Malformed offspring,sibling matings,and selection against inbreeding in the sand lizard (Lacerta agilis)

We demonstrated that sand lizards (Lacerta agilis) are more likely to have malformed offspring when they mate with siblings. Offspring with malformations, such as deformed limbs and heads, have zero survival in a natural population. Normal-looking siblings of malformed hatchlings also had a reduced survival in the wild, compared to offspring from clutches in which all siblings appeared normal. The proportion of malformed hatchlings in the natural population was ca. 10%, in spite of differences in juvenile dispersal between males and females. Male juveniles disperse significantly further from their natal sites than do female juveniles.     

Regulation of microtubule dynamics by Ca2+/calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18.

Oncoprotein 18 (Op18; also termed p19, 19K, p18, prosolin, and stathmin) is a regulator of microtubule (MT) dynamics and is phosphorylated by multiple kinase systems on four Ser residues. In addition to cell cycle-regulated phosphorylation, external signals induce phosphorylation of Op18 on Ser-25 by the mitogen-activated protein kinase and on Ser-16 by the Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr). Here we show that induced expression of a constitutively active mutant of CaMK IV/Gr results in phosphorylation of Op18 on Ser-16. In parallel, we also observed partial degradation of Op18 and a rapid increase of total cellular MTs. These results suggest a link between CaMK IV/Gr, Op18, and MT dynamics. To explore such a putative link, we optimized a genetic system that allowed conditional coexpression of a series of CaMK IV/Gr and Op18 derivatives. The result shows that CaMK IV/Gr can suppress the MT-regulating activity of Op18 by phosphorylation on Ser-16. In line with these results, by employing a chemical cross-linking protocol, it was shown that phosphorylation of Ser-16 is involved in weakening of the interactions between Op18 and tubulin. Taken together, these data suggest that the mechanism of CaMK IV/Gr-mediated suppression of Op18 activity involves both partial degradation of Op18 and direct modulation of the MT-destabilizing activity of this protein. These results show that Op18 phosphorylation by CaMK IV/Gr may couple alterations of MT dynamics in response to external signals that involve Ca2+.     

The biosynthesis of neutrophil and eosinophil granule proteins

Composition of azurophil and specific granules from human polymorphonuclear neutrophils and granules from eosinophils is presented. Biosynthesis of the granule proteins is discussed in detail with particular emphasis on neutrophil myeloperoxidase (MPO) and eosinophil cationic protein (ECP).     

Monoamines in rat thyroid parafollicular cells and the effect of vitamin D2-induced degranulation

Summary Thyroid parafollicular cells of normocalcemic and vitamin D₂-treated rats were investigated by electron microscopy and with the histochemical fluorescence technique of Hillarp and Falck.Administration of high doses of vitamin D₂ caused hypercalcemia and an extensive degranulation of the parafollicular cells.The formation and storage of monoamines in granulated and degranulated parafollicular cells was investigated by fluorescence microscopy after injection of monoamine precursors (DOPA, 5-HTP), alone or in combination with Ro 4-4602, nialamide or reserpine.No fluorescence was observed in parafollicular cells of untreated rats. l-DOPA and l-5-HTP (but not the corresponding D-amino acids) were taken up by a process closely linked to the decarboxylation of the amino acids to the corresponding amines (dopamine and 5-hydroxytryptamine). Treatment with vitamin D₂ did not seem to affect the formation of amines in the parafollicular cells or the formation and storage of amines in other cell systems investigated. The amine itself (dopamine) was not taken up by the parafollicular cells.In normocalcemic rats, the amine formed was retained in the cytoplasm of the parafollicular cells by a partially reserpine-resistant mechanism. The storage of amines is concluded to occur in association with the calcitonin-containing granules.In parafollicular cells of vitamin D₂-treated rats, a certain amount of amine was bound in the cytoplasm in the absence of typical granules. As a considerable amount of calcitonin is known to remain in the thyroid of vitamin D₂-treated rats, the present observations may indicate an association between the amine and the polypeptide hormone calcitonin, whether the latter is confined to typical granules or not.The present study was supported by grants B72-12X-3352-02 and B72-14X-2207-06B from the Swedish Medical Research Council and by grants from Magnus Bergwall's Foundation, Gustav and Majen Lundgren's Foundation, Wilhelm and Martina Lundgren's Foundation and from the Faculty of Medicine, University of Göteborg, Sweden. For skilful technical assistance we are indebted to Mrs. Kirsten Collin and Mr. Pär-Anders Larsson.     

