Distribution and toxicity of mercury in rats after oral administration of mercury-contaminated whale red meat marketed for human consumption.

Toothed-whales and dolphins have been hunted for human consumption in Japan, and their muscles (red meats) are highly contaminated with mercury (Hg). We investigated the distribution and toxicity of Hg in rats after oral administration of Hg-contaminated whale red meat marketed for human consumption in Japan. Rats were orally administered the red meat homogenate for seven consecutive days (0.5 g red meat/kg-bw/day). The red meat administered to rats contained 81microg/g of total mercury (T-Hg) and 13.4 microg/g of methyl mercury (M-Hg). This dose corresponds to the human consumption of 210 g red meat/60 kg-bw/week, exceeding by about 29 times the provisional tolerable weekly intake of M-Hg at 1.6 microg/kg-bw/week set by JECFA [JECFA, 2003. Joint FAO/WHO expert committee on food additives. 61st meeting, Rome]. Twenty-four hours after the last administration, the distribution of T-Hg in rat organs and biochemical parameters in serum were analyzed. The administration of red meat significantly elevated T-Hg concentrations in the liver, kidney, erythrocytes, cerebral cortex and medulla oblongata from the control levels but did not elevate the T-Hg concentration in serum, showing the typical distribution pattern of M-Hg, not of inorganic Hg. The administration slightly but significantly increased GTP activity and P concentration and decreased BUN concentration in serum, although no abnormalities were observed in rat body weight gain and movement during the 7 days. The occasional consumption of red meat from small cetaceans, therefore, could pose a health problem for not only pregnant women but also for the general population.

Analysis of a mod B mutant in Dictyostelium discoideum using mRNA differential display.

Three differential cDNAs of Dictyostelium, not detected in the mod B mutant defective in O-glycosylation, were isolated by using an mRNA differential display. These cDNAs encode a protein tyrosine kinase, an adenylyl cyclase and a putative protein kinase C inhibitor whose expression is developmentally regulated.

A novel glycine-rich protein is associated with starch grain accumulation during anther development.

LIM14, originally identified as a lily gene associated with microsporogenesis, encodes a protein which has two distinct domains, one with glycine-serine repeats and the other with a hydrophobic signal peptide at the N-terminus. The putative LIM14 protein, however, is distinct from the glycine-rich cell wall proteins which have been described before. RNA analyses indicated that the LIM14 transcript is specifically detected in the anther from zygotene to young pollen stage. By using antibodies raised against recombinant LIM14 protein, we detected anther-specific 15 kDa protein. Immunofluorescence microscopy demonstrated that the LIM14 protein is associated with starch grains in the anther wall cells just prior to microspore mitosis and then accumulates at a higher level with the starch grains of immature pollen. We tagged LIM14 with the GUS and GFP reporter genes and introduced them into tobacco BY-2 cells. Analysis of the transformed cells revealed that the chimeric proteins are functional and specifically targeted to plastids. These results indicate that LIM14 is an anther-specific protein that may play a role in starch accumulation and amyloplast differentiation during anther development and pollen formation.

Large-scale sequencing of the rabbit corneal endothelial cDNA library.

PURPOSE: This study sought to identify novel or active genes in corneal endothelial cells with description of the gene-expression profile. METHODS: We performed the single-pass sequencing of 1,000 clones from a rabbit corneal endothelial cDNA library. Inserts of the library were amplified by polymerase chain reaction (PCR), sequenced, and compared with several databases. We used four database similarity search programs: FASTA, BLASTN, TBLASTX, and BLASTX. RESULTS: Of the sequences generated, 618 (61.8%) showed sequence homology with known genes, whereas 192 (19.2%) matched previous reported expression sequence tags (ESTs) and 174 (17.4%) showed no sequence similarity. Among the homologous clones to known genes are collagen type VIII, secreted protein acidic and rich in cysteine (SPARC), lysyl oxidase, phosphatidylcholine-2-acylhydrolase, and thrombospondin. Several matched ESTs, and no matched clones that showed high frequency were also detected. CONCLUSION: Large-scale sequencing can be useful in obtaining a profile of the active genes. Several ESTs showed relatively frequent expression, suggesting that these genes may have important functions in the corneal endothelium.

Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in rat retina.

We have cloned and characterized a new diacylglycerol kinase (DGK) isozyme which is expressed in the retina and the brain of rat. The cDNA contains an open reading frame of 567 amino acid residues with a predicted protein of 64 kDa and shows very high homology to human DGK epsilon. The new DGK isozyme contains two distinctive zinc-finger structures and a putative catalytic domain. This DGK expressed predominantly in the inner and outer nuclear layers of retina. This expression pattern is different from those of the previously cloned DGKs including the human DGK epsilon, suggesting that this DGK isozyme has potential importance in visual functions as was the case in Drosophila retinal cells.

Studies on cardiac ingredients of plants. XIII: Chemical modification of gitoxin to cardiotonic compounds without vascular effect.

