Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Paenisporosarcina indica sp. nov., a psychrophilic bacterium from a glacier, and reclassification of Sporosarcina antarctica Yu et al., 2008 as Paenisporosarcina antarctica comb. nov. and emended description of the genus Paenisporosarcina.

A Gram-stain-positive, aerobic, spore-forming, rod-shaped bacterium, PN2(T), was isolated from a soil sample collected near the Pindari glacier. It contained anteiso-C15 : 0, iso-C15 : 0 and C16 : 1ω7c alcohol as the predominant fatty acids, MK-7 as the major menaquinone and A4α type (l-Lys-d-Glu) peptidoglycan. Based on these characteristics, strain PN2(T) was assigned to the genus Paenisporosarcina. Phylogenetic analysis based on 16S rRNA gene sequence placed strain PN2(T) within the genus Paenisporosarcina and showed a sequence similarity of 98.5-99.0 % with members of this genus. Paenisporosarcina macmurdoensis CMS 21w(T), Paenisporosarcina quisquiliarum SK 55(T) and Sporosarcina antarctica N-05(T) were identified as the most closely related species with 16S rRNA gene sequence similarities of 98.6 %, 99.0 % and 98.4 %, respectively. The values for DNA-DNA relatedness between strain PN2(T) and P. macmurdoensis, P. quisquiliarum and S. antarctica were below the 70 % threshold value (32.0 %, 42.0 % and 38.0 % respectively). In addition, strain PN2(T) exhibited a number of phenotypic differences from P. macmurdoensis, P. quisquiliarum and S. antarctica. Based on the cumulative differences, strain PN2(T) was identified as representing a novel species and the name Paenisporosarcina indica sp. nov. was proposed. The type strain of Paenisporosarcina indica sp. nov. is PN2(T) (LMG 23933(T) = JCM 15114(T)). Furthermore, based on the morphological and chemotaxonomic characteristics, the species Sporosarcina antarctica was reclassified as a species of the genus Paenisporosarcina and renamed Paenisporosarcina antarctica comb. nov. In addition, an emended description of the genus Paenisporosarcina is presented.

Pre-clinical toxicity & immunobiological evaluation of DNA rabies vaccine & combination rabies vaccine in rhesus monkeys (Macaca mulatta).

BACKGROUND & OBJECTIVES: Pre-clinical toxicology evaluation of biotechnology products is a challenge to the toxicologist. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed recombinant DNA anti-rabies vaccine [DRV (100 µg)] and combination rabies vaccine [CRV (100 µg DRV and 1.25 IU of cell culture-derived inactivated rabies virus vaccine)], which are intended for clinical use by intramuscular route in Rhesus monkeys. METHODS: As per the regulatory requirements, the study was designed for acute (single dose - 14 days), sub-chronic (repeat dose - 28 days) and chronic (intended clinical dose - 120 days) toxicity tests using three dose levels, viz. therapeutic, average (2x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in monkeys. The selection of the model i.e. monkey was based on affinity and rapid higher antibody response during the efficacy studies. An attempt was made to evaluate all parameters which included physical, physiological, clinical, haematological and histopathological profiles of all target organs, as well as Tiers I, II, III immunotoxicity parameters. RESULTS: In acute toxicity there was no mortality in spite of exposing the monkeys to 10XDRV. In sub chronic and chronic toxicity studies there were no abnormalities in physical, physiological, neurological, clinical parameters, after administration of test compound in intended and 10 times of clinical dosage schedule of DRV and CRV under the experimental conditions. Clinical chemistry, haematology, organ weights and histopathology studies were essentially unremarkable except the presence of residual DNA in femtogram level at site of injection in animal which received 10X DRV in chronic toxicity study. No Observational Adverse Effects Level (NOAEL) of DRV is 1000 ug/dose (10 times of therapeutic dose) if administered on 0, 4, 7, 14, 28 th day. INTERPRETATION & CONCLUSIONS: The information generated by this study not only draws attention to the need for national and international regulatory agencies in formulating guidelines for pre-clinical safety evaluation of biotech products but also facilitates the development of biopharmaceuticals as safe potential therapeutic agents.

Submitral aneurysm of the left ventricle.

Submitral aneurysm is a rare cardiac pathology of uncertain origin with varied clinical manifestations. Recent studies have revealed a congenital basis of this pathology, although genetic link has been suspected because of the racial predilection. The other suggested aetiologies are infection and inflammation. The case reported here is that of a young female with a large submitral aneurysm presenting in a state of cardiogenic shock. In addition, the presence of raised inflammatory parameters indicates that the cause of origin of this aneurysm is related to inflammation.

