Biomedical subjects

G S Reddy

Publications and source records attributed to G S Reddy.

At least 19 recordsLinked to original sources

PubMed2006

Nonclinical toxicology study of recombinant-plasmid DNA anti-rabies vaccines.

The absence of standard guidelines from National and International regulatory agencies for the safety evaluation of biotechnology products challenges the ingenuity of toxicologists. At present, the development of standard pre-clinical toxicology protocols for such products is on an individual case basis. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed DNA based anti-rabies vaccine in India. The test compounds were DNA rabies vaccine [DRV (100 microg)] and combination rabies vaccine (CRV (100 microg DRV and 1/50 dose of cell culture vaccine)), intended for clinical use by intramuscular route on 1, 7, 14 and 28 day. As per the regular mandatory requirements, the study has been designed to undertake acute (single dose--10 days), sub-chronic (repeat dose--28 days) and chronic (intended clinical dose--120 days) toxicity tests using three dose levels viz. therapeutic, average (2 x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in Swiss Albino mice. The selection of the rodent model viz. Swiss Albino mice is based on affinity and rapid higher antibody response during the efficacy studies. Apart from physical, physiological, clinical, hematological and histopathology profiles of all target organs, the tier-I immunotoxicity parameters have also been monitored. There were no observational adverse effects even at levels of 10x therapeutic dose administration of DRV and CRV. The procedure also emphasizes on the designing of protocols for the products developed by recombinant technique.

Animals↗

PubMed2005

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.

Animals↗

PubMed2005

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.

Forecasting↗

PubMed2003

Anti-proliferative effects of 20-epi-vitamin-D3 analogue, KH1060 in human neuroblastoma: induction of RAR-beta and p21(Cip1).

We determined the in vitro biological activities of 1 alpha, 25-dihdroxyvitamin D(3) (1,25-D(3)) and its analogue, 20-epi-22-oxa-24a, 26a, 27a-trihomo-1 alpha, 25 (OH)(2) vitamin D(3) (KH1060) in six human neuroblastoma (NB) cell lines (SH-SY5Y, NB69, SK-N-AS, IMR5, CHP-134, NGP). The ability of these compounds to inhibit cell growth and DNA synthesis was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and BrdU incorporation, respectively. The induction of cell death was monitored by caspase-3 activity. Their antineoplastic effect was assessed by clonal proliferation in soft agar. KH1060 was more effective than 1,25 D(3) in inhibiting cell growth and DNA synthesis. The IC-(50) (inhibition of 50% cell viability) indicated that KH1060 was about 10-20-fold more potent than 1,25 D(3). This growth inhibition was also accompanied by induction of caspase-3 activity, indicating that these compounds induce cell death in a caspase-dependent fashion. Moreover, KH1060 exerted potent antineoplastic activity by suppressing the clonal proliferation of the six NB cells. For the first time we demonstrate that KH1060 induces the expression of retinoic acid receptor-beta and p21(Cip1) suggesting that these proteins in part mediate the growth inhibitory effects. Taken together, all the six NB cells were more susceptible to growth inhibition by KH1060 than 1,25-D(3), suggesting its possible use in NB to potentiate the action of retinoids, which are in clinical use for this disease.

Antineoplastic Agents↗

PubMed2003

Ambient air quality status in Raniganj-Asansol area, India.

