Development of LOX-1 Antibody Modified Immuno-liposomes as Drug Carriers to Macrophages in Atherosclerotic Lesions.

We developed a drug delivery system for atherosclerotic lesions using immuno-liposomes. We focused on enhancing the delivery efficiency of the liposomes to macrophages in atherosclerotic lesions by antibody modification of lectinlike oxidized low-density lipoproteins (LDL) receptor 1 (LOX-1). The cellular accumulation of the liposomes in foam cells induced by oxidized LDL (oxLDL) in Raw264 mouse macrophages was evaluated. The cellular accumulation of LOX-1 antibody modified liposomes in oxLDL-induced foam cells and untreated Raw264 cells was significantly higher compared with that of unmodified liposomes. The liposomes were also administered intravenously to Apoeshl mice as an atherosclerosis model. Frozen sections were prepared from the mouse aortas and observed by confocal laser microscopy. The distribution of LOX-1 antibody modified liposomes in the atherosclerotic regions of Apoeshl mice was significantly greater compared with that of unmodified liposomes. The results suggest that LOX-1 antibody modified liposomes can target foam cells in atherosclerotic lesions, providing a potential route for delivering various drugs with pharmacological effects or detecting atherosclerotic foci for the diagnosis of atherosclerosis.

Screening of RAPD markers linked to the photoperiod-sensitivity gene in rice chromosome 6 using bulked segregant analysis.

Bulked segregant analysis was used to determine randomly amplified polymorphic DNA (RAPD) markers in a specific interval in the middle of chromosome 6 of rice for tagging the photoperiod sensitivity gene. Two pools of F2 individuals (japonica cv. Nipponbare and indica cv. Kasalath) were constructed according to the genotypes of three restriction fragment length polymorphism (RFLP) markers located at both ends and the middle of the targeted interval. Then another pair of pools were constructed based on the "graphical genotype," which was made with our high density linkage map. RAPD analysis was performed using these DNA pools as templates, and polymorphic fragments were detected and mapped. Using 80 primers, either singly or pairwise, we tested 2,404 primer pairs and established 14 markers tightly linked to the photoperiod sensitivity gene. The obtained RAPD markers were converted into sequence-tagged sites by cloning and sequencing of the polymorphic fragments and they can be used directly for construction of physical maps. This bulked segregant method can be applied for any species and any region of interest in which detailed linkage maps or physical maps are needed.

Mapping of sequence-tagged sites in rice by single strand conformation polymorphism.

The conditions for efficient single-strand conformation polymorphism (SSCP) detection were examined for its application to mapping of DNA regions in the rice genome. Temperature for electrophoresis and glycerol concentrations in gel affected SSCP patterns significantly. The optimal detection conditions for SSCP also depends on the nucleotide sequences of fragments analyzed. Fragments over 300 bp show complicated patterns depending on their nucleotide sequences and were not suitable for SSCP analysis. Seventy primer pairs were designed from the sequence data available to amplify DNA regions as sequence tagged sites (STSs), and 39 of these STSs were found to generate SSCP between japonica rice (Nipponbare) and indica rice (Kasalath) in at least one of the experimental conditions. The maps of DNA fragments amplified from 186 F2-plant DNAs with 17 primer pairs were successfully determined. This direct mapping method of the amplified DNA fragments with PCR is simple and quite sensitive, and can be used to set markers in the gap regions of a genetic linkage map.

Determination of RAPD markers in rice and their conversion into sequence tagged sites (STSs) and STS-specific primers.

We produced 102 randomly amplified polymorphic DNA (RAPD) markers mapped on all 12 chromosomes of rice using DNAs of cultivars Nipponbare (japonica) and Kasalath (indica) and of F2 population generated by a single cross of these parents. Sixty random primers 10 nucleotides long were used both singly and in random pairs and about 1,400 primer-pairs were tested. Using both agarose gel and polyacrylamide gel electrophoresis enabled us to detect polymorphisms appearing in the range from < 100 bp to 2 kb. The loci of the RAPD markers were determined onto the framework of our RFLP linkage map and some of these markers were mapped to regions with few markers. Out of the 102 RAPD markers, 20 STSs (sequence-tagged sites) and STS-specific primer pairs were determined by cloning, identifying and sequencing of the mapped polymorphic fragments.

Characterization and genetic mapping of simple sequence repeats in the rice genome.