Evaluating the substrate-envelope hypothesis: structural analysis of novel HIV-1 protease inhibitors designed to be robust against drug resistance.

Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to "lock" into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors.

Comparison of bacterial diversity in proglacial soil from Kafni Glacier, Himalayan Mountain ranges, India, with the bacterial diversity of other glaciers in the world.

Two 16S rRNA gene clone libraries (KF and KS) were constructed using two soil samples (K7s and K8s) collected near Kafni Glacier, Himalayas. The two libraries yielded a total of 648 clones. Phyla Actinobacteria, Bacteroidetes, Chloroflexi Firmicutes, Proteobacteria, Spirochaetae, Tenericutes and Verrucomicrobia were common to the two libraries. Phyla Acidobacteria, Chlamydiae and Nitrospirae were present only in KF library, whereas Lentisphaerae and TM7 were detected only in KS. In the two libraries, clones belonging to phyla Bacteroidetes and Proteobacteria were the most predominant. Principal component analysis (PCA) revealed that KF and KS were different and arsenic content influenced the differences in the percentage of OTUs. PCA indicated that high water content in the K8s sample results in high total bacterial count. PCA also indicated that bacterial diversity of KF and KS was similar to soils from the Pindari Glacier, Himalayas; Samoylov Island, Siberia; Schrimacher Oasis, Antarctica and Siberian tundra. The eleven bacterial strains isolated from the above two soil samples were phylogenetically related to six different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase, lipase and urease activities were detected in the majority of the strains. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria.

Bacterial diversity of soil in the vicinity of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones.

Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria.

Bacterial biodiversity from Roopkund Glacier, Himalayan mountain ranges, India.

The bacterial diversity of two soil samples collected from the periphery of the Roopkund glacial lake and one soil sample from the surface of the Roopkund Glacier in the Himalayan ranges was determined by constructing three 16S rRNA gene clone libraries. The three clone libraries yielded a total of 798 clones belonging to 25 classes. Actinobacteria was the most predominant class (>10% of the clones) in the three libraries. In the library from the glacial soil, class Betaproteobacteria (24.2%) was the most predominant. The rarefaction analysis indicated coverage of 43.4 and 41.2% in the samples collected from the periphery of the lake thus indicating a limited bacterial diversity covered; at the same time, the coverage of 98.4% in the glacier sample indicated most of the diversity was covered. Further, the bacterial diversity in the Roopkund glacier soil was low, but was comparable with the bacterial diversity of a few other glaciers. The results of principal component analysis based on the 16S rRNA gene clone library data, percentages of OTUs and biogeochemical data revealed that the lake soil samples were different from the glacier soil sample and the biogeochemical properties affected the diversity of microbial communities in the soil samples.

HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants.

The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.

Design and synthesis of HIV-1 protease inhibitors incorporating oxazolidinones as P2/P2' ligands in pseudosymmetric dipeptide isosteres.

A series of novel HIV-1 protease inhibitors based on two pseudosymmetric dipeptide isosteres have been synthesized and evaluated. The inhibitors were designed by incorporating N-phenyloxazolidinone-5-carboxamides into the hydroxyethylene and (hydroxyethyl)hydrazine dipeptide isosteres as P2 and P2' ligands. Compounds with (S)-phenyloxazolidinones attached at a position proximal to the central hydroxyl group showed low nM inhibitory activities against wild-type HIV-1 protease. Selected compounds were further evaluated for their inhibitory activities against a panel of multidrug-resistant protease variants and for their antiviral potencies in MT-4 cells. The crystal structures of lopinavir (LPV) and two new inhibitors containing phenyloxazolidinone-based ligands in complex with wild-type HIV-1 protease have been determined. A comparison of the inhibitor-protease structures with the LPV-protease structure provides valuable insight into the binding mode of the new inhibitors to the protease enzyme. Based on the crystal structures and knowledge of structure-activity relationships, new inhibitors can be designed with enhanced enzyme inhibitory and antiviral potencies.

Luciferase Activity of Insect Fatty Acyl-CoA Synthetases with Synthetic Luciferins.

