Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.     

Improved immunocytochemical detection of daunomycin

Improved immunocytochemical (ICC) detection of the anthracycline anticancer antibiotic daunomycin (DM) has been achieved by use of hydrogen peroxide oxidation prior to ICC staining for DM. The new method greatly enhanced the localization of DM accumulation in cardiac, smooth and skeletal muscle of rats after a single i.v. dose of the drug. DM accumulated in the nuclei as well as in the sarcoplasm, where it occurred in the form of small granules, which were particularly evident in cardiac muscle cells. The distribution of the granules coincided with that of mitochondria. Uptake of DM in nuclei and mitochondria of heart muscle cells may help to improve our understanding of the cardiac toxicity of DM and related anthracyclin antibiotics. A number of ELISA tests were carried out in order to elucidate the mechanims of H₂O₂−assisted antigen retrieval. A possible mechanism is that DM is reduced and converted to its semiquinone and/or hydroquinone derivative in vivo. Oxidation by hydrogen peroxide acts to convert these derivatives back to the native antigen. The improved ICC methodology using oxidation to recreate native antigens from reduced metabolites may be helpful also with respect to the localization of other drugs.     

Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K(+) channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca(2+)-activated K(+) channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K(+)) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain.     

Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects

Some Bence Jones proteins (BJPs) can display catalytic activity. Although the catalytic activity of BJPs might be associated with the pathogenesis of disease, this relationship has not yet been established. We tested the effects of seven BJPs on LLC-PK1 cells to assess their pathogenicity. Two out of the seven BJPs showed cytotoxic activity, as assessed by microscopic analysis, the WST method and TUNEL staining. Moreover, the cytotoxic BJPs were excreted by patients who presented with renal impairment. The cytotoxic BJPs displayed 20- to 40-fold higher catalytic activities (kcat of 3.5-2.2 min(-1)) in hydrolyzing a chromogenic substrate compared to the other BJPs. By treating the cytotoxic BJPs with diisopropylfluorophosphate, they lost not only their catalytic activity, but also the cytotoxic effects. These results indicate a direct link between cytotoxicity and the catalytic activity of the BJPs. The catalytic activity of BJPs contributes to the pathogenesis, as well as to development, of symptoms of multiple myeloma. Inhibition of the catalytic activity of BJPs may form the basis of a novel treatment for multiple myeloma patients with renal dysfunction.     

Reversed Sex Change in The Haremic Protogynous Hawkfish Cirrhitichthys falco in Natural Conditions

Bi‐directional sex change has recently been reported in a range of reef fishes, including haremic species that were earlier thought to be protogynous (female to male). However, the occurrence of this phenomenon and the social conditions driving the reversion of males to females (reversed sex change) have been poorly documented under natural conditions. Reversed sex change is predicted to occur in low‐density populations where facultative monogamy is common. However, few studies have evaluated this over a long period in such populations. We documented the occurrence of bi‐directional sex change during a 3‐yr demographic survey of a population characterised by small harem sizes in haremic hawkfish Cirrhitichthys falco. New males were derived following a change in sex of functional females (secondary males; n = 3) and juveniles always matured first as females (n = 3). Thus, C. falco exhibited a typical protogynous sexual pattern, consistent with a range of haremic fish species. We observed reversed sex change in two males. In both cases, all the females disappeared from their harems and the neighbouring males expanded their territories to encompass the territories of the sex changers. However, bachelor males did not always revert to females. A dominant male experienced bachelor status twice but regained mating opportunities following the immigration of a female into his territory or by taking a female from a neighbouring harem. Thus, we conclude that bachelor males use reversed sex change as a facultative tactic to regain reproductive status in a haremic mating system. In addition, we discuss the influence of harem size upon occurrence of reversed sex change.     

Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo(r)), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo(r) marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo(r) mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C(20:3n-6)) and eicosatetraenoic acid (C(20:4n-3)), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C(20:4n-6)) and eicosapentaenoic acid (C(20:5n-3)), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.     

Enzymatic fluorometric microplate assay for quantitative analysis of 3-hydroxybutyric acid in mouse plasma

3-Hydroxybutyric acid (3HB) is a ketone body and acts as an indicator of energy balance and a central regulator of energy homeostasis. We report the application of a sensitive fluorometric assay for the quantitative determination of 3HB. The assay is based on the oxidation of 3HB by 3HB dehydrogenase and on the diaphorase-resazurin amplifying system. This simple assay enables the measurement of changes in 3HB levels in the blood of normal mice by very small volume sample collection. Therefore, this assay will be useful for in vivo studies of small animals.     

Discovery of novel 5-alkynyl-4-anilinopyrimidines as potent, orally active dual inhibitors of EGFR and Her-2 tyrosine kinases

5-Alkenyl or 5-alkynyl-4-anilinopyrimidines were prepared and evaluated for in vitro inhibition of EGFR/Her-2 kinase activity and the growth of tumor cell lines (BT474 and N87). Several of these compounds inhibited the growth of BT474 and N87 at concentrations below 200nM. Structure-activity relationship studies revealed a critical role for the 5-alkynyl moieties. The representative compound 19 exhibited significant antitumor potency in a mouse xenograft model.