This investigation presents the assessment of ambient air quality with respect to suspended particulate matter (SPM), sulphur dioxide (SO2) and oxides of nitrogen (NOx) at four sites (RGC, SRS, BBC and BCC) in the Raniganj-Asansol area in West Bengal, India. Ambient air was monitored with a sampling frequency of twenty four hours (3 x 8 hours) at each site on every alternate day (3 days a week) covering a period of one year. A total of 429 samples were collected from RGC, 429 from SRS and 435 each from the BBC and BCC sites. Meteorological parameters such as temperature, relative humidity, wind-speed and wind-direction were also recorded simultaneously during the sampling period. Monthly and seasonal variation of these pollutants have been observed and recorded. The annual average and range values have also been calculated. Results of the investigation indicates that the 95th percentile values of SPM levels exceed the limits (200 microg m(-3)) at RGC, SRS and BBC sites and is within the limit of 500 microg m(-3) at the BCC sites. The 95th percentile values of SO2 levels did not exceed the reference level at any of the monitoring stations. The 95th percentile values of NOx are found to be exceeding the limit (80 microg m(-3)) at RGC, SRS and BBC sites but is within the prescribed limit of 120 microg m(-3) at the BCC site. Further, it has been observed that the concentrations of the pollutants are high in winter in comparison to the summer or the monsoon seasons. Results of the investigation indicates that industrial activities, indiscriminate open air burning of coal by the local inhabitants for cooking as well as coking purposes, vehicular traffic, etc. are responsible for the high concentration of pollutants in this area.

Air Pollutants↗

PubMed2001

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.

Animals↗

PubMed2001

Calcitroic acid is a major catabolic metabolite in the metabolism of 1 alpha-dihydroxyvitamin D(2).

Calcitroic acid (1 alpha-hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3)) is known to be the major water-soluble metabolite produced during the deactivation of 1 alpha,25-dihydroxyvitamin D(3). This deactivation process involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1 alpha,25-dihydroxyvitamin D(3), 1 alpha,25-dihydroxyvitamin D(2) is also known to undergo side-chain oxidation; however, to date there has been no evidence suggesting that 1 alpha,25-dihydroxyvitamin D(2) undergoes side-chain cleavage. To investigate this possibility, we studied 1 alpha,25-dihydroxyvitamin D(2) metabolism in HPK1A-ras cells as well as the well characterized perfused rat kidney system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase HPLC analysis. The major aqueous-soluble metabolite from both the kidney and cell incubations comigrated with authentic calcitroic acid on two reverse-phase HPLC columns of different chemistry. The putative calcitroic acid from the cell and kidney incubations was methylated and found to comigrate with methylated authentic standard on straight-phase and reverse-phase HPLC columns. The identity of the methylated metabolite from cell incubations was also confirmed by mass spectral analysis. These data show, for the first time, that calcitroic acid is a major terminal product for the deactivation of 1 alpha,25-dihydroxyvitamin D(2). Intermediates leading to the formation of the calcitroic acid in the 1 alpha,25-dihydroxyvitamin D(2) metabolism pathway are currently being studied.

Animals↗

PubMed2001

Solid-phase synthesis of pyrrolo[2,1-c][1,4]benzodiazepine-5,11-diones.

The solid-phase synthesis of biologically important pyrrolo[2,1-c][1,4]benzodiazepine-5,11-diones using Wang resin through amide formation and reductive cyclization procedures is described. Further, N10-substituents have been introduced in the final products and these have been cleaved from the solid support in good yields.

Anti-Anxiety Agents↗

PubMed2001

Tissue specific metabolism of 1alpha,25-dihydroxy-20-epi-vitamin D3 into new metabolites with significant biological activity: studies in rat osteosarcoma cells (UMR 106 and ROS 17/2.8).