We searched partial sequences of over 22,706 rice cDNA and 1220 genomic DNA clones to find and characterize simple sequence repeats (SSRs) in the rice genome. The most frequently found repeated SSR motif in both cDNA and genomic DNA sequences was d(CCG/CGG)n. The second most frequently found SSR was d(AG/CT)n. In contrast with mammalian genomes, in which d(AC/GT)n sequences are the most abundant, d(AG/GT)n sequences were not frequently observed in rice. Sequences containing d(CCG/CGG)n, d(AG/CT)n repeats, and other SSRs were chosen for polymorphism detection. It was predicted that 17 of 20 SSRs in cDNA sequences were located in 5'-untranslated regions near initiation codons. Twenty-two loci can be mapped on our RFLP linkage map by these SSRs. Six markers were tested with 16 japonica rice varieties as templates for PCR. Two markers exhibited amplified fragment length polymorphism among these rice varieties, implying that SSRs are polymorphic among rice varieties which have similar genetic backgrounds. Even these polymorphic SSRs are located within or around genes which code ubiquitous proteins.

Sequence of the Bacillus subtilis homolog of the Escherichia coli cell-division gene murG.

The Bacillus subtilis homology of the Escherichia coli murG gene [encoding UDP-N-acetylglucosamine:N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase] was cloned in E. coli K-12 and sequenced. The murG homolog encodes a protein of M(r) 39,936 [363 amino acid (aa) residues] of which 108 aa residues (29.8%) are identical with the E. coli murG product.

Antioxidant enzymes and lipoperoxide in blood in uremic children and adolescents.

To determine whether oxidant-antioxidant balance is altered in chronic renal failure, antioxidant enzymes and lipid peroxide in peripheral blood cells and lipid peroxide in plasma were measured. Nine children and adolescents maintained on hemodialysis (HD), 9 on continuous ambulatory peritoneal dialysis (CAPD), and 14 controls were studied. Lipid peroxide was assayed fluorimetrically as thiobarbituric acid-reactive substances, superoxide dismutases by radioimmunoassays. Both manganese and copper-zinc superoxide dismutases in lymphocytes and monocytes in the HD and CAPD patients, and manganese superoxide dismutase in polymorphs in the HD patients were higher than in the controls. Copper-zinc superoxide dismutase, glutathione peroxidase, and catalase in erythrocytes were unaltered. The lipid peroxide level in plasma in the dialyzed patients was increased, whereas those in polymorphs and lymphocytes were unaltered. Triglyceride and total cholesterol in plasma in the dialyzed patients were also increased. The plasma lipid peroxide in the patients correlated with the triglyceride and total cholesterol level. This is the first study in which manganese superoxide dismutase is measured in nucleated cells of the patients with chronic renal failure. The present results suggest that increased superoxide dismutases protect against oxidative stress induced by chronic renal failure in nucleated cells but in neither erythrocytes nor plasma.

Histopathological findings in proliferative membrane from a patient with sarcoid uveitis.

BACKGROUND: Sarcoid uveitis is occasionally accompanied by proliferative changes, such as retinal neovascularization and vitreous hemorrhage. Steroid administration, retinal photocoagulation, and vitrectomy may be indicated in such proliferative cases. CASE: A 19-year-old woman presented with proliferative sarcoid uveitis accompanied by recurrent vitreous hemorrhage. OBSERVATIONS: At the initial examination, bilateral vitreous opacity, retinal exudates, mild vitreous hemorrhage, retinal vasculitis, and neovascularization of the retina and optic disc were observed. Although prednisolone was administered and panretinal photocoagulation was performed several times, recurrent vitreous hemorrhage continued. Since the vitreous hemorrhage was not absorbed, pars plana vitrectomy and lensectomy were performed. After surgery, neovascularization and intraocular inflammation decreased, and the corrected visual acuity in the right eye improved to 20/50. Histopathologic analysis of the proliferative membrane removed during surgery revealed substantial neovascularization and numerous neutrophils in the vessels. CONCLUSIONS: Based on these findings, an inflammatory reaction as well as retinal ischemia were thought to be involved in the proliferative changes in this patient.

[A case of sarcoidosis with proliferative retinopathy].

