Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset)

Vitamin D₃ (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D₃ due to lack of sunlight exposure. The minimum dietary vitamin D₃ requirement and the maximum amount of vitamin D₃ that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D₃ provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D₃ (25(OH)D₃), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D₃ in captive marmosets, but 25(OH)D₃ is not the final product of vitamin D₃ metabolism. In addition to serum 25(OH)D_3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the less well understood metabolite, 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) to characterize the marmoset's ability to metabolize dietary vitamin D₃. We present vitamin D₃ metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D₃ per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D₃ (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)₂D₃ (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)₂D₃ between the colonies. Serum 1,25(OH)₂D₃ increased and 25(OH)D₃ decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D₃ into the active metabolite 1,25(OH)₂D₃; excess 25(OH)D₃ is metabolized into 24,25(OH)₂D₃. This ability explains the tolerance of high levels of dietary vitamin D₃ by marmosets, however, our data suggest that these high dietary levels are not required.     

Pulling or drilling, does size or species matter? An experimental study of prey handling in Octopus dierythraeus ()

The influence of both predator and prey size on the shift from a pulling to a drilling predatory response was examined in the intertidal octopus Octopus dierythraeus, using an experimental program. Additionally, selective drilling, where particular regions of the prey are targeted, was examined for a variety of bivalve and gastropod prey. O. dierythraeus always initially attempted to pull bivalves apart. Shells that were eventually drilled were always subjected to significantly more pulling attempts than those that could be pulled apart, indicating that octopus are willing to expend more energy to access the flesh quickly. There was no defined threshold where bivalve size caused an octopus to switch from a pulling to a drilling response. Instead, there was a broad size range where the octopus could adopt either handling method and it varied for each individual. Octopus may only able to pull open bivalves before the molecular ratchet or ‘catch’ mechanism that many bivalves possess is engaged. This might explain the lack of a relationship between either octopus or bivalve size and the success of pulling, as it is likely that when the bivalves were presented to individual octopus they were either setting the ‘catch’ mechanism, or had already engaged it. O. dierythraeus demonstrated selective drilling on a variety of molluscan prey, with penetration sites differing between prey species. O. dierythraeus targeted the valve periphery, which was the thinnest part of the shell, therefore minimizing handling time. O. dierythraeus always drilled gastropods, but did not target the thinnest regions of the shells, with drill site varying according to the morphology of the prey. Elongate species with pronounced aperture lips were drilled in the apical region, close to the columella on the side of the opercula whereas nonelongate species were drilled immediately above the aperture. The location of drilling sites may represent a trade-off between targeting the most effective places to inject paralyzing secretions and the mechanically simplest places to drill.     

Dictyosome vesicle production and plasma membrane turnover in auxin-stimulated outer epidermal cells of coleoptile segments fromAvena sativa (L.)

Summary Dark grown coleoptile segments were floated on solutions of IAA alone and of IAA and the secretion inhibitors cytochalasin and monensin. The secretion inhibitors prevented normal elongation of the tissue segments, the monensin inhibition being virtually complete while cytochalasin gave a 40% reduction over the first six hours with little further further elongation in the following 18 hours. Vesicle production was assessed in outer epidermal cells after 6 hours of IAA-stimulated elongation using the vesicle accumulation method following a cytochalasin-block of vesicle transport. The results were compared with the area of plasma membrane required to enable cell elongation to proceed at the observed rate. The area of vesicle membrane delivered to the cell surface exceeded this requirement to such an extent that at least 65% of the delivered membrane must be recycled back into the cytoplasm. Expressed in terms of the whole cell, the plasma membrane turnover rate was found to be once every 200 minutes. It is concluded that limitation of elongation by secretion inhibitors is more likely to reflect a requirement for the vesicle contents than the vesicle membrane. These results are compared with those obtained from other secretory systems using a similar approach.Abbreviations IAA indole acetic acid - DMSO dimethyl sulphoxide - D dictyosome - ER endoplasmic reticulum - V vesicle     

Stimulation of phospholipid hydrolysis and arachidonic acid mobilization in human uterine decidua cells by phorbol ester.

