1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.     

Cytotoxicity and cytokinetic effects of mitomycin C and/or photochemotherapy in a human colon adenocarcinoma cell line.

1. The cytotoxicity and cytokinetic effects of Mitomycin C (MC) and/or photochemotherapy (PCT) in cultured human colon adenocarcinoma (WiDr) cells were investigated using colony formation to determine cell survival and DNA flow cytometry to analyze cell kinetics. 2. A low concentration of MC (0.01 micrograms/ml) caused accumulation of cells in late S and early G2 phase; higher concentrations (0.05-0.5 micrograms/ml) induced accumulation of the cells in mid and early S phase. 3. The effects of the lowest concentration of MC (0.01 micrograms/ml) were reversible upon removal of the drug, whereas a higher concentration of MC (0.1 micrograms/ml) resulted in a permanent inhibition of cell cycle progression. 4. The sensitivity of Photofrin II-loaded cells to PCT can be enhanced significantly by the addition of MC. 5. The MC-induced accumulation of the cells in S phase may be one reason for the increased cytotoxicity of PCT combined with MC. 6. The data suggest that MC may also inhibit repair of PCT-induced DNA damage.     

Causes and consequences of adaptive seasonal sex ratio variation in house sparrows

1. Here we examine how sex ratio variation in house sparrow broods interacts with other demographic traits and parental characteristics to improve the understanding of adaptive significance and demographic effects on variation in sex ratio. 2. The sex ratio in complete broods did not deviate significantly from parity (54.9% males). 3. There was sex-specific seasonal variation in the probability of recruitment. Male nestlings that hatched late in the breeding season had larger probability of surviving than early hatched males. 4. An adaptive adjustment of sex ratio should favour production of an excess of males late in the breeding season. Accordingly, the proportion of male offspring increased throughout the breeding season. 5. A significant nonlinear relationship was present between sex ratio and age of the female. However, there was no relationship between parental phenotype and standardized hatch day that could explain the observed seasonal change in sex ratio. 6. The sex-specific number of offspring recruited by a pair to subsequent generations was closely related to the brood sex ratio. 7. These results indicate an adaptive adjustment of sex ratio to seasonal variation in environmental conditions that affects the offspring fitness of the two sexes differently. Our results also suggest that such a sex ratio variation can strongly influence the demography and structural composition of small passerine populations.     

Direct and indirect mate choice on leks

Indirect mate choice is any behavior that restricts the individual's set of potential mates without discrimination of mate attributes directly, for example, by having preferences about where to mate. We analyzed a 14-year data set from great snipe (Gallinago media) leks for evidence of indirect mate choice based on relative and absolute position of lek territories. We found little or no effect of the centrality of territories on mating and no between-year consistency in the spatial distribution of matings within leks. Instead, the probability of matings occurring at a particular site increased if the current territory owner had mated the previous year. Furthermore, individual females returned in later seasons to mate with the same male as previously rather than at the same site. Previous work found that male interactions and dominance do not control matings and that females are very choosy about which territory they mate in. Here we show that this is because of the male occupying the territory rather than its position. We therefore conclude that direct female mate choice is the main behavioral process affecting variation in mating success among great snipe males, unlike in some lekking mammals where male competition and/or indirect mate choice appears more important.     

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (H?gseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestr?m, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., H?gset, A., Gautvik, V. T., Alestr?m, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.     

Expression of human parathyroid hormone in Escherichia coli

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids. Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E. coli. From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly. The peptide was extracted from E. coli cells and purified by high performance liquid chromatography. In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard. N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH. At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine. This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes.     

A new species of Polemanniopsis (Apiaceae) from Namibia

A new species of woody Apiaceae, Polemanniopsis namibensis B-E. Van Wyk, A. Burke & C. Mannheimer, is described. The species is allied to Polemanniopsis marlothii but differs in the much smaller habit of (up to 0.6 m vs 1.5–4 m in Polemanniopsis marlothii), the white or greyish colour of the twigs (brown in Polemanniopsis marlothii), the 3-foliolate leaves (predominantly 5-foliate in Polemanniopsis marlothii), the smaller inflorescences with persistent involucral bracts (caducous in Polemanniopsis marlothii) and anatomical details of the leaves. The species is endemic to Namibia and is known only from a localised area in the southern Namib desert near Lüderitz, where it is relatively common.     

Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.     

Female calls in lek-mating birds: indirect mate choice, female competition for mates, or direct mate choice?

I tested predictions from ultimate hypotheses of why femalegreat snipe Gallinago media give loud calls when visiting leks,using observational data and playback experiments. One hypothesisis that calls might be used in female—female competitionfor popular males, either (1) in an aggressive context towarda specific female, or (2) toward females in general to defendthe male. It has also been suggested that female calls (3) may not have an adaptive function, the capability of vocalizingbeing explained as a correlated response to selection on malesinging. Further, calls might function as (4) a copulationsolicitation toward a specific male. Finally, calls might havea function in mate choice, either (5) in indirect mate choiceas a fertility advertisement to incite male fighting, or (6)in direct mate choice as a mate-sampling aid to provoke quality-revealing responses from males. Disproportionately many female calls wereuttered when no other females were present on a male's territoryand in territories of males without mating success, contradictinghypotheses 1 and 2. Female calls were not associated with copulation;calls generally occurred several days before copulations, contradictinghypotheses 4 and 5. Playback of female calls attracted malesand increased fighting among males, even if females were presentnearby, contradicting hypothesis 3. Males changed their ownsongs in response to playback, and the response was relatedto their mating success. Taken together, the results are onlyconsistent with one of the hypotheses considered—femalecalls may function as a mate-sampling aid used in direct matechoice.