Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

AUDIT,AUDIT-C,and AUDIT-3: Drinking Patterns and Screening for Harmful,Hazardous and Dependent Drinking in Katutura,Namibia

Objectives

To describe alcohol drinking patterns among participants in Katutura, Namibia, and to evaluate brief versions of the AUDIT against the full AUDIT to determine their effectiveness in detecting harmful drinking.

Methods

A cross-sectional survey was conducted in four constituencies and 639 participants, 18 years or older, completed a sociodemographic survey and the AUDIT. The effectiveness of the AUDIT-C (first three questions) and the AUDIT-3 (third question) was compared to the full AUDIT.

Results

Approximately 40% were identified as harmful, hazardous or likely dependent drinkers, with men having a higher likelihood than women (57.2% vs. 31.0%, p<.0001). Approximately 32% reported making and/or selling alcohol from home. The AUDIT-C performed best at a cutoff ≥ 3, better in men (sensitivity: 99.3%, specificity: 77.8%) than women (sensitivity: 91.7%, specificity: 77.4%). The AUDIT-3 performed poorly (maximum sensitivity: < 90%, maximum specificity: <51%). According to AUROC, the AUDIT-C performed better than the AUDIT-3.

Conclusions

A large proportion of participants met criteria for alcohol misuse, indicating a need for screening and referral for further evaluation and intervention. The AUDIT-C was almost as effective as the full AUDIT and may be easier to implement in clinical settings as a routine screening tool in resource-limited settings because of its brevity.     

Formulation and Release Behavior of Doxycycline–Alginate Hydrogel Microparticles Embedded into Pluronic F127 Thermogels as a Potential New Vehicle for Doxycycline Intradermal Sustained Delivery

The aim of this work was the formulation and characterization of alginate (ALG)–doxycycline (DOX) hydrogel microparticles (MPs) embedded into Pluronic F127 thermogel for DOX intradermal sustained delivery. ALG–DOX MPs were formed by adding a solution of the drug into a 1.5% polymer solution while stirring. The MPs were cross-linked by dispersion into a 1.2% CaCl₂ solution. Free MPs were characterized in terms of size, drug content, and release behavior by HPLC and UV–vis. DOX and hydrogel MPs were embedded into PF127, PF127-HPMC, and PF127-Methocel thermogels. The thermogels were characterized in terms of gelling time, morphology, and release behavior. A target release period of 4–7 days was considered optimal. The hydrogel MPs were about 20 μm in size with 90% of the population <59 μm. Drug content was about 35% (w/w). DOX released rapidly from the MPs, 90% within 2 days. An expected faster release was observed for free DOX from the thermogels with 80–90% of drug released after 3.5–4 h even in the presence of 1% HPMC or Methocel. The release was sustained after embedding the MPs into PF127 and PF127-HPMC thermogels. In particular, the PF127-HPMC thermogel showed an almost linear release, reaching 80% after 3 days and 90% up to 6 days. Although a further characterization and formulation assessment is required to optimize MP characteristics, ALG/DOX-loaded hydrogel MPs, when embedded into a PF127-HPMC thermogel, show a potential for achieving a 7-day sustained release formulation for DOX intradermal delivery.     

Amino acid 95 causes strong alteration of peptide position PΩ in HLA-B*41 variants

There have been several attempts over the years to identify positions in the peptide-binding region (PBR) of human leukocyte antigens (HLA) that influence the specificity of bound amino acids (AAs) at each position in the peptide. Originally, six pockets (A-F) were defined by calculating the surface area of the PBR on the crystal structure of HLA-A2 molecules. More recent crystallographic analyses of a variety of HLA alleles have led to broader pocket definitions. In this study, we examined the peptide-binding specificity of HLA-B*41 alleles and compared our results with the available pocket definitions. By generating recombinant HLA-B molecules and studying the eluted peptides by mass spectrometry and pool sequencing, we detected two different POmega peptide motifs within the B*41 group: Leu vs Val/Pro. Specificity was dependent on the presence of Leu (B*4102, B*4103, and B*4104) vs Trp (B*4101, B*4105, and B*4106) at AA position 95 in the HLA molecule, whose impact on POmega has been a subject of controversy in current pocket definitions. In contrast, the Arg97Ser mutation did not affect pocket F binding specificity in B*41 subtypes although residue 97 was previously identified as a modulator of peptide binding for several HLA class I alleles. According to most pocket definitions, this study shows that the Asn80Lys substitution in B*4105 impels the peptide's POmega anchor toward more promiscuity. Our sequencing results of peptides eluted from HLA-B*41 variants demonstrate the limitations of current pocket definitions and underline the need for an extended peptide motif database for improved understanding of peptide-major histocompatibility complex interactions.     

Methyl substitution of the 25-hydroxy group on 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) reduces potency but allows bone selectivity

The discovery of 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) as a bone selective and bone anabolic form of vitamin D has stimulated an investigation of structure/function of bone selectivity. Four new 2-substituted-19-norvitamin D analogs 3-6 have been developed to study the structure-activity relationship at C-25. As predicted, removing the 25-hydroxy group (compound 3) from the very potent analog 2MD and its 2-methyl derivatives (5 and 6) dramatically reduces in vitro activities, but biological potency is nearly fully restored in vivo likely due to in vivo 25-hydroxylation. The introduction of a methyl group at C-25 (compound 4) that blocks in vivo 25-hydroxylation reduces biological activity both in vitro and in vivo. However, analog 4 retains bone selectivity making it interesting as a possible therapeutic for bone loss diseases.     

Spindle microtubules: getting attached at both ends

A recent study describes a novel role for the centrosomal protein Cep57 in attaching spindle microtubules to both kinetochores and centrosomes, suggesting similar mechanisms may be used for generating these two distinct linkages in mitosis.     

Structure-activity relationships of 19-norvitamin D analogs having a fluoroethylidene group at the C-2 position

We have synthesized four new geometric isomers of 1alpha,25-dihydroxy-2-(2'-fluoroethylidene)-19-norvitamin D analogs 1 and 2 having a 20R- and 20S-configuration, whose structures are correlated with 2MD possessing high potencies in stimulating bone formation in vitro and in vivo. The E-isomers of (20R)- and (20S)-2-fluoroethylidene analogs 1a and 1b were comparable with the natural hormone 1alpha,25-(OH)(2)D(3) in binding to the vitamin D receptor (VDR), while two Z-isomers 2a and 2b were about 15-20 times less active than the hormone. In inducing expression of the vitamin D responsive element-based luciferase reporter gene, the E-isomers 1a and 1b were 1.2- and 8.6-fold more potent than the hormone, respectively, while the Z-isomers 2a and 2b had 27-55% of the potency. On the basis of the biological activities and a docking simulation based on X-ray crystallographic analysis of the VDR ligand-binding pocket, the structure-activity relationships of the fluorinated 19-norvitamin D analogs are discussed.