Nitrated gitoxins (4) and bufotoxin homologues with various lengths of alkyl chain at C-3 of the steroid nucleus (10) were prepared from gitoxin (1). The pharmacological activities of the resulting compounds (4 and 10) were evaluated by measurement of inhibitory effect on NA+, K(+)-adenosine triphosphatase (ATPase) prepared from dog kidney, positive inotropic effect (PIE) on isolated guinea-pig papillary muscle preparations, and the effect on smooth muscle using the mesenteric artery from spontaneously hypertensive rats. Most of the compounds showed a smaller contractile effect on the arterial muscle. Among these compounds, gitoxin 3"-nitrate (4g) exhibited the most desirable biological activities, such as PIE comparable to that of 1, 1.25 times wider concentration-dependent range than 1, and lack of contractile activity on vascular muscle.

Plus/minus screening of rabbit corneal endothelial cDNA library.

Plus/minus screening of the rabbit corneal cDNA library was performed using corneal and iris RNA as probes. Thirteen clones were isolated: three ferritin H-chains, a NADH-ubiquinone oxidoreductase B22 subunit, an alpha 1 type VIII collagen, a 25 KDa FKBP-506 binding protein (FKBP25), a thrombospondin 2, and six unknown clones. Although proteins translocated from these isolated mRNA are not corneal specific, they play an important role in the cornea. None of the isolated known mRNAs maps to chromosome 1, 16, or 20. These clones, thrombospondin excepted, were not observed in the high frequency clones in the profile of the aortic endothelial cDNA library.

Recovery of alveolar bone by the guided bone regeneration technique.

Bone defects associated with the removal of endosteal implants often create challenging problems for clinicians. The guided bone regeneration (GBR) technique has been recently used in the treatment of such defects, and promising results have been obtained. This report describes the use of Gore-Tex tissue augmentation material (GTAM) membranes after the removal of Bioceram screw implants in two patients who wore the implants for 10 years and seven years, respectively. After removal, the residual bone defects were treated by the GBR technique. Bone regeneration was confirmed in the defects around the newly placed implants, and decreased bone mass was avoided in both patients. In case 1, a free gingival graft was used to increase the area of attached gingiva around the implants, since the status of peri-implant soft tissue is also crucial to the outcome of therapy. This technique was very effective in maintaining an adequate width of attached gingiva.

Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster.

The nucleotide sequence of a 25.7 kilobase Drosophila melanogaster genomic DNA segment containing a gene for a ryanodine receptor/calcium release channel homologue has been determined. Computer analysis and partial cDNA cloning revealed 26 exons comprising the protein-coding sequence in this gene. The predicted protein is homologous in amino acid sequence and shares characteristic structural features with the mammalian ryanodine receptors. In blot hybridization analysis, a approximately 16 kilobase RNA species was identified abundantly in a 6-12 h embryo as the transcript from this gene. In situ hybridization to polytene chromosomes indicated that this gene locates at band position 44F on the second chromosome.

Drosophila retinal degeneration A gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc-finger motifs and ankyrin repeats.

The Drosophila visual mutant, carrying the retinal degeneration A gene (rdgA), has photoreceptor cells that degenerate within a week after eclosion. Morphological studies suggested that this mutant harbors abnormalities in membrane turnover of the photoreceptor cells. Biochemically, the rdgA mutant lacks an eye-specific and membrane-associated diacylglycerol kinase (DGK; EC 2.7.1.107) activity in a gene-dosage-dependent manner, suggesting that rdgA gene encodes a DGK. We report the molecular cloning and characterization of a DGK gene, which maps to the rdgA locus. This gene, designated as DGK2, has a single open reading frame that encodes 1454 amino acids. Like porcine DGK, DGK2 has two cysteine-rich zinc-finger motifs as well as a DGK catalytic domain. The DGK2 protein contains four ankyrin-like repeats at the C-terminal region, suggesting that DGK2 is likely anchored to the membrane or cytoskeleton. Northern blot analysis and tissue in situ hybridization to adult sections revealed that DGK2 is expressed exclusively in the adult retina and that the amount of its mRNA is reduced in some of the rdgA mutant alleles. Furthermore, in two rdgA alleles, rdgA1 and rdgA2, nonsense and missense mutations occur within their DGK2 gene, respectively. Thus, we conclude that rdgA encodes an eye-specific DGK, the absence of which leads to rhabdomere degeneration due to defective phospholipid turnover.

Plant saponins can affect DNA recombination in cultured mammalian cells.

Some plant extracts and products are known to affect mammalian cells, tissues and organisms as they contain a toxic substance or a metabolic stimulant. Our biochemical investigations revealed that some plant saponins can increase the cellular DNA repair activity and the general recombinase activity measured by in vitro assay (1). In the experiments described here, HeLa cells were cultured for several days with plant saponins or flavonoids and analyzed to measure i) recombination activity of the cell extract by induction of Tcr colonies from two mutant DNAs (mutants 1 and 2, which are both tetracycline sensitive) after transformation into E. coli recA-, and ii) repair synthesis of nuclear DNA followed by incorporation of 3H-thymidine. Saikosaponins a, b1, d, ginsenosides Rb1, Re, Rh and flavonoid baicalin caused a significant stimulation of intermolecular recombination. It is worth noting that none of the plant saponins and flavonoids had any inhibitory or toxic effect at concentrations less than 25 micrograms/ml in the culture media.