Membrane glycoproteins involved in hepatocyte adhesion to collagen type I

Liver membrane glycoproteins with affinity for immobilized collagen type I were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution of the separated proteins. Electroeluted glycoproteins with ability to neutralize the inhibitory effect of anti-CollCAM antibodies on hepatocyte adhesion to collagen were collected from several consecutive runs and used to raise a high titer antiserum, denoted anti-CollCAM II. IgG from this antiserum inhibited the attachment of hepatocytes to dishes coated with collagen type I, but not to fibronectin- or collagen type IV-coated dishes. When the antibodies were immobilized to Sepharose CL-4B they bound three sets of glycoproteins with apparent Mr's of 105,000, 115,000, and 130,000 as analyzed by SDS-PAGE under nonreducing (NR) conditions. Upon reduction (R) the glycoproteins migrated with apparent Mr's of 115,000, 130,000, and 160,000, respectively. The Mr 105,000-115,000 (NR) glycoproteins effectively neutralized the inhibitory effect exerted by both anti-CollCAM and anti-CollCAM II antibodies, on hepatocyte spreading and attachment to collagen type I substrates. Peptide mapping suggested the Mr 160,000 (R) species to be different from the Mr 115,000 (R).     

Characterization of the receptor for lymphotoxin; a spontaneous internalization without recycling and ligand-induced downregulation in HL-60 cells

The receptor system for lymphotoxin (LT) was investigated by use of the human promyelocytic HL-60 cell line. Utilizing subcellular fractionation, internalization of cell surface bound recombinant LT (rLT) and transfer to lysosomes followed by degradation was demonstrated. Binding of rLT to its cell surface receptor induced a downregulation of the receptor which was persistent for at least 6 h. Downregulation of the receptors from the cell surface by activation of protein kinase C with the diacylglycerol OAG was completely reversible but the recovery of the binding capacity was dependent on protein synthesis as it was inhibited by cycloheximide. Treatment with cycloheximide alone resulted in a loss of binding capacity with a half life of approximately 2 h, suggesting a spontaneous consumption of receptors. Affinity cross-linking revealed three ligand-receptor complexes with Mr of 85, 105 and 125 kDa. Because of a strong tendency for ligand polymerization these results suggest oligomer binding of the ligand to a single receptor molecule with an apparent Mr of 70 kDa. N-linked glycosylation constituted 4 to 5 kDa of the total molecular weight. In conclusion, we demonstrated a spontaneous internalization of the receptor for LT without recycling, and that ligand binding resulted in an irreversible downregulation of the receptor.     

Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes

Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells.     

Evidence for efficacy of drugs affecting bone metabolism in preventing hip fracture.

OBJECTIVE--To examine the effects of taking drugs affecting bone metabolism on the risk of hip fracture in women aged over 50 years. DESIGN--Retrospective, population based, case-control study by questionnaire. SETTING--14 centres in six countries in southern Europe. SUBJECTS--2086 women with hip fracture and 3532 control women matched for age. MAIN OUTCOME MEASURES--Number of drugs affecting bone metabolism taken and length taken for. RESULTS--Women taking drugs affecting bone metabolism had a significantly decreased risk of hip fracture. After adjustment for differences in other risk factors, the relative risk of hip fractures was 0.55 (95% confidence interval 0.31 to 0.85) in women taking oestrogens, 0.75 (0.60 to 0.94) in those taking calcium, and 0.69 (0.51 to 0.92) in those taking calcitonin. The fall in risk was not significant for anabolic steroids (0.6 (0.29 to 1.22)). Neither vitamin D nor fluorides were associated with a significant decrease in the risk of hip fracture. The effect on hip fracture risk increased significantly with increasing duration of exposure (risk ratio 0.8 (0.61 to 1.05) for less than median exposure v 0.66 (0.5 to 0.88) for greater than median exposure). Drugs were equally effective in older and younger women, with the exception of oestrogen. CONCLUSIONS--Oestrogen, calcium, and calcitonins significantly decrease the risk of hip fracture. Short term intervention late in the natural course of osteoporosis may have significant effects on the incidence of hip fracture.