In a recent study, we investigated the metabolism of 1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), a potent synthetic vitamin D3 analog in the isolated perfused rat kidney and proposed that the enhanced biological activity of 1alpha,25(OH)2-20-epi-D3 is in part due to its metabolism into stable bioactive intermediary metabolites derived via the C-24 oxidation pathway (Siu-Caldera et al. [1999] J. Steroid. Biochem. Mol. Biol. 71:111-121). It is now well established that 1alpha,25(OH)2D3 and its analogs are metabolized in target tissues not only via the C-24 oxidation pathway but also via the C-3 epimerization pathway. As the perfused rat kidney does not express the C-3 epimerization pathway, we could not identify other possible bioactive metabolites of 1alpha,25(OH)2-20-epi-D3 such as 1alpha,25(OH)2-20-epi-3-epi-D3, derived via the C-3 epimerization pathway. Therefore, we studied the metabolism of 1alpha,25(OH)2-20-epi-D3 in rat osteosarcoma cells (UMR 106) which express both the C-24 oxidation and the C-3 epimerization pathways. Our results indicate that 1alpha,25(OH)2-20-epi-D3 is metabolized in UMR 106 cells into several metabolites which included not only the previously known metabolites of the C-24 oxidation pathway but also three new metabolites which were labeled as metabolites X, Y1, and Y2. Metabolite X was unequivocally identified as 1alpha,25(OH)2-20-epi-3-epi-D3. Even though definite structure identification of the metabolites, Y1 and Y2 was not achieved in our present study, we determined that the metabolite Y1 is produced from 1alpha,25(OH)2-20-epi-D3 and the metabolite Y2 is produced from 1alpha,25(OH)2-20-epi-3-epi-D3. We also noted the production of both 1alpha,25(OH)2-20-epi-3-epi-D3 and the two metabolites Y1 and Y2 in different rat osteosarcoma cells (ROS 17/2.8) which express only the C-3 epimerization pathway but not the C-24 oxidation pathway. Furthermore, we investigated the metabolism of 1alpha,25(OH)2-20-epi-D3 in the isolated perfused rat kidney in an earlier study. The results of this study indicated that the rat kidney unlike rat osteosarcoma cells did not produce either 1alpha,25(OH)2-20-epi-3-epi-D3 or the metabolites Y1 and Y2. Thus, it appears that the metabolites Y1 and Y2, like 1alpha,25(OH)2-20-epi-3-epi-D3, are produced only in specific tissues. Preliminary biological activity of each new metabolite is assessed by measuring its ability to generate VDR-mediated gene transcription. 1alpha,25(OH)2-20-epi-3-epi-D3 was found to be almost equipotent to 1alpha,25(OH)2-20-epi-D3 while the metabolites, Y1 and Y2 were found to be less active. The metabolite Y1 when compared to the metabolite Y2 has higher biological activity and its potency is almost equal to 1alpha,25(OH)2D3. In summary, we report for the first time tissue specific metabolism of 1alpha,25(OH)2-20-epi-D3 into several bioactive metabolites which are derived not only via the previously established C-24 oxidation and C-3 epimerization pathways but also via a new pathway. (c) 2001 Wiley-Liss, Inc.

Animals↗

PubMed2001

The biological activities of 1alpha,25-dihydroxyvitamin D3 and its synthetic analog 1alpha,25-dihydroxy-16-ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS-2 cells are modulated by 17-beta estradiol and dependent on stage of differentiation.

We compared the effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and its analog, 1alpha,25-dihydroxy-16-ene-vitamin D3 [1alpha,25(OH)2-16-ene-D3], as well as their interactions with 17-beta estradiol (E2) on osteoblastic function in our human normal (HOB) and osteosarcoma SaOS-2 cell models representing two different stages of differentiation, the more differentiated HOB+DEX cells and SaOS+DEX cells, and the corresponding less differentiated HOB-DEX and SaOS-DEX cells. The differential effects of 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 and the modulation by E2 on ALP activity in HOB-DEX and HOB+DEX cells were small but significant. The most significant effects were seen in SaOS+DEX cells, in which 1alpha,25(OH)2-16-ene-D3 was 100-fold more potent than 1alpha,25(OH)2D3, the maximal enhancement being exerted at 0.1 nM and 10 nM, respectively. E2 enhanced the stimulatory effects of both compounds, with ALP being increased 2-fold at 0.1 nM (p<0.001). Osteocalcin (OC) production in HOB-DEX cells was stimulated 1.3 to 1.4-fold by 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 at a concentration of 0.01 nM, with E2 inhibiting the effect of 1alpha,25(OH)2-16-ene-D3. In SaOS-DEX and SaOS+DEX cells, 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-D3 stimulated OC production 1.6-fold at 0.1 nM with E2 slightly enhancing the effect of 1alpha,25(OH)2D3. Western blot analysis of 1alpha,25(OH)2D3 receptor (VDR) levels showed that in SaOS+DEX cells, the effect of 1alpha,25(OH)2D3 was larger than that of 1alpha,25(OH)2-16-ene-D3. These results show that 1alpha,25(OH)2-16-ene-D3 is biologically active in human osteoblasts.

