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.     

Role of protein synthesis in the accumulation of ferritin mRNA during exposure of cells to iron.

The iron-induced biosynthesis of ferritin is regulated at the translational level via multiple mechanism. A prolonged exposure of cells to iron leads to a marked increase in ferritin mRNA levels caused by stabilization of the message. Here we show that this stabilization requires the synthesis de novo of an iron-inducible protein factor.     

Salicylate hydroxylation as an early marker of in vivo oxidative stress in diabetic patients.

In vivo metabolism of salicylic acid produces two main hydroxylated derivatives (2,5- and 2,3-dihydroxybenzoic acid). The former can be produced by enzymatic pathways through the cytochrome P-450 system, while the latter is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2,3-dihydroxybenzoate, following oral administration of salicylate in its acetylated form (aspirin), has been proposed for assessment of oxidative stress. In this article we report plasma levels of 2,3- and 2,5-dihydroxybenzoates following the administration of 1 g aspirin and plasma levels of thiobarbituric acid-reactive material (TBARM) in well-controlled diabetic patients and in healthy subjects. 2,3-Dihydroxybenzoate levels were significantly higher (23%) in diabetic patients than in controls (63.4 +/- 20.1 versus 49.0 +/- 6.8 nM; p < .05). On the other hand, TBARM values were not significantly different between groups. These results suggest that the method is useful to reveal in vivo oxidative stress independently from the peroxidation of lipids, and they support the hypothesis that oxygen radicals are involved in the pathogenesis of chronic complications of diabetes.     

Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.     

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).     

Refinement of the structure of bovine seminal ribonuclease

We report here the refinement at 2.5-Å resolution of the x-ray crystal structure of bovine seminal ribonuclease, a dimeric covalent enzyme. The protein, which crystallizes with one molecule in the asymmetric unit, consists of two subunits of identical chemical sequences, related by an almost exact binary axis. The tertiary structure of the subunits is similar to that of the pancreatic enzyme, which shows similar catalytic properties. The refinement was carried out using the restrained least-squares procedure both in the reciprocal and real spaces. The assemblage of the subunits in the dimer is described and discussed.     

Postnatal Development of a Granule Cell-Enriched, Neurone-Specific Glycoprotein, gp50, in Normal and Thyroid-Deficient Rats

Glycoprotein gp50 is a neurone-specific, granule cell-enriched glycoprotein that is also a major component of isolated synaptic membranes. Here, we describe the use of a monoclonal antibody, mab SM gp50, to study the postnatal development of gp50 in the brain of normal and thyroid-deficient rats. Radioimmunoassay, enzyme-linked immunosorbent assay, and Western blotting show that gp50 is not detectable in brain until postnatal day 4 (P4) in both forebrain and cerebellum. In forebrain, the rate of increase of gp50 levels is maximal between P12 and P20. It is somewhat later in cerebellum, where peak levels are attained between P30 and P35. Immunocytochemical studies show little detectable gp50-like immunoreactivity before P16, and the staining is still weak, relative to adult tissue, at P25. The intense staining of the granule cell layer characteristic of adult cerebellum predominantly appears after P25. Development of gp50 is severely retarded in the cerebellum of thyroid-deficient rats, particularly during the second and third postnatal weeks. However, by the fourth postnatal week, gp50 levels in normal and hypothyroid animals are comparable. The results indicate that significant alterations in the pattern of gp50 expression continue to occur at a late stage of cerebellar development. In particular, the increase in immunocytochemical staining of the granule cells after P25 is striking in that by this time most major events associated with cerebellar development are essentially complete.     

Development of human monoclonal antibodies: A review

This article describes the current status in the development of human monoclonal antibodies. Over the last ten years a lot of information about the human immune system has emerged. Combining these with the many new (bio-)technologies it is plausible that the long awaited breakthrough of this technology is close. This paper focuses on the classical cell-biological methods of achieving stable, antibody-producing human cell lines via cell fusion methods or virus derived transformations of human B-lymphocytes, as well as genetic engineering methods e.g. DNA libraries or phage display technology. The available in vitro immunization methods are critically reviewed and their impact on this topic is discussed. Therapeutic applications for cancer treatment or passive immunization against infectious diseases with antibodies derived by both ways are also reviewed.     

Exclusively paternal X chromosomes in a girl with short stature

A 13 1/2 year-old girl with short stature and very few Turner stigmata revealed 45,X/46,XX mosaicism with 90%–100% 46,XX cells in three sequential blood lymphocyte cultures. Molecular investigation of the parental origin of her X chromosomes revealed homozygosity for paternal X markers and an absence of maternal markers. Luteinizing hormone response to growth hormone releasing hormone was increased. Impaired gonadal function and shortness of stature in this case could be a result of the mild mosaicism with a 45,X cell line and/or is a consequence of the paternal-only origin of her X chromosomes.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .