Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples

C-3-substituted 25-hydroxyvitamin D₃ analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3β-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue–DBP complex. When compared to 25-hydroxyvitamin D₃-DBP, the binding of some antibodies to the analogue–DBP complex of the 25-hydroxyvitamin D₃ derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

Energy consumption by embryos of a viviparous lizard, Eulamprus tympanum, during development

Energy consumption during development has been measured in many oviparous lizards, but not in viviparous lizards in utero. It has always been assumed that energy consumption by embryos of viviparous lizards during development is similar to that of oviparous species. Estimation of energy consumption of viviparous lizards in vivo are confounded by the possible influence of pregnancy on maternal metabolism. Here we separated maternal and embryonic metabolism in measurements of pregnant Eulamprus tympanum throughout pregnancy. Our data support the hypothesis that the energetic cost of development in viviparous lizards (19.8 kJ g⁻¹) is similar to that in oviparous lizards (mean 16.2 kJ g⁻¹), at least for a species with a simple placenta. An increase in maternal metabolism of 29% above that for non-pregnant E. tympanum goes to maintain pregnancy, and represents an important component of the reproductive effort in E. tympanum.     

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc 1 proteins. These results show that evolution at the DBP level can be considered as a continous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.     

The impact of vitamin D3 supplementation on muscle function among HIV-infected children and young adults: a randomized controlled trial

Objectives:

We tested the hypothesis that daily vitD₃ supplementation increases neuromuscular motor skills, jump power, jump energy, muscular force, and muscular strength.

Methods:

This was a secondary analysis of a randomized controlled trial of 12-months of oral 7,000 IU/day vitD₃ supplementation or placebo among 56 persons living with HIV aged 9-25 years. Neuromuscular motor skills were quantified using the Bruininks-Oseretsky Test of Motor Proficiency. Power was quantified using peak jump power, and energy was quantified using peak jump height. Muscular force was quantified using isometric ankle plantar- and dorsiflexion, isokinetic knee flexion and extension. Muscular strength was quantified using isometric handgrip strength.

Results:

After 12-months, serum 25-hydroxyvitamin D [25(OH)D] was higher with supplementation versus placebo (β=12.1 ng/mL; P<0.001). In intention-to-treat analyses, supplementation improved neuromuscular motor skills versus placebo (β=1.14; P=0.041). We observed no effect of supplementation on jump power, jump energy, muscular force, or muscular strength outcomes versus placebo.

Conclusions:

Among HIV-infected children and young adults supplementation with daily high-dose vitD₃ increased concentration of serum 25(OH)D and improved neuromuscular motor skills versus placebo.     

Distribution of vitamin D binding protein expressing neurons in the rat hypothalamus

We observed immunostaining for vitamin D binding protein (DBP) in rat hypothalamus. Part of the supraoptic and of the paraventricular neurons showed DBP immunoreactivity, in part colocalized with Arg-vasopressin. DBP was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. A portion of ependymal cells, the choroids plexus epithelium and some of the endocrine cells in the anterior pituitary lobe contained DBP immunoreactivity. In situ hybridization of semithin sections with a synthetic oligonucleotide probe to DBP mRNA resulted in staining of magnocellular hypothalamic neurons, but not of ependymal cells or anterior lobe cells. Our observations indicate an intrinsic expression of DBP in the rat hypothalamus. DBP may be synthesized and transported along with the classical neurohypophyseal hormones. The multiple locations of DBP-expressing neurons indicate multiple functional properties: DBP may be released from in the posterior lobe, it may act as a hypophyseotropic factor and as a central neuroactive substance.     

Patterns of plant visitation by nectar-feeding lizards

Geckos in the genus Hoplodactylus visit flowers to feed on nectar. I examined the patterns of flower visitation exhibited by two gecko species (H. maculatus and H. duvauceli) having access to two plant species: pohutukawa (Metrosideros excelsa: Myrtaceae) and flax (Phormium tenax: Agavaceae). Individual geckos were not observed to visit both plant species; individuals visiting flax tended to revisit the same plant. Geckos visiting pohutukawa were larger than those visiting flax and exhibited an early night peak in plant visitation, while lizards on flax displayed a more even pattern of activity throughout the night. On flax, geckos were more likely to be found on plants with a greater number of male flowers. Male flax flowers were of greater diameter than female flowers and produced nectar at higher rates and with greater concentrations of sugars. Experimental manipulation of pohutukawa nectar volumes suggested that the distribution of geckos is influenced by the pattern of nectar availability.     

