Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport.

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-promoting phorbol esters resulted in inhibition of nuclear-envelope NTPase activity and in a concomitant decrease of mRNA (actin) efflux rate from isolated rat liver nuclei. Protein kinase C, but not nuclear envelope NI-like or NII-like protein kinase, was found to be solubilized from the envelope by Triton X-100, whereas the presumable poly(A)-binding site [the 106 kDa polypeptide, representing the putative carrier for poly(A)+mRNA transport] remained bound to this structure. RNA efflux from detergent-treated nuclei lost its susceptibility to phorbol esters. Addition of purified protein kinase C to these nuclei restored the effect of the tumour promoters. Protein kinase C was found to bind also to isolated rat liver nuclear matrices in the absence but not in the presence of ATP. The NII-like nuclear-envelope protein kinase co-purified together with the 106 kDa polypeptide which specifically binds to poly(A) in an ATP-labile linkage.     

Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

Allocation of a transgenic c-myc integration site to mouse chromosome 8B3-C1

A recombinant plasmid containing the mouse c-myc gene was injected into mouse pronuclei. The transgenic line 478 contains about 100 copies of the transgene integrated into one chromosome site. By in situ hybridization, the integration site was localized to chromosome 8B3-C1.     

Pachytene analysis in males heterozygous for a familial translocation (9;12;13) (q22; q22; q32) ascertained through a child with partial trisomy 9

A family with four male and three female heterozygotes for a three-way translocation (9;12;13) (q22; q22; q32) in three generations was ascertained through a chromosomally imbalanced newborn with an additional derivative chromosome 9 resulting from nondisjunction. Three heterozygous males from two generations with apparent differences in their fertility status were investigated using pachytene spreads and testicular histology. Pachytene analysis in all three individuals, the fertile (II-2) as well as the subfertile (III-7) and infertile (III-9), showed a hexavalent with central nonpairing around the translocation breakpoints in nearly all spermatocytes. Thus, the observed hexavalent configurations in pachytene do not seem to have caused impaired fertility. This rather may have been the result of sperm carrying unbalanced chromosome sets. However, the observed difference in fertility between the heterozygous fertile male in generation II and his two heterozygous sons remains unexplained.     

Neonatal screening for congenital adrenal hyperplasia using 17-hydroxyprogesterone assay in filter paper blood spots

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.     

Biochemical correlates of fatigue. A brief review

Muscle fatigue, defined as a decreased force generating capacity, develops gradually during exercise and is distinct from exhaustion, which occurs when the required force or exercise intensity can no longer be maintained. We have reviewed several biochemical and ionic changes reported to occur in exercising muscle, and analysed the possible effects these changes may have on the electrical and contractile properties of the muscle. There is no evidence that substrate depletion can account for the decreased force generating capacity, but this factor may be important for the rate of energy turnover and be a major determinant for endurance. Increased concentration of inorganic phosphate and hydrogen ions will depress the force generating capacity, but since fatigue can develop gradually without accumulation of these ions they can only be important when aerobic ATP production is insufficient to support the contractions. Evidence is presented showing that a disturbed balance of K+ alone might cause depolarisation block at high stimulation frequencies, but extracellular K+ accumulation does not increase gradually during prolonged dynamic or static exercise, and is therefore not closely related to fatigue. The repeated release of Ca2+ from the sarcoplasmic reticulum (SR) during muscular activity is suggested of Ca2+ by the mitochondria, increasing with stimulation frequency and duration and possibly also deteriorating mitochondrial function. We therefore speculate that decreased Ca2+ availability for release from SR might contribute to a gradual decline in force generating capacity during all types of exercise.     

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

The lipopolysaccharide of the phototrophic bacterium Ectothiorhodospira vacuolata

The lipopolysaccharide of Ectothiorhodospira vacuolata was obtained by the phenol-water procedure. It contained a 3-O-methyl-hexose, glucose, galacturonic and glucuronic acids. The finding of d-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate (tentatively identified) suggested a core-structure. The lipid fraction of the lipopolysaccharide contained phosphate and both, 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The major fatty acids were amine-bound 3-OH-10:0 and 3-OH-12:0 and esterbound 14:0 and 16:0 Sodium deoxycholate gel-electrophoresis, showing a single band only, indicated R-type character of the lipopolysaccharide of Ectothiorhodospira vacuolata.Abbreviations DOC sodium deoxycholate - GC/MS combined gasliquid chromatography - PAGE polyacrylamide gel-electrophoresis     

Age- and sex-related differences in lipid peroxidation of mouse cardiac and skeletal muscles

1. The purpose of the present study was to characterize age- and sex-related changes in lipid peroxidation capacities and enzymatic antioxidants of cardiac and skeletal muscles in NMRI-mice (Mus musculus). 2. Lipid peroxidation rates (unstimulated and enzymatic/iron-stimulated) strongly decreased in skeletal muscle during ageing. 3. Unstimulated lipid peroxidation rate but not that of stimulated, also decreased in cardiac muscle. 4. The total level of Fe2+/ascorbate-stimulated non-enzymatic lipid peroxidation was not, however, affected by ageing. 5. The activity of catalase slightly increased in cardiac muscle and that of glutathione peroxidase in skeletal muscle during ageing. 6. Unstimulated lipid peroxidation rate was significantly higher in the skeletal muscle of male than female mice. 7. Correspondingly, the Fe2+/ascorbate-stimulated lipid peroxidation capacities of microsomal and mitochondrial fractions of skeletal muscle were significantly higher in male mice. 8. The activity of glutathione peroxidase as well as the concentration of lipofuscin were higher in the cardiac muscles of female than male mice.