Insulin-sparing and fungible effects of E4orf1 combined with an adipocyte-targeting sequence in mouse models of type 1 and type 2 diabetes.

Obesity impairs glycemic control and causes insulin resistance and type 2 diabetes. Adenovirus 36 (Ad36) infection can increase the uptake of excess glucose from blood into adipocytes by increasing GLUT4 translocation through the Ras-Akt signaling pathway, which bypasses PI3K-Akt-mediated insulin receptor signaling. E4orf1, a viral gene expressed early during Ad36 infection, is responsible for this insulin-sparing effect and may be an alternative target for improving insulin resistance. To deliver the gene to adipocytes only, we connected the adipocyte-targeting sequence (ATS) to the 5' end of E4orf1 (ATS-E4orf1). In vitro transfection of ATS-E4orf1 into preadipocytes activated factors for GLUT4 translocation and adipogenesis to the same extent as did Hemagglutinin (HA)-E4orf1 transfection as positive reference. Moreover, the Transwell migration assay also showed that ATS-E4orf1 secreted by liver cells activated Akt in preadipocytes. We used a hydrodynamic gene delivery technique to deliver ATS-E4orf1 into high-fat diet-fed and streptozotocin-injected mice (disease models of type 2 and type 1 diabetes, respectively). ATS-E4orf1 improved the ability to eliminate excess glucose from the blood and ameliorated liver function in both disease models. These findings suggest that ATS-E4orf1 has insulin-sparing and fungible effects in type 2 and 1 diabetes independent of the presence of insulin.

Epidermal regeneration by ent-16α, 17-dihydroxy-kauran-19-oic acid isolated from Siegesbeckia pubescens.

OBJECTIVES: Keratinocyte stem/progenitor cells (KSCs) are known to regenerate epidermal tissue which they perform through to their great regenerative capacity. MATERIALS AND METHODS: Because stimulation of resident KSCs may regenerate epidermal tissue, we devised a strategy to find an appropriate KSC activator from natural products and to develop it as a skin-rejuvenating agent. RESULTS: Ent-16α, 17-dihydroxy-kauran-19-oic acid (DHK) isolated from Siegesbeckia pubescens exhibited a KSC-stimulating effect during screening of natural products. DHK increased proliferation and migration of KSCs using the Akt/ERK pathway. We further examined the mechanism of KSC stimulation and found that phosphorylation of Y1068 epithelial growth factor receptor (EGFR) was significantly increased. Functional inhibition of EGFR using neutralizing antibody and a chemical inhibitor, AG1478, attenuated DHK-induced KSC stimulation. In a 3D culture model of KSCs, DHK treatment significantly induced establishment of fully stratified epidermis and increased numbers of p63-positive cells. Likewise, DHK treatment significantly accelerated healing of epidermal wounds created by laser and dermatome, and increased p63-positive cells, in animal models. CONCLUSION: Collectively, these results indicate that DHK regenerates epidermal tissue mainly through EGFR phosphorylation. As DHK has diverse advantages over recombinant growth factors for commercialization (that is long-term stability and skin permeability), DHK might be applied to wound-healing agents and to a basic materials used in cosmetics.

Cysteine effects on the pharmacokinetics of etoposide in protein-calorie malnutrition rats: increased gastrointestinal absorption by cysteine.

Protein-calorie malnutrition (PCM) occurs frequently in advanced cancer patients and has a profound impact on the toxicity of many drugs. Thus, the pharmacokinetics of etoposide were evaluated in control, control with cysteine (CC), PCM, and PCM with cysteine (PCMC) rats. Etoposide was administered intravenously (2 mg/kg) or orally (10 mg/kg). Changes in hepatic and intestinal cytochrome P450s (CYPs) and effects of cysteine on intestinal P-glycoprotein (P-gp)-mediated efflux were also measured. In PCM rats, the CL(NR) (AUC(0-∞)) of intravenous etoposide was significantly slower (greater) than that in controls, because of the significant decrease in the hepatic CYP3A subfamily and P-gp. In PCMC rats, the slowed CL(NR) of etoposide in PCM rats was restored to the control level by cysteine treatment. PCMC rats showed a significantly greater AUC(0-6 h) of oral etoposide than PCM rats, primarily because of the increased gastrointestinal absorption of etoposide as a result of the inhibition of intestinal P-gp by cysteine. The gastrointestinal absorption of an oral anticancer drug, which is a substrate of P-gp, may be improved by co-administration of cysteine in advanced cancer patients if the present rat data can be extrapolated to patients.

Diazoxide acts more as a PKC-epsilon activator, and indirectly activates the mitochondrial K(ATP) channel conferring cardioprotection against hypoxic injury.

BACKGROUND AND PURPOSE: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection. EXPERIMENTAL APPROACH: In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. KEY RESULTS: Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD. CONCLUSIONS AND IMPLICATIONS: Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.

Altered TRPC7 gene expression in bipolar-I disorder.