Long-chain fatty acyl-CoA synthetases (ACSLs) are homologues of firefly luciferase but are incapable of emitting light with firefly luciferin. Recently, we found that an ACSL from the fruit fly Drosophila melanogaster is a latent luciferase that will emit light with the synthetic luciferin CycLuc2. Here, we have profiled a panel of three insect ACSLs with a palette of >20 luciferin analogues. An ACSL from the nonluminescent beetle Agrypnus binodulus (AbLL) was found to be a second latent luciferase with distinct substrate specificity. Several rigid luciferins emit light with both ACSLs, but styryl luciferin analogues are light-emitting substrates only for AbLL. On the other hand, an ACSL from the luminescent beetle Pyrophorus angustus lacks luciferase activity with all tested analogues, despite its higher homology to beetle luciferases. Further study of ACSLs is expected to shed light on the features necessary for bioluminescence and substrate selectivity.

Discovery of HIV-1 protease inhibitors with picomolar affinities incorporating N-aryl-oxazolidinone-5-carboxamides as novel P2 ligands.

Here, we describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors incorporating N-phenyloxazolidinone-5-carboxamides into the (hydroxyethylamino)sulfonamide scaffold as P2 ligands. Series of inhibitors with variations at the P2 phenyloxazolidinone and the P2' phenylsulfonamide moieties were synthesized. Compounds with the (S)-enantiomer of substituted phenyloxazolidinones at P2 show highly potent inhibitory activities against HIV-1 protease. The inhibitors possessing 3-acetyl, 4-acetyl, and 3-trifluoromethyl groups at the phenyl ring of the oxazolidinone fragment are the most potent in each series, with K(i) values in the low picomolar (pM) range. The electron-donating groups 4-methoxy and 1,3-dioxolane are preferred at P2' phenyl ring, as compounds with other substitutions show lower binding affinities. Attempts to replace the isobutyl group at P1' with small cyclic moieties caused significant loss of affinities in the resulting compounds. Crystal structure analysis of the two most potent inhibitors in complex with the HIV-1 protease provided valuable information on the interactions between the inhibitor and the protease enzyme. In both inhibitor - enzyme complexes, the carbonyl group of the oxazolidinone ring makes hydrogenbond interactions with relatively conserved Asp29 residue of the protease. Potent inhibitors from each series incorporating various phenyloxazolidinone based P2 ligands were selected and their activities against a panel of multidrug-resistant (MDR) protease variants were determined. Interestingly, the most potent protease inhibitor starts out with extremely tight affinity for the wild-type enzyme (K(i) = 0.8 pM), and even against the MDR variants it retains picomolar to low nanomolar K(i), which is highly comparable with the best FDA-approved protease inhibitors.

Occurrence of Bluetongue in ruminants in Tamil Nadu, South India.

Tamil Nadu is located in the South-Eastern part of Indian peninsula, between 8.087° and 13.09°N and 76.50° and 80.27°E. Bluetongue (BT) was first reported in this region in sheep during 1982 with regular occurrence thereafter. In 1989-1990, 1997-1998 and 2005-2006, there was wide spread occurrence of BT resulting in huge mortality of sheep. The present study had the goal of isolating the BTV from outbreaks in sheep occurred in Tamil Naadu between 2003-2011 and comparing the VP2 gene sequences of the BTV isolates involved in such outbreaks. Serotypes 1, 2, 16, and 23 of the Bluetongue virus (BTV) have been isolated from sheep during BT outbreaks. BTV-16 has also been isolated in goats and cattle in the region; BTV-2 isolated in Tamil Nadu has homology with BTV-2 isolated in Africa; whereas the BTV-23 isolated in this area has homology with BTV-23 from South East Asia, indicating that both Eastern and Western topotypes of BTV are circulating in ruminant population in Tamil Nadu.

Garlic and Resveratrol Attenuate Diabetic Complications, Loss of ß-Cells, Pancreatic and Hepatic Oxidative Stress in Streptozotocin-Induced Diabetic Rats.

The study was aimed at finding the effect of garlic and resveratrol on loss of ß-cells and diabetic complication in streptozotocin (STZ)-induced Type-I diabetic rats. Rats were injected with single dose STZ (50 mg/kg, i.p.) for induction of type 1 diabetes (Dia) and compared with control group. Rats from third (Dia+Gar), fourth (Dia+Resv), and fifth (Dia+Met) groups were fed raw garlic homogenate (250 mg/kg/day), resveratrol (25 mg/kg/day), and metformin (500 mg/kg/day) orally, respectively, for a period of 4 weeks. Diabetic group had decreased serum insulin and hydrogen sulfide levels along with increased blood glucose and glycated hemoglobin, triglyceride, uric acid, and nitric oxide levels. Significant (p < 0.05) increase in pancreatic and hepatic TBARS, conjugated dienes, nitric oxide, and AGE level and significant (p < 0.05) decrease in SOD, catalase, H2S, GSH level were observed in diabetic group. Administration of garlic, resveratrol, and metformin significantly (p < 0.05) normalized most of the altered metabolic and oxidative stress parameters as well as histopathological changes. Administration of garlic, resveratrol, and metformin in diabetic rat decreases pancreatic ß-cell damage and hepatic injury. Our data concluded that administration of garlic showed more promising effect in terms of reducing oxidative stress and pathological changes when compared to resveratrol and metformin groups.