Adult↗

PubMed2001

Isolation, identification and biological activity of 24R,25-dihydroxy-3-epi-vitamin D3: a novel metabolite of 24R,25-dihydroxyvitamin D3 produced in rat osteosarcoma cells (UMR 106).

We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] as a metabolite of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] produced in rat osteosarcoma cells (UMR 106). We now report the isolation of 24R,25-dihydroxy-3-epi-vitamin D3 [24R,25(OH)2-3-epi-D3] as a metabolite of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] by high-performance liquid chromatography (HPLC) with chiral column and its structure assignment by proton nuclear magnetic resonance (1H-NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. We also demonstrated the production of 24R,25(OH)2-3-epi-D, in two other cell lines [human colon carcinoma cells (Caco-2) and porcine kidney cells (LLC-PK1)] which were previously shown to convert 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3. It can be seen that the production of 24R,25(OH)2- 3-epi-D3 from 24R,25(OH)2D3 is lower than that of 1alpha,25(OH)2-3-epi-D3 from 1alpha,25(OH)2D3 in all the cells studied. 24R,25(OH)2-3-epi-D3 was found to be inactive in terms of its ability to bind to the vitamin D receptor (VDR), in inhibiting proliferation and in inducing differentiation of human promyelocytic leukemia cells (HL-60). Thus, our study indicates that the C-3 epimerization pathway is common to both 1alpha,25(OH)2D3 and 24R,25(OH)2D3 and may play an important role in modulating the concentration and the biological activity of these two major vitamin D3 metabolites in target tissues.

24,25-Dihydroxyvitamin D 3↗

PubMed2001

Evaluation of rabies virus neutralizing antibody titres induced by intramuscular inoculation of rabies DNA vaccine in mice and Bonnet monkeys (Macaca radiata).

A rabies DNA vaccine consisting of plasmid DNA expressing the rabies virus surface glycoprotein was injected (im) twice at two week interval to outbred swiss mice or Bonnet monkeys (Macaca radiata) and the levels of rabies virus neutralizing antibody (VNA) titres were examined over a one year period. In mice, the VNA titre was maintained above the minimum protective level (0.5 I.U./ml) up to 10 months after primary immunization, while in monkeys, the titre dropped below the protective level by 6 months. An anamnestic B cell response was seen in both mice and monkeys following the administration of a booster dose, 10 and 6 months after the primary immunization, respectively. These results indicate that im injection of rabies DNA vaccine induces VNA in nonhuman primates and mice unlike intradermal (id) immunization, which was shown to induce VNA only in mice but not in monkeys. This is the first report on the induction of VNA in nonhuman primates by im inoculation of rabies DNA vaccine.

Animals↗

PubMed2000

1alpha,25-Dihydroxy-3-epi-vitamin D3 a physiological metabolite of 1alpha,25-dihydroxyvitamin D3: its production and metabolism in primary human keratinocytes.

