Resveratrol transcriptionally regulates miRNA-18a-5p expression ameliorating diabetic nephropathy via increasing autophagy.

OBJECTIVE: To investigate the effects of resveratrol on autophagy in the chronically diabetic nephropathy and to study the effects of the different expression of microRNAs after resveratrol (RSV) treated in db/db mice (diabetic mice). MATERIALS AND METHODS: Db/m (non- diabetic) and db/db mice were randomly divided into intra gastric RSV treatment group or control group. Renal tissues were prepared for HE/PAS staining. In vitro, mouse podocytes cell lines were grown in different mediums with different dose of resveratrol treatment. microRNA (miRNA) gene chips assay was performed for differentially expressed miRNAs screening. Western blot was used to detect protein levels. RESULTS: In vivo, RSV significantly decreased urinary albumin, serum creatinine, mesangial area and glomerular size in db/db mice. After RSV treatment, LC3-II/LC3-I and synaptopodin were increased while cleaved-caspase 3 was decreased in kidney tissues. In vitro, podocytes treated with RSV exhibited significantly increased LC3-II/LC3-I and decreased cleaved caspase 3. Moreover, this effect of RSV can be enhanced by rapamycin (RAPA, an activator of autophagy) but partially reversed by 3-MA (an autophagy inhibitor). Further, we found that miR-18a-5p was significantly upregulated after RSV treatment in db/db mice. Overexpression of miR-18a-5p in podocytes resulted in significant inhibition of cleaved-caspase 3 protein, and increased the ratio of LC3-II/LC3-I. Dual luciferase report assay validated that Atactic telangiectasis mutation (ATM) was a target of miR-18a-5p. In podocytes, downregulation of cleaved caspase 3 and the enhanced ratio of protein LC3-II/LC3-I were detected in cells transfected with ATM siRNA. CONCLUSIONS: Role of miRNA-18a-5p in the regulation of autophagy via targeting ATM may represent a promising therapeutic target for preventing and attenuating diabetic nephropathy.

Development of novel approach to diagnostic imaging of lung cancer with 18F-Nifene PET/CT using A/J mice treated with NNK.

Development of novel methods of early diagnosis of lung cancer is one of the major tasks of contemporary clinical and experimental oncology. In this study, we utilized the tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer in A/J mice as an animal model for development of a new imaging technique for early diagnosis of lung cancer. Lung cancer cells in A/J mice overexpress nicotinic acetylcholine receptors. Longitudinal CT scans were carried out over a period of 8 months after NNK treatment, followed by PET/CT scans with 18F-Nifene that binds to α4-made nicotinic receptors with high affinity. PET/CT scans of lungs were also obtained ex vivo. CT revealed the presence of lung nodules in 8-month NNK-treated mice, while control mice had no tumors. Imaging of live animals prior to necropsy allowed correlation of results of tumor load via PET/CT and histopathological findings. Significant amount of 18F-Nifene was seen in the lungs of NNK-treated mice, whereas lungs of control mice showed only minor uptake of 18F-Nifene. Quantitative analysis of the extent and amount of 18F-Nifene binding in lung in vivo and ex vivo demonstrated a higher tumor/nontumor ratio due to selective labeling of tumor nodules expressing abundant α4 nicotinic receptor subunits. For comparison, we performed PET/CT studies with 18F-FDG, which is used for the imaging diagnosis of lung cancer. The tumor/nontumor ratios for 18F-FDG were lower than for 18F-Nifene. Thus, we have developed a novel diagnostic imaging approach to early diagnosis of lung cancer using 18F-Nifene PET/CT. This technique allows quantitative assessment of lung tumors in live mice, which is critical for establishing tumor size and location, and also has salient clinical implications.

Storage hexamer utilization in Manduca sexta.