A 19-year-old female manifested severe bilateral panuveitis with neovascularization in the iris, optic disc, and retina. Fluorescein fundus angiography showed dye leakage from the optic disc and retinal blood vessels, and a large non-perfused area was present in the peripheral retina of the right eye. Sarcoidosis was diagnosed histologically by conjunctival and skin biopsy. Although the patient was given a large dose of a corticosteroid systemically and received panretinal photocoagulation, a dense vitreous hemorrhage and cataract were apparent in the right eye. The right visual acuity decreased to hand motions. A pars plana lensectomy and vitrectomy were performed. After vitrectomy, inflammation and neovascularization regressed and the visual acuity improved to 20/100. Proliferative membrane obtained during vitrectomy was histopathologically studied by light and electron microscopy. Many new vessels containing neutrophils were observed. A direct effect of inflammation as well as ischemia in the retina may have been the stimulus for the proliferative changes.

High-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations determined in serum of newborns, infants, children, adolescents, and adults by use of a micromethod for combined precipitation ultracentrifugation.

Cholesterol (C) concentrations in the two major subfractions of high-density lipoproteins (HDL2-C and HDL3-C) in sera from both sexes, ages ranging from newborns to adults, were measured by use of a micromethod for combined precipitation-ultracentrifugation. Sera were obtained from 91 boys, 68 girls, 15 healthy men, and 14 women. The HDL2-C concentration was higher in women than in men; the HDL3-C concentration was similar in these two groups. This sex-related difference, generally seen in adults, was found to begin at ages 11-15 y. The value of HDL2-C in females increased with age in a stepwise manner, whereas that in males increased up to ages 6-10 y but tended to decline thereafter. The HDL3-C concentration was higher in the adults than in the children. This micromethod for separating operationally defined HDL subfractions is of value for lipoprotein research in children.

Bacillus subtilis spoVE gene is transcribed by sigma E-associated RNA polymerase.

Expression of the Bacillus subtilis sporulation gene spoVE was examined by runoff transcription assay with an RNA polymerase preparation obtained from vegetative and sporulating cells. Transcripts from tandem promoters (P1 and P2 promoters) located just upstream of the spoVE structure gene were detected. The transcription of spoVE initiated within an hour after the onset of sporulation and coincided with the presence of RNA polymerase associated with a 33-kDa protein. Amino acid sequence analysis of the 33-kDa protein revealed that it is a sigma factor, sigma E. Reconstitution analysis of sigma E purified from the sporulating cell extracts and vegetative core RNA polymerase showed that sigma E recognizes the P2 promoter. SpoVE protein could not be synthesized in the transcription-translation coupled system prepared from vegetative cells (M. Okamoto, S. Fukui, and Y. Kobayashi, Agric. Biol. Chem. 49:1077-1082, 1985). However, addition of sigma E-associated RNA polymerase to the coupled system restored SpoVE protein synthesis. These results indicate that spoVE expression in sporulating cells is controlled essentially by sigma E-associated RNA polymerase.

Isolation and characterization of rice phytochrome A mutants.

To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.

In vivo expression of the Bacillus subtilis spoVE gene.

In vivo expression of the Bacillus subtilis spoVE gene was studied by S1 nuclease mapping and spoVE gene fusion analysis. Transcription of spoVE is induced at about the second hour of sporulation from two closely spaced promoters designated P1 and P2. Examination of the precise transcription initiation site by high-resolution primer extension mapping indicated that the nucleotide sequences of the -10 and -35 regions of both P1 and P2 were similar to those of promoters recognized by E sigma E. Moreover, S1 nuclease mapping and translational spoVE-lacZ fusion studies with various spo mutants suggest that the expression of spoVE P2 requires the spoIIG gene product, sigma E. The sporulation of a wild-type strain was inhibited severely in the presence of a multicopy plasmid, pKBVE, carrying the spoVE promoter, indicating the possible titration of a transcriptional regulatory element(s).

Antioxidant enzyme status and lipid peroxidation in various tissues of diabetic and starved rats.

