Determination of vitamin D and its metabolites in plasma from normal and anephric man

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.     

Photoreception inEuglena gracilis: Fluence-rate and time dependence of photoaccumulation and photodispersal

The fluence-rate and time dependence for photoaccumulation and photodispersal ofEuglena gracilis was measured for the wild-type strain and three white mutants. For wavelengths of 453 or 463 nm the threshold for photoaccumulation was close to 6×10⁻²Wm⁻². Photoaccumulation increased steadily with time and reached a maximum after about 4 hr. Red light elicited substantial photoaccumulation in the wild type and photodispersal in the white, non-photosynthetic mutant 1224-5/9f. The chromophore mediating the red-light response needs to be a non-photosynthetic pigment which remains presently unidentified. A whiteEuglena mutant, FB, which had retained a reduced stigma and a paraflagellar body, showed weak photoaccumulation. Two white mutants, 1224-5/1f and 1224-5/9f, both of which lacked the stigma and positive phototaxis, displayed during the first 90 min of irradiation photodispersal; after longer irradiations they showed instead photoaccumulation. These results contradict a widely held belief that the presence of a stigma represents a stringent requirement for photoaccumulation. Our results imply that phototaxis is not a prerequisite for photoaccumulation. Exogenous flavins and 5,10-methenyl-tetrahydrofolate (MTHF) influenced in a wavelength-dependent manner photoaccumulation and photodispersal. In the wild type FAD and riboflavin (RB) caused at 453 nm an increase of the responsiveness for photoaccumulation. The photoaccumulation of the white mutant FB, was sensitized by FMN and FAD. In the white mutant 1224-5/9f exogenous flavins lowered the threshold for photodispersal. FMN, which absorbs only blue light, altered also the responsiveness to red light: in the wild type FMN reduced photoaccumulation and in the white mutant 1224-5/9f it reduced photodispersal.     

Hemoglobin M Iwate is caused by a C→T transition in codon 87 of the human α1-globin gene

Summary DNA restriction, molecular cloning, and sequencing methods have been used to characterize the mutation leading to the methemoglobinemia HbM Iwate. It could be demonstrated that the HbM Iwate defect is caused by a point mutation involving a transition from C to T in the first position of codon 87 of the ₁-globin gene. Furthermore, the HbM Iwate mutation can directly be identified upon RsaI digestion. This direct detection of the mutation on the gene level is of significant advantage for differential diagnostic purposes.     

Mechanisms of adaptation of plants to acid soils

Major constraints for plant growth on acid mineral soils are toxic concentrations of mineral elements like Al, of H⁺, and/or low mineral nutrient availability either as a result of solubility (e.g. P and Mo), low reserves, and impaired uptake (e.g. Mg²⁺) at high H⁺ concentrations. Inhibition of root growth particularly by Al leads to more shallow root systems, which may affect the capacity for mineral nutrient acquisition and increase the risk of drought stress. Of the two principal strategies (tolerance and avoidance) of plants for adaptation to adverse soil conditions, the strategy of avoidance is more common for adaptation to acid mineral soils. Examples are (i) root-induced changes in the rhizosphere such as pH increase, (ii) release of chelators for Al, higher activity of ectoenzymes (acid phosphatases), and (iii) increase in root surface area via mycorrhizae. In order to have a better understanding of the principles of the mechanisms by which plants adapt to acid mineral soils more attention should thus be given to conditions at the root-soil interface.     

Direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes

The practical feasibility and generic applicability of the direct integration of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated. Pilot-scale purification of the enzyme L-asparaginase from unclarified Erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrifugal clarification and batch adsorption. In addition to improved yield and quality of product, the process time during primary stages of purification was greatly diminished. Two cation exchange adsorbents, CM HyperD LS (Biosepra/Life Technologies) and SP UpFront (custom made SP form of a prototype stainless steel/agarose matrix, UpFront Chromatography) were physically and biochemically evaluated for such direct product sequestration. Differences in performance with regard to product capacity and adsorption/desorption kinetics were demonstrated and are discussed with respect to the design of adsorbents for specific applications. In any purification of L-asparaginase (pI = 8.6), product-debris interactions commonly diminish the recovery of available product. It was demonstrated herein, that immediate disruptate exposure to a fluidised bed adsorbent promoted concomitant reduction of product in the liquid phase, which clearly counter-acted the product-debris interactions to the benefit of product yield.     

Secondary H/D isotope effect on hydrogen-bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides

An isotopomer-selected NOE (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the partner hydroxyl, whereas a NOE for this group can originate from cross-relaxation with the protio isotopomer of this hydroxyl only. Hence, the isotopically shifted component of this group's signal does not appear in a ROE difference spectrum obtained with selective excitation of the partner hydroxyl. This method is also applicable in those cases when only one of two mutually hydrogen-bonded groups exhibits resolvable isotope shifts. Furthermore, it is shown that isotope shifts may occur even for pairs of OH groups that are not mutually hydrogen-bonded, if these participate in hydrogen bonds with other hydroxyls and thereby affect conformational equilibria. The ISNOE experiment enables one to distinguish between these two sources of isotope shifts. Since the OO distance for hydrogen-bonded hydroxyls in sugars is known to lie between 2.7 and 3.0 Å , the hydrogen bonds established by ISNOE can be used in conformational analysis as reliable, motionally non-averaged distance constraints for the conformations containing these bonds.     

Temperature inducible protochlorophyllide reduction in darkness in a pigment mutant of Scenedesmus obliquus

Accumulation of chlorophyll and protochlorophyllide (PChlide) was followed during beterotrophic growth of the pigment mutant C-2A' of Scenedesmus obliquus L. in the darkness at 30 and 20°C. At 30°C the cells remained yellow with accumulation of protochlorophyllide, whereas they became green at 20°C with only traces of protochlorophyllide. The capacity of mutant cells to reduce PChlide to chlorophyllide (Chlide) in the dark with or without addition of 5-aminolevulinic acid as measured in isolated membranes, was high in cells grown at 20°C but negligible at 30°C. The high capacity to reduce PChlide created in cells growing at 20°C was only slightly diminished by exposure of cells to 38°C for 3 h. Mechanisms of temperature-sensitive chlorosis in algae and higher plants are discussed in relation to the results with pigment mutant C-2A' of Scenedesmus obliquus . It is assumed that either an activator of NADPH protochlorophyllide oxidoreductase (EC 1.6.99.1) or a different enzyme system can be activated by lower temperature as by light.     

The role of olfaction throughout juvenile development: Functional adaptations in elasmobranchs

Seven elasmobranch species, a group known for their highly‐developed sense of smell, were examined for developmental changes in the number of olfactory lamellae, the size of the surface area of the sensory olfactory epithelium and the mass of both the olfactory rosettes (primary input to the CNS), and the olfactory bulbs. Within each species, juveniles possessed miniature versions of the adult olfactory organs, visually not distinguishable from these and without any obvious structural differences (e.g., with respect to the number of lamellae and the extent of secondary folding) between differently sized individuals. The size of the olfactory organs was positively correlated with body length and body mass, although few species showed proportional size scaling. In Aetobatus narinari and Aptychotrema rostrata, olfactory structures increased in proportion to body size. With respect to the growth of the olfactory bulb, all species showed allometric but not proportional growth. Olfaction may be of particular importance to juveniles in general, which are often subjected to heavy predation rates and fierce inter/intraspecific competition. Accordingly, it would be advantageous to possess a fully functional olfactory system early on in development. Slow growth rates of olfactory structures could then be attributed to a greater reliance on other sensory systems with increasing age or simply be regarded as maintaining an already optimized olfactory system. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.