Hysteresis of heart rate and heat exchange of fasting and postprandial savannah monitor lizards (Varanus exanthematicus)

Reptiles are ectothermic, but regulate body temperatures (T(b)) by behavioural and physiological means. Body temperature has profound effects on virtually all physiological functions. It is well known that heating occurs faster than cooling, which seems to correlate with changes in cutaneous perfusion. Increased cutaneous perfusion, and hence elevated cardiac output, during heating is reflected in an increased heart rate (f(H)), and f(H), at a given T(b), is normally higher during heating compared to cooling ('hysteresis of heart rate'). Digestion is associated with an increased metabolic rate. This is associated with an elevated f(H) and many species of reptiles also exhibited a behavioural selection of higher T(b) during digestion. Here, we examine whether digestion affects the rate of heating and cooling as well as the hysteresis of heart rate in savannah monitor lizards (Varanus exanthematicus). Fasting lizards were studied after 5 days of food deprivation while digesting lizards were studied approximately 24 h after ingesting dead mice that equalled 10% of their body mass. Heart rate was measured while T(b) increased from 28 to 38 degrees C under a heat lamp and while T(b) decreased during a subsequent cooling phase. The lizards exhibited hysteresis of heart rate, and heating occurred faster than cooling. Feeding led to an increased f(H) (approximately 20 min(-1) irrespective of T(b)), but did not affect the rate of temperature change during heating or cooling. Therefore, it is likely that the increased blood flows during digestion are distributed exclusively to visceral organs and that the thermal conductance remains unaffected by the elevated metabolic rate during digestion.     

The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3), suppresses autoimmune disease in several animal models including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The molecular mechanism of this immunosuppression is at present unknown. While 1alpha,25-dihydroxyvitamin D(3) is believed to function through a single vitamin D receptor, there are reports of other vitamin D receptors as well as a "nongenomic" mode of action. We have prepared the EAE model possessing the vitamin D receptor null mutation and determined if 1alpha,25-dihydroxyvitamin D(3) can suppress this disease in the absence of a functional vitamin D receptor. Vitamin D receptor null mice develop EAE although the incidence rate is one-half that of wild-type controls. The administration of 1alpha,25-dihydroxyvitamin D(3) had no significant effect on the incidence of EAE in the vitamin D receptor null mice, while it completely blocked EAE in the wild-type mice. We conclude that 1alpha,25-dihydroxyvitamin D(3) functions to suppress EAE through the well-known VDR and not through an undiscovered receptor or through a "nongenomic" mechanism.     

Comparison of nutrient transport across the placenta of lizards differing in placental complexity

We have reviewed published and new quantitative data on the net uptake of nutrients by embryos of oviparous and viviparous lizards that vary in chorioallantoic placental complexity to better understand the evolution of complex placentae. We assessed net nutrient uptake during embryonic development by measuring the total dry mass, or the mass of separate nutrients, in the egg at about the time of ovulation and in the neonate. There is no significant difference in the fresh egg to neonate dry mass ratio of oviparous and viviparous species that have simple placentae, indicating that there is little, if any, net uptake of nutrients by viviparous species with simple chorioallantoic placentae. In contrast, there is significant uptake of dry matter and individual nutrients across the placenta of species with complex chorioallantoic placentae. Species of the genus Niveoscincus have a range of placentae and nutrient uptakes, even among populations of one species, suggesting that further studies among populations of single species are required. Data are available for relatively few clades, and all the data for the three most complex chorioallantoic placental types are derived from a single genus. Thus, further research on new genera of lizards is required to overcome the potentially confounding effects of phylogeny in our analyses.     