The effect of short term (2-wk) diabetes induced by streptozotocin and starvation (1-wk) on antioxidant enzymes and lipid peroxidation in the liver, kidney and heart of rats was investigated. The activity of mitochondrial oxidative markers was increased in diabetic liver and kidney, while the activity in tissues of starved rats tended to be decreased. Immunoreactive manganese superoxide dismutase was increased only in diabetic liver and was unchanged or decreased in the rest of the tissues. Glutathione peroxidase activity was increased in tissues of diabetic but not starved rats. The changes in copper-zinc superoxide dismutase and catalase in diabetic rats were similar to those in starved rats. In both groups, copper-zinc superoxide dismutase was decreased in liver, while catalase activity was decreased in liver and kidney, and increased in heart. The lipid peroxide level was increased in diabetic kidney and in the heart of starved rats, and decreased in the rest of the tissues. Insulin treatment in diabetic rats and refeeding in starved rats restored most of the abnormalities toward normal. These results suggest that accelerated mitochondrial oxidative metabolism not accompanied by induction of manganes superoxide dismutase results in oxidative injury in the hypertrophied kidney at an early stage of diabetes and possibly contributes to the development of nephropathy. Peroxidative myocardial damage in starved rat appears to be mediated by a catabolic process.

Concentration of lipid peroxide in serum lipoproteins of insulin-dependent diabetic children.

Lipids, apolipoproteins and lipid peroxide were measured in the sera of 29 children with insulin-dependent diabetes mellitus. Ten non-diabetic children served as controls. High-density lipoprotein (HDL) was separated by heparin-MnCl2 precipitation. Lipid peroxides in HDL and non-HDL fractions were estimated by fluorimetric measurement of thiobarbituric acid-reactive substances. The patients were normolipidemic, and their HDL-cholesterol was increased. Apo A1 level in the patients was similar to that in the controls, while levels of apo A2 and apo B were decreased in the patients. Concentrations of lipid peroxides in the whole serum and non-HDL were unaltered, while that in the HDL was higher in the patients than in the controls. Hemoglobin AIc in the patients correlated with the triglyceride and urinary excretion rate of N-acetylglucosaminidase (NAG). The NAG correlated with the triglycerides. The triglycerides correlated with the atherogenic index, apo B and total cholesterol. The lipid peroxides in the non HDL correlated with the triglyceride, atherogenic index, and NAG. That in the HDL correlated with the HDL-cholesterol, apo A1 and endogenous creatinine clearance, and inversely with the atherogenic index and apo B. Lipid peroxides in HDL and non-HDL appeared to play different physiological roles from each other, and they have provided evidence suggesting that diabetic microvascular injury is mediated by reactive oxygen species.

Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene.

The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice.

An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1, results in a defect in homologous chromosome pairing during meiosis.

To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis. The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene. The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae. Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues. In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages. The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1. The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene.

The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets.

The rice retrotransposon Tos17 is highly activated by tissue culture. To evaluate the impact of transposition of Tos17 on the rice genome and examine its utility for insertional mutagenesis, more than 100 sequences flanking newly transposed Tos17 copies were characterised. The 5-bp target-site duplications flanking Tos17 did not show any consensus sequence, and preferred nucleotides, A/T and G/C, were only found at the second and third nucleotides from both ends of the target site duplications, respectively, indicating that Tos17 has relatively low target-site specificity at the nucleotide sequence level. Integration targets were widely distributed over the chromosomes; however, preferential integration into the sucrose synthase 2 gene and into Tos17 itself was demonstrated by PCR screening using pooled DNA prepared from the mutant population. Hybridisation studies indicated that Tos17 preferentially integrates into low-copy-number regions of the genome. In agreement with this result, about 30% of flanking sequences examined showed significant homology to known genes. Taken together, these results show that Tos17 can have a significant impact on the rice genome and can be used as a tool for efficient insertional mutagenesis.

Sequence-tagged sites (STSs) as standard landmarkers in the rice genome.

Generating sequence-tagged sites (STSs) is a prerequisite to convert a genetic map to a physical map. With the help of sequence information from these STSs one can also isolate specific genes. For these purposes, we have designed PCR primer sets, of 20 bases each, by reference to sequences of restriction fragment length polymorphism (RFLP) landmarkers consisting of rice genomic clones. These markers were evenly distributed over the 12 chromosomes and were shown to be single copy by Southern-blot analysis. With improved PCR protocols, 63 standard STS landmarkers in the rice genome were generated. Similarity searches of all partial sequences of RFLP landmarkers by the FASTA algorithm showed that 2 of the 63 RFLP landmarkers, G357 and G385, contained part of the ORFs of aspartate aminotransferase and protein kinase, respectively.