In preparing for metamorphosis insects store in their hemolymph and fat bodies a major nutrient reserve of 500-kDa hexamerins. At least three hexamerins serve this function in Lepidoptera, including arylphorin (ArH) and two high methionine proteins (M-MtH and V-MtH). Six day-old adults of Manduca sexta are shown here to have consumed over 99% of their pupal reserves of ArH and in the case of males, 99.8% of M- and V-MtH. In support of egg formation, however, females at this stage retain over 25% of their pupal reserves of the high methionine proteins. Demonstrated here are three factors contributing to the methionine protein reserves in day-6 adult females. (1) Pupal stores of the methionine proteins average 1.67 times larger in females than in males. (2) A fraction of this pupal store remains undiminished during pharate adult development: centrifugation of homogenates partitions the hexamerins into a fraction that is soluble in PBS and a smaller, particle-associated fraction that is not. Pharate adults consume most of the soluble fraction and relatively little of the particulate fraction, which then constitutes over half of the methionine protein reserves of post-eclosion females. (3) Both soluble and particle-associated reserves double in the week following eclosion and this suggests that adult females may resume the synthesis of V- and M-MtH. Though differing in amino acid sequence and antigenic properties, V-MtH and M-MtH showed no significant differences in their storage and utilization profiles.

Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation.

Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction. Hexamerin specializations are compared here in the Cecropia moth (Hyalophora cecropia), which produces eggs during the pupal-adult molt, and the Monarch butterfly (Danaus plexippus), which produces eggs under long-day conditions after adult eclosion. In both sexes of both species, reserves of arylphorin (ArH) were exhausted by the end of metamorphosis. In Cecropia, the same was true for the high-methionine hexamerins, V-MtH and M-MtH. But in short day Monarch females 20-30% of the pupal reserves of V-MtH and M-MtH survived metamorphosis, persisting until long-day conditions were imposed to stimulate egg formation. Differences in storage sites have been documented in other lepidopterans, with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph. Similar differences could explain how a fraction of the MtH's, but not of ArH, escapes utilization during metamorphosis in a species with post-eclosion egg formation. No differences in utilization schedules were detected between V- and M-MtH, despite divergent compositions and antigenic reactivity.

Equivalence of riboflavin-binding hexamerin and arylphorin as reserves for adult development in two saturniid moths.

The riboflavin-binding hexamerin (RbH) and arylphorin (ArH) were compared as storage reservoirs for adult development in Hyalophora cecropia. The two hexamerins were metabolically labeled with [3H]leucine and [35S]methionine, isolated by column chromatography, and separately injected into pupae whose diapause had been terminated by chilling. By the time of eclosion at least 98% of both hexamerins had been cleared from the hemolymph. Every reproductive and somatic tissue tested contained trichloroacetic acid-precipitable label; consistent differences between the two hexamerins were not detected in the distribution of their label to these tissues. While incorporation of intact hexamerins was not ruled out, hydrolysis and reincorporation of the liberated amino acids were indicated by label in vitellogenin and lipophorin, and by differences in 35S/3H ratios, which ranged from over 1.0 in chorions to 0.4 in wings, as compared with 0.75 in the injected hexamerins. Injection of [35S,3H]RbH from H. cecropia into A. luna, a species in the same subfamily whose pupae lack this hexamerin, resulted in a pattern of isotope incorporation similar to that yielded by RbH in the donor species. In neither species was there indication of a developing adult tissue that distinguished between RbH and ArH as precursor reservoirs for morphogenesis. This equivalence helps explain how many species of Lepidoptera are able to complete metamorphosis and reproduce without expressing an RbH gene. Evidence is also presented that ArH stored in the fat body protein granules during pupation may be utilized differently from that remaining in pupal hemolymph.

Methionine-rich hexamerin and arylphorin as precursor reservoirs for reproduction and metamorphosis in female luna moths.

The storage proteins of Lepidoptera include a pair of methionine-rich hexamerins (MtH) that are more abundant in female pupae than in males. Their inferred support of female reproduction could be achieved either by enhancing general pools of amino acids, or by hydrolyzing MtH at times and/or sites that direct its constituents to the synthesis of egg proteins. The two models were tested in Actias luna, a saturniid moth that makes its eggs during adult development. MtH and arylphorin (ArH), the third major storage protein of this species, were labeled metabolically with [35S]-methionine and [3H]-leucine, and injected individually into wandering stage caterpillars. Isotope distributions at eclosion indicated that both hexamerins supported egg formation as well as adult tissue protein synthesis. In the absence of evidence for targeting, MtH appears to support egg formation in A. luna by enhancing the amino acid pools derived from ArH. Analysis of 35S labeling and of 35S/3H ratios indicated, however, that ArH is consumed over a period that extends somewhat later in adult development than MtH. Differences in timing should prove to be much greater in Lepidoptera that delay egg formation until after eclosion.

Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias luna.

When Hyalophora cecropia hemolymph was injected into wandering Actias luna larvae, a methionine-rich hexamerin was selectively transferred to the host's fat body, and completely cleared from the hemolymph by the time of pupal eclosion. Donor arylphorin was 30-40% removed from the hemolymph, and riboflavin-binding hexamerin was even less completely cleared. During the pupal-adult molt, these rates were reversed: methionine-rich hexamerin disappeared no faster than bovine serum albumin, while riboflavin-binding hexamerin was rapidly and completely cleared from the hemolymph, even though A. luna hemolymph lacks a homologue of this protein; arylphorin, again, was cleared at an intermediate rate. Selective clearing of the three hexamerins occurred at similar stages in H. cecropia, their species of origin. Developmentally programmed clearing, with selectivity at least partially conserved between genera, was also demonstrated with transfused vitellogenin: in A. luna females that were forming yolk, H. cecropia vitellogenin was cleared more rapidly than bovine serum albumin; but in younger females, and in males at all stages of metamorphosis, this Mr 510,000 molecule was instead an indicator of nonselective, large protein clearing. Nonselective clearing was more complete during adult development than during pupation. It also showed signs of being more effective for small than for large proteins, insensitive to carbohydrate conjugates, and unsaturated at the protein levels used.

Synthesis, purification and stability of no carrier added radioiodinated 1,1-bis(4-hydroxyphenyl)-2-iodo-2-phenylethylene (IBHPE), a prototype triphenylethylene estrogen-receptor binding radiopharmaceutical.

A triphenylethylene compound [1,1-bis(4-hydroxyphenyl)-2-iodo-2-phenylethylene; IBHPE] has been labeled by halodestannylation with 123I at a specific radioactivity of 13,200 Ci/mmol (by in vitro receptor assay) after HPLC purification. The corresponding 80mBr-labeled compound (BrBHPE), which has a 3-fold higher affinity for the estrogen receptor, was previously prepared and examined as a potential therapeutic radiopharmaceutical exploiting Auger electron toxicity. Stability of IBHPE was a concern because free iodide was generated when HPLC solvents were removed with a stream of nitrogen in a glass vial; however, decomposition was minimal when polypropylene vials were used, and ethanol solutions of [123I]IBHPE were stable for several days at 0-4 degrees C. Tissue distribution studies of IBHPE after intraperitoneal injection to mature female rats showed highest estradiol-inhibitable uptake in the peritoneal estrogen-receptor rich tissues (uterus, ovaries and vagina) at 30 min. Specific uptake (percent dose per gram) in the pituitary, and peritoneal target tissue-to-blood ratios were greater at 2 h than 30 min. In immature female rats, uterus-to-blood ratios of greater than 50, progressively lowered by increasing diethylstilbestrol levels, were obtained. These data demonstrate good binding of IBHPE to the estrogen receptor in vivo, in spite of extensive non specific binding in in vitro estrogen receptor assays. Most of the label in the uterus at 1 h after injection was still unchanged IBHPE. Our results suggest that IBHPE or related 123I-labeled iodovinyl triphenylethylenes could have diagnostic or therapeutic radiopharmaceutical utility.

Juvenile hormone induced vitellogenin synthesis in the monarch butterfly.

The vitellogenin (yolk protein) of the monarch butterfly has been identified by electrophoretic and immunochemical techniques. In freshly emerged female adults ligature of the neck prevents appearance of this protein in the hemolymph; it prevents yolk deposition and egg maturation as well. These processes are restored by injection of juvenile hormone; the restoration involves induction of vitellogenin synthesis, as shown by incorporation of [(3)H]leucine.

Vitellogenic blood protein synthesis by insect fat body.

The female fat body of a moth and a roach incorporated labeled amino acids in vitro into substances precipitable by antibodies formed in response to the sex-limited vitellogenic blood proteins of these species. The fat body of males failed to do so, as did that of females before the appearance of these proteins in the blood.