Differential behaviour of 25-hydroxyvitamin D3 24-hydroxylase and 1-hydroxylase in response to protein synthesis inhibitors and cytochrome P-450 inhibitors

To characterize 25-hydroxyvitamin D3 24-hydroxylase and 25-hydroxyvitamin D3 1-hydroxylase, the activities of the two enzymes were measured in the presence of two types of inhibitors. The effect of protein synthesis inhibitors on 25-hydroxyvitamin D3-stimulated 24-hydroxylase activity in 1-hydroxylating rat kidneys perfused in vitro was tested. Actinomycin D (4 microM) and cycloheximide (10 microM) each abolished 25-hydroxyvitamin D3 24-hydroxylase synthesis when added at the start of perfusion but not when added 4 h later; they did not affect 25-hydroxyvitamin D3 1-hydroxylase activity. The effects of cytochrome P-450 inhibitors on the two enzyme activities were then studied in vivo. Metyrapone and SKF-525A (50 mg/kg body weight) each inhibited 25-hydroxyvitamin D3 24-hydroxylase at 6 and 24 h; in contrast 1-hydroxylase increased and was 5 times the control value at 24 h. Finally, the in vitro effects of six cytochrome P-450 inhibitors at concentrations ranging from 10(-7) to 10(-3) M on enzyme activities in renal mitochondrial preparations were compared. Both enzymes were inhibited by all of the inhibitors, but inhibition of 25-hydroxyvitamin D3 24-hydroxylase was consistently greater than that of 25-hydroxyvitamin D3 1-hydroxylase. These studies demonstrate that 24-hydroxylation and 1-hydroxylation respond differently to protein synthesis inhibitors and to cytochrome P-450 inhibitors. The findings are consistent with the hypothesis that the two enzyme activities are associated with different cytochrome P-450 moieties.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150m and cultured for 40days. Up to 100m MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 m MJ peaked after 10days at 48mgg^–1 dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Revisions requested; 2 July 2004; Revisions received 30 June 2004; 3 September 2004     

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50–400 M); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 M MJ. In the second experiment, 200 M MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.     

Origin of insulin secreted from islet-like cell clusters derived from murine embryonic stem cells

Islet-like cell clusters (ILCCs) were derived from murine embryonic stem cells using a slightly modified version of the protocol originally described by Lumelsky et al. in 2001. Analysis with enzyme-linked immunosorbent assays (ELISAs) that distinguish human from murine insulin demonstrated that insulin released from these ILCCs, upon initial in vitro glucose challenge, was of non-murine origin and in fact corresponded to the species of insulin, human or bovine, that had been added to the culture media used to derive ILCCs. This finding convincingly supports the hypothesis that ILCCs are not synthesizing insulin de novo, but rather simply regurgitating insulin taken up during tissue culture. In further experiments, ILCCs were derived in media in which insulin had been replaced by IGF-I with which it shares a common signaling pathway. These ILCCs failed to release any detectable insulin. In contrast, ILCCs produced by various protocols stained positive (dithizone and immunoselective antibodies) for intracellular insulin and, in some cases, C-peptide. Despite the presence of at least some level of de novo, synthesized insulin in ILCCs, the majority of insulin released by ILCCs was sequestered from the exogenous medium.     

Quality assessment of tandem mass spectra based on cumulative intensity normalization

A large proportion of MS/MS spectral analyses do not result in significant matches because their spectral quality is too poor to produce meaningful identification. Throughput of peptide identification can be greatly improved, if one can filter out, in advance, spectra that would lead to wrong identification. We introduce here an innovative approach to assess spectral quality utilizing a new spectral feature called Xrea, based on cumulative intensity normalization.     

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P₀. Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P₀ and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK _c 1) gene was increased specifically in the incompatible interaction with TMV-P₀. The expression of CaPK _c 1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK _c 1 responds to several defense-related abiotic stresses in addition to TMV infection. The nucleotide sequence data reported in this paper were submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number DQ114474.