Recent studies of metabolism using pharmacological substrate concentrations of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] in several tissues including primary cultures of human keratinocytes, bovine parathyroid cells and bone cells led to the identification of 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)(2)-3-epi-D3] as a major natural metabolite of 1alpha,25(OH)(2)D3. In the present study, we demonstrate that human keratinocytes incubated with 25-hydroxy[26,27-(3)H] vitamin D3 produce 1alpha,25(OH)(2)-3-epi-D3 along with 1alpha,25(OH)(2)D3. The production of 1alpha,25(OH)(2)-3-epi-D3 is also identified in human keratinocytes incubated with physiological substrate concentrations of 1alpha,25(OH)(2)D3. Unlike 24-hydroxylase, the major enzyme involved in the further metabolism of 1alpha,25(OH)(2)D3 in human keratinocytes, the enzyme(s) responsible for the production of 1alpha,25(OH)(2)-3-epi-D3 is constitutive and is not inhibited by ketoconazole. It is also noted that 1alpha,25(OH)(2)-3-epi-D3 is further metabolised in human keratinocytes into several as yet unidentified metabolites, the production of which is inhibited to a great extent by SDZ 89-443, an inhibitor of 24-hydroxylase. This finding indicates that the 24-hydroxylase like in the case of 1alpha,25(OH)(2)D3, also plays a major role in the metabolism of 1alpha,25(OH)(2)-3-epi-D3. The results obtained from the metabolism studies performed in parallel among 25OHD3, 1alpha,25(OH)(2)D3 and 1alpha,25(OH)(2)-3-epi-D3 indicate that 1alpha,25(OH)(2)-3-epi-D3 and its metabolites exhibit higher metabolic stability. In summary, we demonstrate for the first time that 1alpha,25(OH)(2)-3-epi-D3 is a physiological metabolite of 1alpha,25(OH)(2)D3 in human keratinocytes. Also, 1alpha,25(OH)(2)-3-epi-D(3) is further metabolised in human keratinocytes mainly through the activity of 24-hydroxylase. Furthermore, our finding of the relative metabolic stability of 1alpha,25(OH)(2)-3-epi-D3 and especially its metabolites when compared to 1alpha,25(OH)(2)D3 and its metabolites provides an important explanation for its previously observed potent inhibitory effect on keratinocyte growth in spite of its low affinity to vitamin D receptor.

Calcitriol↗

PubMed2000

1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1alpha,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the previously well established main side chain modification pathway, is initiated by hydroxylation at C-24 of the side chain. The C-3 epimerization pathway, the newly discovered A-ring modification pathway, is initiated by epimerization of the hydroxyl group at C-3 of the A-ring. The end products of the metabolism of 1alpha,25(OH)2D3 through the C-24 oxidation and the C-3 epimerization pathways are calcitroic acid and 1alpha,25-dihydroxy-3-epi-vitamin-D3 respectively. During the past two decades, numerous noncalcemic analogs of 1alpha,25(OH)2D3 were synthesized. Several of the analogs have altered side chain structures and as a result some of these analogs have been shown to resist their metabolism through side chain modifications. For example, two of the analogs, namely, 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-D3] and 1alpha,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-D3], have been shown to resist their metabolism through the C-24 oxidation pathway. However, the possibility of the metabolism of these two analogs through the C-3 epimerization pathway has not been studied. Therefore, in our present study, we investigated the metabolism of these two analogs in rat osteosarcoma cells (UMR 106) which are known to express the C-3 epimerization pathway. The results of our study indicate that both analogs [1alpha,25(OH)2-16-ene-23-yne-D3 and 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3] are metabolized through the C-3 epimerization pathway in UMR 106 cells. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-D3 [1alpha,25(OH)2-16-ene-23-yne-3-epi-D3] was confirmed by GC/MS analysis and its comigration with synthetic 1alpha,25(OH)2-16-ene-23-yne-3-epi-D3 on both straight and reverse-phase HPLC systems. The identity of the C-3 epimer of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 [1alpha,25(OH)2-16-ene-23-yne-20-epi-3-epi-D3] was confirmed by GC/MS and 1H NMR analysis. Thus, we indicate that vitamin D analogs which resist their metabolism through the C-24 oxidation pathway, have the potential to be metabolized through the C-3 epimerization pathway. In our present study, we also noted that the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-20-epi-D3 is about 10 times greater than the rate of C-3 epimerization of 1alpha,25(OH)2-16-ene-23-yne-D3. Thus, we indicate for the first time that certain structural modifications of the side chain such as 20-epi modification can alter significantly the rate of C-3 epimerization of vitamin D compounds.

