In Vivo Metabolism of the Vitamin D Analog, 22-Oxacalcitriol: Evidence for Side-chain Truncation and 17-Hydroxylation

After intravenous administration of the vitamin D₃ analog, 22-oxacalcitriol (OCT), to normal rats plasma metabolites were investigated by HPLC, GC-MS and LC-MS. Five side-chain oxidation metabolites, 24R(OH)OCT, 24S(OH)OCT, (25R)-26(OH)OCT, (25S)-26(OH)OCT and 24oxoOCT, were identified by comparison with the corresponding synthetic compounds. These side-chain oxidation metabolites were similar to those of calcitriol [1,25(OH)₂ vitamin D₃] described previously. Besides these five metabolites, two unique side-chain cleavage metabolites, 20S(OH)-hexanor-OCT and 17,20S(OH)₂-hexanor-OCT, were identified as main metabolites in plasma by GC-MS and LC-MS using a specific chemical reaction. Our studies suggest that OCT is extensively metabolized and circulates in blood as a number of metabolites as well as unchanged OCT. This metabolism includes both unique pathways of C₂₃-O₂₂ cleavage and 17-hydroxylation, in addition to the side-chain oxidation metabolites similar to those of 1,25-(OH)₂D₃.     

Contribution of micro-organisms to the carbon dynamics in black spruce (Picea mariana) forest soil in Canada

In order to clarify the role of micro-organisms in the carbon cycle of the boreal forest ecosystem, the vertical distribution of soil carbon, soil microbial biomass and respiratory activity was studied in a black spruce forest near Candle Lake in Saskatchewan, Canada. The total amount of carbon contained in moss and soil layers (to the depth of 50cm beneath the mineral soil surface) was 7.2kgm^–2, about 47% of which was in the L and FH horizons of the soil. Soil microbial biomass per dry weight of soil was largest in the L horizon, while the biomass per ground area was largest in the FH horizon. Soil respiration rate, measured using a portable infrared gas analyzer, was highest in the FH horizon, exceeding 50% of the total soil respiration. Low but significant CO₂ emission was detected even in deeper soil horizon (E horizon). We also examined the respiration rate of cut roots and the effect of root excision on respiration. The contribution of root respiration to total soil respiration, calculated from root biomass and respiration rate of cut roots, was about 54%. The amount of carbon evolved through microbial respiration during the snow-free season (June–October) was estimated as 221gCm^–2. Micro-organisms in the L horizon showed high respiratory activity as compared with those in deeper soil horizons.     

Direct Angiotensin II Type 2 Receptor Stimulation Ameliorates Insulin Resistance in Type 2 Diabetes Mice with PPARγ Activation

Objectives

The role of angiotensin II type 2 (AT₂) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT₂ receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue.

Methods

T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[³H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined.

Results

Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored β cell damage in diabetic pancreatic tissue.

Conclusion

The present study demonstrated that direct AT₂ receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic β cells.     

TX-1123: an antitumor 2-hydroxyarylidene-4-cyclopentene-1,3-dione as a protein tyrosine kinase inhibitor having low mitochondrial toxicity

A series of 2-hydroxyarylidene-4-cyclopentene-1,3-diones were designed, synthesized, and evaluated with respect to protein tyrosine kinase (PTK) inhibition, mitochondrial toxicity, and antitumor activity. Our results show that the cyclopentenedione-derived TX-1123 is a more potent antitumor tyrphostin and also shows lower mitochondrial toxicity than the malononitrile-derived AG17, a potent antitumor tyrphostin. The O-methylation product of TX-1123 (TX-1925) retained its tyrphostin-like properties, including mitochondrial toxicity and antitumor activities. However, the methylation product of AG17 (TX-1927) retained its tyrphostin-like antitumor activities, but lost its mitochondrial toxicity. Our comprehensive evaluation of these agents with respect to protein tyrosine kinase inhibition, mitochondrial inhibition, antitumor activity, and hepatotoxicity demonstrates that PTK inhibitors TX-1123 and TX-1925 are more promising candidates for antitumor agents than tyrphostin AG17.     

Coexistence of fast-type and slow-type C-proteins in neonatal chicken breast muscle

Two different C-protein variants which selectively react with either monoclonal anti-fast C-protein antibody (MF-1) or monoclonal anti-slow C-protein antibody (ALD-66) were separated from neonatal chicken pectoralis muscle by hydroxylapatite column chromatography. Myofibrils isolated from the neonatal chicken muscle reacted with both monoclonal antibodies as examined by an indirect immunofluorescence method. These observations strongly indicate that both fast-type and slow-type C-proteins are expressed in the neonatal chicken skeletal muscle. Both of them are intermingled and assembled in the same myofibrils.     

Changes in Organelle Movement in the Nuclear Region during the Cell Cycle of Adiantum Protonema

Movements of organelles in the nuclear region as the cell cycleprogresses in single-celled protonemata of Adiantum capillus-veneriswere examined by digital image processing techniques and microscopyof particle movement. Organelles in the nuclear region werenot very crowded and moving directionally along the longitudinalaxis of the filamentous cell in the G₁ and S phases. They beganto gather and accumulate in the nuclear region in early G₂ phase,after which directional movement changed to undirectional Brownianmotion-like movement in late G₂ phase. Movement of organelleslocated on the lateral surface of the nucleus slowed after premitoticpositioning of nucleus and lasted until the nucleolus disappeared.Movement of organelles in the cytoplasm surrounding the nucleoplasmresumed just after the nucleolus disappeared, whereas organelleslocated in the outer regions of the apical and basal surfacesof the nucleus moved rapidly during prophase but did not moveduring metaphase, movement being resumed after chromosome separation.Thus, organelle movement in the nuclear region showed temporaland spatial change during the cell cycle. (Received August 24, 1983; Accepted December 28, 1983)     

Phytochrome Control of Its Own Synthesis in Pisum sativum

An analysis of phytochrome synthesis in Pisum seedlings by measuringthe activity of polysomal polyadenylated RNA (poly-A⁺-RNA) codingfor phytochrome apoprotein showed phytochrome control of itsown synthesis; brief red-light irradiation of pea seedlingsinhibited the activity of the RNA, and the red-light effectwas red/far-red reversible. ⁴ Permanent address: Biology Department, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 13, 1984; Accepted September 17, 1984)     

Photomovement in Dunaliella salina: Fluence rate-response curves and action spectra

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450–460 nm. These action spectra are not compatible with the hypothesis that flavoproteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak a 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.Diagrams of the optical set-ups used for measuring the responses at the single-cell level and of the plans for building the phototaxometer described in this paper are available to the interested readerWe thank Mr. M. Kubota for a tremendous amount of technical assistance and Mr. R. Nagy for building the phototaxometer. We thank T. Kondo, Professor H. Imaseki and the members of the Laboratory of Biological Regulation, NIBB, for their help and support in various aspects of this research. This research was supported, in part, from grants from the Okazaki Large Spectrograph (Project Nos. 86-535, 87-518, 88-523), the Japanese Society for the Promotion of Science, and the College of Agriculture and Life Sciences at Cornell University to R. W.