Novel [2Fe-2S]-type redox center C in SdhC of archaeal respiratory complex II from Sulfolobus tokodaii strain 7

The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously in Escherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S]2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaii respiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms.     

Truncation of CDK5 activator p35 induces intensive phosphorylation of Ser202/Thr205 of human tau

Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer's disease. CDK5 is a kinase that phosphorylates the tau protein, and its endogenous activator, p35, regulates its activity. Recently, calpain was found to digest p35 to its truncated product, p25. Several lines of evidence suggest that p25-CDK5 has much more powerful kinase activity and that it may cause abnormal hyperphosphorylation of tau. In this study, we have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins. Although the kinase activity of CDK5 in phosphorylating tau was significantly higher in the presence of p25, the affinity of CDK5 for tau was not different. Phosphopeptide mapping revealed enhanced phosphorylation of Ser(202)/Thr(205) residues by p25-CDK5 (amino acid residues of tau are numbered according to the longest isoform of human tau). These results suggest that cleavage of p35 to p25 greatly enhances the kinase activity of CDK5 and increases the phosphorylation of Ser(202)/Thr(205). Considering the fact that phosphorylation of Ser(202)/Thr(205) antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease.     

Endosomal/lysosomal targeting of a single helper T-cell epitope of an intracellular bacterium by DNA immunisation induces a specific T-cell subset and partial protective immunity in vivo

We evaluated here the effect of the intracellular targeting of a helper T-cell (Th) epitope, literiolysin O 215-226 derived from Listeria monocytogenes, on induction of a specific Th by gene gun immunisation. Immunisation of C3H/He mice with pE215LAMP plasmid encoding the Th epitope fused with the endosomal/lysosomal targeting signal of lysosome-associated membrane protein (LAMP)-1 gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pE215LAMP-immunised mice showed moderate, but significant protective immunity against listerial challenge. These results suggest that the intracellular targeting of a Th epitope to endosomal/lysosomal compartments by DNA immunisation is useful for eliciting a specific Th subset in vivo.     

Synthesis and biological activities of fluorinated chalcone derivatives

We have designed and synthesized new 5-lipoxygenase inhibitors, fluorinated 3,4-dihydroxychalcones, and evaluated their biological activities with respect to antiperoxidation activity and in vitro antitumor activities. All fluorinated chalcones tested showed 5-lipoxygenase inhibition on rat basophilic leukemia-1 (RBL-1) cells and inhibitory action on Fe(3+)-ADP induced NADPH-dependent lipid peroxidation in rat liver microsomes. The potencies were comparable or better to that of the lead 3,4-dihydroxychalcone. 6-Fluoro-3,4-dihydroxy-2',4'-dimethoxy chalcone (7) was the most effective compound in the in vitro assay using a human cancer cell line panel (HCC panel) consisting of 39 systems.     

Inhibition of luteinizing hormone receptor expression during the development of caprine corpora lutea by administration of gonadotropin-releasing hormone antagonist

The present study was conducted to examine effects of a potent GnRH antagonist (GA), which suppresses release of luteinizing hormone (LH), on LH receptor expression during the development of the caprine corpus luteum (CL). Goats were divided into control and GA-treated groups. The goats were treated with saline or GA (50 microg/kg, s.c.) on days 0 (day of ovulation), 4 and 8 (control only), and CL collected on a subset of goats (n = 3 for each day) on days 0 (no saline), 4, 8, or 14 (control only). Ribonuclease protection assay and [(125)I]-hCG binding assay were performed to quantitate mRNA and protein of the LH receptor in the CL, respectively. On day 4, CL weight, levels of LH receptor mRNA and protein in the GA-treated group were similar to those of the control group. By day 8, CL weight and levels of LH receptor mRNA and protein in the GA-treated group were reduced relative to those of the control group (P < 0.05). There was no difference of affinity of the LH receptor between both groups on day 8. These results suggest that the treatment with GA inhibits gene and protein expressions of the LH receptor during the development of CL in the goat, and thus, support an idea that endogenous LH participates in the increase of its own receptor.     

Improved background correction for spotted DNA microarrays.

Most microarray scanning software for glass spotted arrays provides estimates for the intensity for the "foreground" and "background" of two channels for every spot. The common approach in further analyzing such data is to first subtract the background from the foreground for each channel and to use the ratio of these two results as the estimate of the expression level. The resulting ratios are, after possible averaging over replicates, the usual inputs for further data analysis, such as clustering. If, with this background correction procedure, the foreground intensity was smaller than the background intensity for a channel, that spot (on that array) yields no usable data. In this paper it is argued that this preprocessing leads to estimates of the expression that have a much larger variance than needed when the expression levels are low.     