Animals↗

PubMed2000

Conceptually new 20-epi-22-oxa sulfone analogues of the hormone 1alpha,25-dihydroxyvitamin D(3): synthesis and biological evaluation.

New C,D-ring side-chain-modified sulfone 4a, with natural 1alpha, 3beta-hydroxyl groups but lacking the 25-hydroxyl group characteristic of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1), has been prepared and characterized. Novel synthetic features include: (1) chemoselective oxidation of only a primary silyl ether in a primary-secondary bis-silyl ether intermediate and (2) smooth reductive etherification without interference by a neighboring sulfonyl group. Sulfone 4a, but not its 1beta, 3alpha-diastereomer 4b, is powerfully antiproliferative and transcriptionally active in vitro but desirably noncalcemic in vivo. Although sulfone 4a, designed to resemble Leo Pharmaceutical Co.'s KH-1060 (3), is recognized by catabolic enzymes, the selective biological profile of sulfone 4a is likely not due to its metabolites that are formed in only minor amounts.

Animals↗

PubMed2000

Synthesis and biological activities of the two C(23) epimers of 1alpha,23,25-trihydroxy-24-oxo-19-nor-vitamin D(3): novel analogs of 1alpha,23(S),25-trihydroxy-24-oxo-vitamin D(3), a natural metabolite of 1alpha,25-dihydroxyvitamin D(3).

In a previous report, we indicated that 1alpha,23(S), 25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S), 25(OH)(3)-24-oxo-D(3)], a natural metabolite of 1alpha, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is almost equipotent to 1alpha,25(OH)(2)D(3) in suppressing parathyroid hormone (PTH) secretion (Lee et al., 1997. Biochemistry 36, 9429-9437). Also, 1alpha,23(S),25(OH)(3)-24-oxo-D(3) has been shown to possess only weak in vivo calcemic actions. Thus, vitamin D(3) analogs structurally related to 1alpha,23(S),25(OH)(3)-24-oxo-D(3) may have therapeutic value. Furthermore, biologic activity studies of various synthetic analogs of 1alpha,25(OH)(2)D(3) showed that the removal of carbon-19 (C-19) reduces the calcemic activity of 1alpha, 25(OH)(2)D(3.) Therefore, in an attempt to produce vitamin D(3) analogs with a better therapeutic index, we synthesized C(23) epimers of 1alpha,23,25(OH)(3)-24-oxo-19-nor-vitamin D(3) [1alpha,23, 25(OH)(3)-24-oxo-19-nor-D(3)]. The two epimers were compared to 1alpha,25(OH)(2)-19-nor-D(3) and 1alpha,25(OH)(2)D(3) in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) was as potent as 1alpha, 25(OH)(2)-19-nor-D(3) but was less potent than 1alpha,25(OH)(2)D(3). In the same assay 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) exhibited greater potency than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) was less potent than 1alpha,25(OH)(2)-19-nor-D(3) but was equipotent to 1alpha, 25(OH)(2)D(3). More importantly, in the same assays, 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was more potent than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) and was equipotent to 1alpha, 25(OH)(2)-19-nor-D(3). Also, the vitamin D receptor-mediated transcriptional activity of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was almost equal to that of 1alpha, 25(OH)(2)-19-nor-D(3), but higher than that of 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). This finding explains in part the greater in vitro biologic activities of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3). In summary, our results indicate that 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) and to a lesser extent 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) are potent 19-nor vitamin D(3) analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal growth and induce differentiation of HL-60 cells in vitro.

Animals↗

PubMed2000

Arthrobacter flavus sp. nov., a psychrophilic bacterium isolated from a pond in McMurdo Dry Valley, Antarctica.