Tidal eddies in a semi-enclosed basin: a model study.

A modeling study has been carried out to support a Marine Management Plan for the Gulf of Kachchh, India and here the hydrodynamic part of the programme is described. The hydrodynamic model accurately predicts the tides and tidal currents present in the Gulf and these have been validated with the measured data, albeit at only a few locations. The time averaged residual currents obtained from the model for one lunar cycle clearly reproduce the complex, small-scale, topographically induced flows with several eddies. The existence of a dynamic barrier along Sikka-Mundra section, which divides the Gulf into two distinct dynamic systems, is very evident. The model is further used to predict the movement of surface floating particles launched at different locations in the Gulf, as an aid to determining floating pollutants. The results indicate that industries discharging wastes upstream of the barrier should use extreme caution, as these will remain in the vicinity for at least one lunar cycle.

Preexposure efficacy of a novel combination DNA and inactivated rabies virus vaccine.

Several strategies are being examined to enhance the potency of DNA rabies vaccine (DRV) so that it can be used for both prophylaxis and postexposure therapy of rabies. In this study, we report a novel combination rabies vaccine (CRV) containing a low dose of cell culture-derived inactivated rabies virus vaccine and DRV. Mice immunized with CRV develop higher levels of rabies virus-neutralizing antibodies (RVNA) than those immunized with DRV and are completely protected against peripheral as well as intracerebral rabies virus challenge. The quantity of inactivated rabies virus vaccine required for enhancing the potency of DRV can be 625-fold lower than that of a standard dose of inactivated rabies virus vaccine. CRV induces higher levels of RVNA than DRV in cattle as well. Thus, we demonstrate for the first time that co-inoculation of DNA vaccine and a low dose of inactivated virus vaccine can be developed into a novel cost-effective vaccination strategy for combating rabies in particular, and infectious diseases in general.

1,25-Dihydroxy-24-OXO-16ene-vitamin D3, a renal metabolite of the vitamin D analog 1,25-dihydroxy-16ene-vitamin D3, exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo.

The hormone, 1,25-(OH)2D3, is metabolized into 1,25-(OH)2-24-OXO-D3, in kidney prior to conversion to its final inactive product, calcitroic acid. Similarly, 1,25-(OH)2-24OXO-16eneD3, is produced in the kidney from the Vitamin D analog, 1,25-(OH)2-16eneD3, but resists further hydroxylation. The analog's metabolite was synthesized and its biologic activity compared to the parent compound. Naive SJL/J mice, 4 weeks old, were immunized with neuroantigen in adjuvant to induce experimental autoimmune encephalomyelitis [EAE]. Treatment with 1,25-(OH)2-24OXO-16eneD3 was given at 0.05, 0.15 and 0.3 microgram I.P., on alternate days, starting 3 days prior and for up to 5 days post immunization and compared to a similar treatment with 0.1 microgram 1,25-(OH)2D3 or 1,25-(OH)2-16eneD3. Suppression of EAE was observed with 0.15 microgram 1,25-(OH)2-24OXO-16eneD3, comparable to the suppression induced with the parent compound and more potent than 1,25-(OH)2D3. However, no hypercalcemia was seen in mice treated with 0.15 microgram of OXO-metabolite (9.7 +/- 0.6 vs 9.3 +/- 1.1 mg/dl, treated vs controls), in contrast to 1,25-(OH)2D3 and 1,25-(OH)2-16eneD3 (11.2 +/- 1.0 and 11.0 +/- 0.9 mg/dl respectively; p < 0.001). In summary, our results suggest that 1,25-(OH)2-24OXO-16eneD3, a stable intermediary metabolite of the vitamin D analog, 1,25-(OH)2-16eneD3 exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo.

Human osteoblasts in culture metabolize both 1 alpha, 25-dihydroxyvitamin D3 and its precursor 25-hydroxyvitamin D3 into their respective lactones.