Vasopressin and oxytocin both stimulated inositol phosphate accumulation in isolated uterine decidua cells. Pretreatment of cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) prevented this agonist-induced phosphoinositide hydrolysis. TPA (0.1 microM) alone had no effect on basal inositol phosphate accumulation, but stimulated phosphoinositide deacylation, as indicated by a 2-fold increase in lysophosphatidylinositol and glycerophosphoinositol. TPA also stimulated a dose-related release of arachidonic acid from decidua-cell phospholipid [phosphatidylcholine (PC) much greater than phosphatidylinositol (PI) greater than phosphatidylethanolamine]. The phorbol ester 4 beta-phorbol 12,13-diacetate (PDA) at 0.1 microM had no effect on arachidonic acid mobilization. The TPA-stimulated increase in arachidonic acid release was apparent by 2 1/2 min (116% of control), maximal after 20 min (283% of control), and remained around this value (306% of control) after 120 min incubation. TPA also stimulated significant increases in 1,2-diacylglycerol and monoacylglycerol production at 20 and 120 min. Although the temporal increases in arachidonic acid and monoacylglycerol accumulation in the presence of TPA continued up to 120 min, that of 1,2-diacylglycerol declined after 20 min. In decidua cells prelabelled with [3H]choline, TPA also stimulated a significant decrease in radiolabelled PC after 20 min, which was accompanied by an increased release of water-soluble metabolites into the medium. Most of the radioactivity in the extracellular pool was associated with choline, whereas the main cellular water-soluble metabolite was phosphorylcholine. TPA stimulated extracellular choline accumulation to 183% and 351% of basal release after 5 and 20 min respectively and cellular phosphorylcholine production to 136% of basal values after 20 min. These results are consistent with a model in which protein kinase C activation by TPA leads to arachidonic acid mobilization from decidua-cell phospholipid by a mechanism involving phospholipase A-mediated PI hydrolysis and phospholipase C-mediated PC hydrolysis, coupled with further hydrolysis of the 1,2-diacylglycerol product.     

Fluorescence lifetime quenching and anisotropy studies of ribonuclease T1

The time-resolved fluorescence of the lone tryptophanyl residue of ribonuclease T1 was investigated by using a mode-locked, frequency-doubled picosecond dye laser. The fluorescence decay could be characterized by a single exponential function with a lifetime of 3.9 ns. The fluorescence was readily quenched by uncharged solutes but was unaffected by iodide ion. These observations are interpreted in terms of the electrostatic properties of the amino acid residues at the active site of the protein, which would appear to restrict the access of solute species to the tryptophanyl residue. The temperature dependence of the fluorescence lifetime and anisotropy decay time could be rationalized in terms of a model which postulates a significant ordering of the solvent layer immediately surrounding the surface of the protein.     

Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps

The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.     

Redistribution of cell surface transferrin receptors prior to their concentration in coated pits as revealed by immunoferritin labels

Summary Immunocytochemistry has been used to study distribution of cell surface transferrin receptors in erythroid, leukemic (K562) cells. The cells were fixed and labelled with monoclonal (OKT-9) anti-transferrin receptor antibodies; the antibody-labelled receptors were then detected by either immunofluoresceinor immunoferritin-antimouse-antibody conjugates. Typically, the immunoferritin labels were distributed diffusely at the non-coated regions of the cell surface as well as concentrated in the clathrincoated pits. To examine further this pattern of distribution, cells were labelled at 0° C and then warmed to 37° C for zero to 30 min prior to fixation. The majority of the immunoferritin labels were initially dispersed in small groups at the non-coated regions of the cell surface (mean = 6 immunoferritin labels/cluster), but larger groups were common subsequent to incubation at 37° C (mean = 13 immunoferritin labels/cluster). However, the size of immunoferritin labels in the coated pits was unchanged (mean = 12 immunoferritin labels/pit). Immunoferritin labels were typical in coated and uncoated vesicles l min after warming to 37° C, but common in endosomes, multivesicular bodies and lysosomes by 30 min. It appears that single cell-surface receptors form large aggregates prior to their concentration in coated pits. Coated vesicles, uncoated vesicles, and endosomal vacuoles may together form the non-lysosomal compartment where the internalized receptors might be dissociated from the ligands (antibodies).     

The binding site of chicken hepatic lectin

The binding site of the chicken hepatic lectin involved in the clearance of N-acetylglucosamine-terminated serum glycoproteins was explored by a competitive binding assay using 3H-labeled agalacto-orosomucoid and various glycoproteins, polysaccharides, monosaccharides, and glycosides as inhibitors. The binding site is relatively small, involving a terminal nonreducing DGlcNAc structure with an equatorial N-acetamido group on carbon 2 and an equatorial hydroxyl group on carbon 4. Among the mono- and oligosaccharides tested, benzyl alpha DGlcNAc was the best inhibitor, being three times as effective as DGlcNAc; and in general, all alpha-anomeric glycosides were better than beta-glycosides. All oligosaccharides with terminal nonreducing beta DGlcNAc have almost the same inhibitory power, whereas those with nonreducing DGlc or DGal were relatively inactive. Among the serum and blood group glycoproteins, a Smith degraded human H substance with several exposed terminal nonreducing beta DGlcNAc residues was the most active and twice as effective as agalacto-orosomucoid and an A substance, Hog 75 10% precipitate. Almost all hog preparations, some with A or with H activity, were equally effective. A glycopeptide with terminal DGlcNAc was twice as active as one with terminal nonreducing DMan and DGlcNAc residues and almost three times as potent as one with terminal nonreducing DGal; a glycopeptide with terminal sialic acid was inactive. The slopes of the inhibition lines differed, reflecting the heterogeneity of the various determinant groups on the glycoproteins.