CMS 19YT, a psychrophilic bacterium, was isolated from a cyanobacterial mat sample from a pond in Antarctica and was characterized taxonomically. The bacterium was aerobic, gram-positive, non-spore-forming, non-motile, exhibited a rod-coccus growth cycle and produced a yellow pigment that was insoluble in water but soluble in methanol. No growth factors were required and it was able to grow between 5 and 30 degrees C, between pH 6 and pH 9 and tolerated up to 11.5% NaCl. The cell wall peptidoglycan was Lys-Thr-Ala3 (the A3alpha variant) and the major menaquinone was MK-9(H2). The G+C content of the DNA was 64+/-2 mol%. The 16S rDNA analysis indicated that CMS 19YT is closely related to group I Arthrobacter species and showed highest sequence similarity (97.91%) with Arthrobacter agilis. Furthermore, DNA-DNA. hybridization studies also indicated 77% homology between CMS 19YT and A. agilis. It differed from A. agilis, however, in that it was psychrophilic, non-motile, yellow in colour, exhibited a rod-coccus growth cycle, had a higher degree of tolerance to NaCl and was oxidase- and urease-negative and lipase-positive. In addition, it had a distinct fatty acid composition compared to that of A. agilis: the predominant fatty acids were C15:0, anteiso-C15:0, C16:0, iso-C16:0, C17:0, anteiso-C17:0 and C18:0. It is proposed, therefore, that CMS 19YT should be placed in the genus Arthrobacter as a new species, i.e. Arthrobacter flavus sp. nov. The type strain of A. flavus is CMS 19YT (= MTCC 3476T).

Antarctic Regions↗

PubMed2000

Metabolism of [3alpha-3H] 25-hydroxyvitamin D2 in kidneys isolated from normal and vitamin D2-intoxicated rats.

With the availability of A-ring labelled 25OHD2, [3alpha-3H] 25OHD2, we have performed the present study to examine the metabolism of 25OHD2 using physiological substrate concentrations in perfused kidneys isolated from both normal and vitamin D2-intoxicated rats. Our results indicate that [3alpha-3H] 25OHD2 is metabolized into both 24(S),25,28-trihydroxyvitamin D2 [24(S),25,28(OH)3D2] and 24(R),25,26-trihydroxyvitamin D2 [24(R), 25,26(OH)3D2], and the amounts of these two metabolites produced in the kidney of vitamin D2-intoxicated rat were about 3-5 times higher than those produced in the kidney of normal rat. Similar results were also obtained with rat kidney homogenates incubated with [3alpha-3H] 25OHD2. Furthermore, we noted that the production of both 24(S),25,28(OH)3D2 and 24(R),25,26(OH)3D2 in the kidney homogenates of vitamin D2-intoxicated rats increased with the time of incubation and then subsequently decreased. The decrease in both 24(S),25,28(OH)3D2 and 24(R),25,26(OH)3D2 coincided with an increase in the fraction of total radioactivity distributed in the aqueous phase of the kidney homogenates. This finding suggested the possibility of further metabolism of 24(S),25,28(OH)3D2 and 24(R), 25,26(OH)3D2 into polar water-soluble metabolite(s). We then measured the radioactivity in the aqueous phase of kidney homogenates of both normal and vitamin D2-intoxicated rats incubated with [3alpha-3H] 25OHD2. It was noted that the amount of radioactivity in the aqueous phase of kidney homogenates of vitamin D2-intoxicated rats is higher than that present in the aqueous phase of kidney homogenates of normal rats. Thus, our study provides evidence for the first time for the formation of both 24(S),25,28(OH)3D2 and 24(R),25, 26(OH)3D2 under physiological conditions, and the possibility of their further metabolism into as yet unidentified polar water-soluble metabolite(s). As the formation of all these metabolites is increased in the kidney of vitamin D2-intoxicated rats when compared to normal rats, it appears that the increased rate of metabolism of 25OHD2 during hypervitaminosis D2 plays a significant role in the deactivation of 25OHD2.

25-Hydroxyvitamin D 2↗