Thyroxine and vitamin D in the gorgonian Leptogorgia virgulata

The gorgonian coral Leptogorgia virgulata contains thyroxine, or a thyroxine-like substance, referred to here as G-T₄. The G-T₄ levels were significantly higher in colonies collected in the summer vs. winter months. Using immunocytochemical techniques, G-T₄ was localized in the axis, polyp epithelium, and within the electron dense bodies of scleroblasts (spicule-forming cells), as well as on the periphery of spicules. G-T₄ was also localized in the mesoglea between closely adjacent scleroblasts. The effects of exogenous T₄ on the uptake of Ca⁴⁵ was determined in spicule, tissue and axis fractions of L. virgulata. The uptake of Ca⁴⁵ increased in T₄ treated spicules but decreased in the tissue fraction for all time periods tested. The uptake of Ca⁴⁵ into axes was not affected by exogenous T₄ until day 10 of the study. These data suggest that G-T₄ may function in the process of spicule formation. 1,25-dihydroxyvitamin D apparently is synthesized via ultraviolet radiation. Colonies deprived of ultraviolet radiation had significantly more ‘irregular’ spicules than colonies maintained in ultraviolet radiation. Exposure to sunlight therefore may be associated with the process of normal spicule formation.     

Competing for crevices: interspecific conflict influences retreat-site selection in montane lizards

Direct interference competition between sympatric taxa affects habitat use and shelter-site selection in species within most major vertebrate lineages. However, studies on interspecific social interference in reptiles largely have been confined to research on interactions between non-native (invasive) species and native fauna. Does interspecific interference also influence habitat use within natural assemblages of reptiles? We studied five broadly sympatric species of viviparous montane skinks within the genera Egernia and Eulamprus in southeastern Australia. Previous work has shown strong interspecific overlap in abiotic attributes of shelter-sites for these taxa, but no joint occupancy of retreats. Laboratory trials in which we manipulated the identity of co-occurring lizards revealed frequent displacement from “preferred” (hotter) shelters, with interspecific interactions more intense than intraspecific conflicts. The five species displayed a linear interspecific dominance hierarchy, with larger species displacing smaller taxa. Field manipulations confirmed that interspecific interactions between these species affect shelter-site use. We conclude that direct agonistic encounters between individuals of different species strongly influence habitat use by lizards within this assemblage.     

The effect of donor age on the sensitivity of osteoblasts to the proliferative effects of TGF(beta) and 1,25(OH(2)) vitamin D(3)

The loss of osteoblast function in aging bone is one of the major causes of osteopenia, or loss of bone mass. In this study, this loss of function was investigated by examining the proliferative response of rat long bone periosteal osteoblasts to TGF(beta1) and 1,25-dihydroxy vitamin D(3) (1,25-D(3)) as a function of donor age. Using a DNA binding fluorescent dye, DNA levels were measured in osteoblast cultures derived from either young adult (3-4 months) or old (14-15 months) rats following treatment with two concentrations (10(-9) M or 10(-12) M) of either 1,25-D(3) or TGF(beta1) or with vehicle. Cells from young rat bone, when treated with 1, 25-D(3), showed a dose-dependent increase in proliferation when treated with the higher dose and a decrease in proliferation when treated with the lower dose. Osteoblasts isolated from old rats did not respond to 1, 25-D(3) treatment. A similar pattern of response to TGF(beta1) was found. When treated with 10(-9) M TGF(beta1), the rate of proliferation increased for young rat osteoblasts, but the old rat derived cells were unresponsive. The 10(-12) M dose of TGF(beta1) was ineffective for both young and old cells. This study has shown that osteoblasts derived from old donors are impaired in their ability to respond to vitamin D and TGF(beta), two of the major controlling factors of skeletal development and maintenance.     

Paralogous origin of the rhodopsinlike opsin genes in lizards

Rhodopsinlike opsins constitute a distinct phylogenetic group (Yokoyama 1994, Mol. Biol. Evol. 11:32–39). This RH2 group includes the green-sensitive opsins in chicken and goldfish and the blue-sensitive opsin in a nocturnal lizard gecko. In the present study, we isolated and sequenced the genomic DNA clones for the RH2 opsin gene, rh2 _Ac , of the diurnal lizard Anolis carolinensis. This single-copy gene spans 18.3 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Phylogenetic analysis strongly suggests that rh2 _Ac is more closely related to the chicken green opsin gene than to the gecko blue opsin gene. This gene tree differs from the organismal tree, where the two lizard species should be most closely related, implying that rh2 _Ac and the gecko blue-sensitive opsin genes have been derived from duplicate ancestral genes.Correspondence to: S. Yukiyama     

Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats

The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n = 123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 °C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.