Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

High-level expression of naked DNA delivered to rat liver via tail vein injection

Background

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Methods

We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.

Results

We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.

Conclusions

These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.

     

Beraprost sodium,a prostaglandin I2 analogue,protects against advanced gycation end products-induced injury in cultured retinal pericytes

BACKGROUND: Beraprost sodium, a prostaglandin I2 analogue, has been recently reported to exhibit beneficial effects on atherosclerosis in patients with diabetes. However, effects of beraprost sodium on microvascular injury in diabetes remain to be elucidated. We have previously shown that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, caused pericyte apoptosis, thus being involved in the pathogenesis of the early phase of diabetic retinopathy. In this study, we examined whether beraprost sodium can protect against AGE-induced cytotoxicity in cultured retinal pericytes. MATERIALS AND METHODS: Intracellular formation of reactive oxygen species (ROS) was detected using a fluorescent probe. DNA synthesis was determined by measuring [3H]thymidine incorporation into cells. Apoptosis was determined by DNA fragmentations, which were quantitatively measured in an enzyme-linked immunosorbent assay. RESULTS: Beraprost sodium or forskolin, a stimulator of adenylate cyclase, was found to significantly inhibit AGE-induced ROS generation and the subsequent decrease in DNA synthesis in pericytes. Both treatments significantly prevented AGE-induced apoptotic cell death in pericytes. Furthermore, beraprost sodium was found to down-regulate AGE receptor mRNA levels in pericytes. CONCLUSION: The results demonstrated that cyclic AMP-elevating agents such as beraprost sodium and forskolin protected retinal pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that beraprost sodium may have therapeutic potentials in treatment of patients with early diabetic retinopathy.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

crinkle,a novel symbiotic mutant that affects the infection thread growth and alters the root hair,trichome, and seed development in Lotus japonicus

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.     

Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.