Golgi-associated filament networks in duct epithelial cells of rabbit submandibular glands: immunohistochemical light and electron microscopic studies

The duct epithelial cells of rabbit submandibular glands expressed keratin 8, keratin 14, and actin in the supranuclear region, and these cytoskeletal proteins formed ring structures, approximately 1-2.5 microm in diameter. Ultrastructurally, these ring structures were observed as a 'Golgi-associated filament network' surrounding Golgi apparatuses. Double immunofluorescent studies showed that keratin 8 and keratin 14 formed keratin 8/14 filaments, and that these filaments and actin filaments colocalized as components of the Golgi-associated filament network. Our studies suggested that the Golgi-associated filament network maintains the complex structure and location of the Golgi apparatus of the duct epithelium of rabbit submandibular glands. Tubulin was distributed diffusely throughout the cytoplasm of columnar cells, but no special relationship was found between tubulin and the Golgi apparatus.     

Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase

Isoflavonoids are distributed predominantly in leguminous plants, and play pivotal roles in the interaction of host plants with biological environments. Isoflavones in the diet also have beneficial effects on human health as phytoestrogens. The isoflavonoid skeleton is constructed by the CYP93C subfamily of cytochrome P450s in plant cells. The reaction consists of hydroxylation of the flavanone molecule at C-2 and an intramolecular 1,2-aryl migration from C-2 to C-3 to yield 2-hydroxyisoflavanone. In this study, with the aid of alignment of amino acid sequences of CYP93 family P450s and a computer-generated putative stereo structure of the protein, candidates for key amino acid residues in CYP93C2 responsible for the unique aryl migration in 2-hydroxyisoflavanone synthase reaction were identified. Microsomes of recombinant yeast cells expressing mutant proteins of CYP93C2 were prepared, and their catalytic activities tested. The reaction with the mutant in which Ser 310 in the centre of the I-helix was converted to Thr yielded increased formation of 3-hydroxyflavanone, a by-product of the 2-hydroxyisoflavanone synthase reaction, in addition to the major isoflavonoid product. More dramatically, the mutant in which Lys 375 in the end of beta-sheet 1-4 was replaced with Thr produced only 3-hydroxyflavanone and did not yield the isoflavonoid any longer. The roles of these amino acid residues in the catalysis and evolution of isoflavonoid biosynthesis are discussed.     

Formation of thomasidioic acid from dehydrosinapinic acid dilactone under neutral conditions, and a remaining inhibitory activity against peroxynitrite-mediated protein nitration

Dehydrosinapinic acid dilactone (1) was converted to thomasidioic acid (3) and (E,E)-2,3-bis(3,5-dimethoxy-4-hydroxybenzylidene)succinic acid (4) via an alpha,beta-unsaturated gamma-lactone type dimer (2) in phosphate buffer (pH 7.4). A study of the reaction mechanism was accomplished by observing the reaction of methyl ester of 2. The lignans (3, 4) were also prevented the peroxynitrite-mediated protein nitration.     

Targeted gene therapy for rat glomerulonephritis using HVJ-immunoliposomes

Background

Kidney targeted gene transfer has been attempted by many researchers over the last 10 years; however, unfortunately, no reliable technique for gene transfer to the kidney has been established. At experimental level several in vivo gene transfer methods have been reported.

Methods

We were the first to report successful in vivo gene transfer into the kidney using the HVJ‐liposome method. Since then, this method has been modified to achieve highly efficient gene transfer. In this study, we have developed a renal glomerulus‐specific gene transfer method using HVJ‐liposomes with anti‐Thy 1 antibody, OX‐7.

Results

Following systemic delivery of fluoroisothiocyanate (FITC)‐labeled oligodeoxynucleotides (ODN) by HVJ‐liposomes coupled with OX‐7, we observed fluorescence in renal glomeruli from 2 h post‐administration. To examine the efficacy of this delivery system, NF‐κB or scrambled (SD) decoy ODN was administered by HVJ‐liposomes coupled with OX‐7 into a crescent glomerulonephritis, anti‐g lomerular b asement m embrane (GBM) model. Animals given SD decoy ODN developed severe glomerulonephritis by day 7 with heavy albuminuria, glomerular crescent formation and up‐regulated renal expression of IL‐1β and ICAM‐1. In contrast, NF‐κB decoy ODN treatment substantially inhibited the disease with a reduction in alubuminuria, histological damage and the renal expression of inflammatory cytokines.

Conclusions

This study has demonstrated that systemic delivery of HVJ‐liposomes coupled with OX‐7 results in efficient ODN transfer in rat glomeruli. NF‐κB, but not SD decoy ODN administered systemically via HVJ‐liposomes complexed with OX‐7 showed clear therapeutic potential for glomerulonephritis. This novel ODN transfer method combined with decoy strategy has the potential to lead to the establishment of a new therapeutic approach to glomerular diseases. Copyright © 2002 John Wiley & Sons, Ltd.