1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3], the hormonal form of vitamin D3, is further metabolized in the kidney and intestine through the carbon 24 (C-24) oxidation pathway initiated by C-24 hydroxylation, and the carbon 23 (C-23) oxidation pathway initiated by C-23 hydroxylation. The C-24 oxidation pathway leading to the formation of calcitroic acid has been previously reported to be present in bone cells, but the C-23 oxidation pathway leading to the formation of 1 alpha, 25-(OH)2D3-26,23-lactone has not been described in bone cells, even though 1 alpha, 25-(OH)2D3-26,23-lactone is noted to have a significant effect on bone formation. Therefore, in the present study, we investigated the production of 1 alpha, 25-(OH)2D3-26,23-lactone in normal human osteoblasts, and our studies revealed that human osteoblasts possess the activity of both 24- and 23-hydroxylases constitutively. Thus, 1 alpha, 24(R),25-(OH)3D3, 1 alpha, 25-(OH)2-24-oxo-D3, 1 alpha, 23(S), 25-(OH)3-24-oxo-D3, 1 alpha, 23-(OH)2-24,25,26,27-tetranor D3, and calcitroic acid formed through the C-24 oxidation pathway and 1 alpha, 23(S),25-(OH)3D3 and 1 alpha, 25-(OH)2D3-26,23-lactone formed through the C-23 oxidation pathway were detected under basal conditions. Also, the synthesis of these metabolites was increased significantly when the cells were treated with 1 alpha, 25-(OH)2D3 (50 nM) for 24 h before incubation with the tracer. As 25-hydroxyvitamin D3 (25OHD3) follows similar side-chain modifications as 1 alpha, 25-(OH)2D3, the metabolism of 25OHD3 in normal human osteoblasts was studied under basal conditions. We found that 25OHD3 was also metabolized through both C-24 and C-23 oxidation pathways, resulting in significant synthesis of 24(R),25-(OH)2D3 along with 25OH-24-oxo-D3, 23(S),25-(OH)2-24-oxo-D3, 23(S),25-(OH)2D3, and 25OHD3-26,23-lactone. Under the same experimental conditions, we looked for 1 alpha, 25-(OH)2D3 synthesis, as earlier studies have shown production of 1 alpha, 25-(OH)2D3 in human bone cells. During a time-course study ranging from 1-24 h, we found that by 2 h, the 24(R), 25-(OH)2D3 concentration rose and accumulated considerably during the following 24 h, but 1 alpha, 25-(OH)2D3 did not accumulate at any time. However, other 1-hydroxylated metabolites, 1 alpha, 23(S),25-(OH)3D3, 1 alpha, 23(S),25-(OH)3-24-oxo-D3, as well as 1 alpha, 25-(OH)2D3-26,23-lactone were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of vitamin D metabolism in normal human pregnancy.

The increasing serum concentrations of various hormones (PTH, PRL, estrogens, and human placental lactogen) are hypothesized to regulate 1,25-dihydroxyvitamin D [1,25(OH)2D] and possibly 24,25-dihydroxyvitamin D [24,25(OH)2D] production during pregnancy. We examined the correlation between the serum levels of 1,25(OH)2D and the pregnancy-related hormones in 25 normal pregnant women, followed throughout gestation and postpartum. Maternal serum levels of 1,25(OH)2D were high during the first trimester (mean +/- SE, 74 +/- 8 pg/ml), remained high until the time of delivery (95 +/- 14 pg/ml), and then fell to almost normal levels (50 +/- 9 pg/ml) on the third postpartum day. The serum levels of 1,25(OH)2D do not correlate with the serum levels of any of the aforementioned hormones. The increase in serum 1,25(OH)2D in pregnancy has been postulated to be related to the stressed calcium homeostatic mechanisms known to occur in the mother. In twin pregnancy, this maternal calcium homeostatic mechanism(s) conceivably may be stressed to a greater extent. However, serum 1,25(OH)2D levels, measured in 27 women with a twin pregnancy in both the second and third trimesters as well as at delivery, did not differ from the levels observed in women with a singleton pregnancy. There were no significant changes in the serum levels of 24,24(OH)2D or 25-hydroxyvitamin D as pregnancy progressed. However, serum 24,25(OH)2D correlated significantly with both serum 1,25(OH)2D (r - 0.51; p less than 0.001, n = 83) and serum 25-hydroxyvitamin D (r = 0.37; P less than 0.001, n = 94). In conclusion, serum levels of 1,25(OH)2D rise early in the first trimester of pregnancy, fall acutely to normal levels soon after delivery, and are similar in singleton and twin pregnancies. The changes in the serum levels of 1,25(OH)2D do not relate to the changes in the serum levels of any of the pregnancy-related hormones.