotic neurons. In the neocortex, p18INK4c was expressed precisely at those developmental stages when neuroblasts switch from a symmetric to an asymmetric pattern of cell division with concomitant increases in their G1 interval. INK4d RNA was detected from embryonic day 11.5 onward, at higher levels than INK4c and with a distinctly different spatial and temporal pattern. Marked INK4d expression was seen in dorsal root ganglia, spinal cord, and focally throughout the brain, but primarily in postmitotic neurons. Neural expression of INK4d continued postnatally into adulthood in postmitotic cells of the dentate gyrus, the pyramidal layer of the hippocampus, and in discrete regions of the cerebral cortex, cerebellum, thalamus, and brainstem. Downregulation of p19INK4d in the dentate gyrus after kainic acid-induced seizures indicated that its expression could also be modified in nondividing cells by excitotoxic stress. Therefore, p19INK4d may contribute to maintaining the quiescent state, acting as a buffer to prevent reactivation of cyclin D-dependent kinases in terminally differentiated cells.     

Perthes disease--results of a containment-oriented therapy concept

INTRODUCTION: In a retrospective study a treatment concept for Perthes' disease dependent on the containment was applied. PATIENTS/METHODS: 49 hips of 41 children (9 female, 32 male) were treated between 01. 01. 1990 and 31. 12. 1995. In our concept of treatment a varus femoral osteotomy was performed in 28 cases with not contained hips or less than 4/5 coverage of the femoral head (X-ray/MRI). The other 21 well contained hips with 4/5 coverage or more were treated conservatively with physiotherapy and in case of joint effusion and pain additionally with the use of crutches (partial weight bearing) and anti-inflammatory medication. The average age in the non-operative group at the time of first investigation was 4 years and 9 months (3 y./1 m. to 7 y./1 m.) and 6 years and 3 months (4 y/2 m. to 10 y/0 m.) at our last examination (mean follow up 17.7 months, range of 6 to 72 months). At the time of indication for a varus femoral osteotomy the patients had an average age 6 years and 1 month (3 y./6 m. to 10 y./2 m.), the mean age at the last postoperative examination was 7 years and 11 months (4 y./8 m. to 12 y./5 m.) with an average follow up of 21.5 months (6 to 77 months). RESULTS: For the conservatively treated children we achieved good results (still well contained hips with 4/5 coverage, no decrease of function, no increase of pain) in 85.7% (18 of 21 cases). In 85.7% (24 of 28 cases) we found good results (well contained hips, increase of coverage, no decrease of function, no increase of pain) in the operation group. CONCLUSIONS: The presented concept of therapy in Perthes' disease was practicable for all patients and included the possibility of decision for operative or non-operative treatment. In both groups we achieved good results in 85.7% of the cases.     

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.     

Cardiovascular effects produced by R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin in the preoptic area of conscious rats

The effect of ancrod-induced defibrinogenation on thrombosis and bleeding time was determined in anesthetized rats. Functional plasma fibrinogen levels were reduced 42, 71, 94 and 93% by ancrod doses of 5, 10, 20 and 30 U/kg, respectively, while a 2.5 U/kg dose was without significant effect. Ancrod inhibited vena cava thrombosis induced by partial stasis of blood flow combined with mild vascular injury. Thrombus weight was decreased 85 and 93% by the 10 and 20 U/kg doses, but was unaffected at lower doses. In contrast, ancrod doses of up to 30 U/kg did not significantly decrease carotid artery thrombi formed in response to oxidative transmural vessel injury. Ancrod caused a dose-dependent increase in bleeding time measured by puncturing small mesenteric arteries with a hypodermic needle. The bleeding time increase was approximately 38% in response to the 2.5 and 5 U/kg doses, and 182% in response to the 10 U/kg dose. These studies demonstrate that ancrod-induced reductions in plasma fibrinogen more effectively inhibit venous compared to arterial thrombosis, although these activities require doses that also increase bleeding time in small arteries.     

Possible involvement of phosphatidylinositol 3-kinase in regulated exocytosis: studies in chromaffin cells with inhibitor LY294002

Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3-kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine-stimulated chromaffin cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphotyrosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2-containing proteins in stimulated chromaffin cells. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibitor of PI 3-kinase, produced a dose-dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine-labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.