BACKGROUND: As altered storage-operated calcium (Ca(2+)) entry (SOCE) may affect Ca(2+) homeostasis in bipolar disorder (BD), we determined whether changes occur in the expression of TRPC7 and SERCA2s, proteins implicated or known to be involved in SOCE, in B lymphoblast cell lines (BLCLs) from BD-I patients and comparison subjects. METHODS: mRNA levels were determined in BLCL lysates from BD-I, BD-II, and major depressive disorder patients, and healthy subjects by comparative reverse transcriptase-polymerase chain reaction, and BLCL basal intracellular Ca(2+) concentration ([Ca(2+)]B) was determined by ratiometric spectrophotometry using Fura-2, in aliquots of the same cell lines, at 13-16 passages in culture. RESULTS: TRPC7 mRNA levels were significantly lower in BLCLs from BD-I patients with high BLCL [Ca(2+)]B compared with those showing normal [Ca(2+)]B (-33%, p =.017) and with BD-II patients (-48%, p =.003), major depressive disorder patients (-47%, p =.049) and healthy subjects (-33%, p =.038). [Ca(2+)]B also correlated inversely with TRPC7 mRNA levels in BLCLs from the BD-I group as a whole (r = -.35, p =.027). CONCLUSIONS: Reduced TRPC7 gene expression may be a trait associated with pathophysiological disturbances of Ca(2+) homeostasis in a subgroup of BD-I patients.

Altered IMPA2 gene expression and calcium homeostasis in bipolar disorder.

Reduced inositol monophosphatase (IMPase) activity and elevated basal intracellular calcium levels ([Ca(2+)](B)) have been reported in B lymphoblast cell lines (BLCLs) from bipolar I affective disorder (BD-I) patients, which may reflect cellular endophenotypes of this disorder. As the PI cycle couples to intracellular Ca(2+) mobilization, these two putative endophenotypes may be related. Using an RT-PCR assay, mRNA levels were estimated for IMPA1 and 2 genes encoding human IMPase 1 and 2, respectively, in BLCLs phenotyped on [Ca(2+)](B), from patients with a DSM-IV diagnosis of BD-I (n = 12 per phenotype) and from age- and sex-matched healthy subjects (n = 12). IMPA2 mRNA levels were significantly lower in BLCLs from male BD-I patients with high [Ca(2+)](B) (n = 6) compared with healthy male subjects (n = 5) (-52%, P = 0.013), male BD-I patients with normal BLCL [Ca(2+)](B) (n = 8) (-42%, P = 0.003) and female BD-I patients with high [Ca(2+)](B) (n = 6) (-59%, P = 0.0004). A significant negative correlation was observed between IMPA2 mRNA levels and [Ca(2+)](B) in BLCLs from male (P = 0.046), but not female BD-I patients. Sex-dependent differences were also evident in postmortem temporal cortex IMPA2 mRNA levels which, in contrast to BLCLs, were significantly higher in male BD-I subjects compared with male controls (P = 0.025, n = 4/group). Collectively, these observations suggest a potential sex-dependent link between abnormalities in IMPA2 expression and calcium homeostasis in the pathophysiology of BD.

Nocardia keratitis after traumatic detachment of a laser in situ keratomileusis flap.

PURPOSE: Nocardia are gram-positive bacteria existing ubiquitously in the environment; they can cause keratitis. Nocardia asteroides keratitis occurred in the interface between the stromal bed and flap after traumatic detachment of the flap 4 months after an initially uncomplicated laser in situ keratomileusis (LASIK) procedure. METHODS: Nocardia asteroides keratitis was confirmed by culture. Therapy included topical and oral trimethoprim-sulfamethoxazole. RESULTS: Thirteen months after the trauma, the patient's spectacle-corrected visual acuity was 20/20 with a manifest refraction of -2.25 -1.00 x 30 degrees. CONCLUSIONS: The immediate steps of management consisting of surgically lifting the corneal flap, rapid microbial identification, and proper treatment with specific antibiotics resulted in the successful treatment of Nocardia asteroides keratitis in a traumatized eye after LASIK.

Characterization of an auxin-inducible 1-aminocyclopropane-1-carboxylate synthase gene, VR-ACS6, of mungbean (Vigna radiata (L.) Wilczek) and hormonal interactions on the promoter activity in transgenic tobacco.

A genomic clone for VR-ACS6, an isozyme of auxin-inducible ACC synthase of mungbean, was isolated, and its promoter activity was examined in transgenic tobacco. The clone contained 1,612 bp long 5' untranscribed region and its coding sequence consisted of three exons and two introns. Genomic Southern hybridization indicated that VR-ACS6 is a single copy gene. The transcription initiation site was a cytosine present at 231-base upstream the translation start codon. The VR-ACS6 promoter contained DNA sequences homologous to various functionally identified auxin-responsive elements. To demonstrate hormonal response of the promoter region, transgenic tobacco plants carrying the 1,719 bp VR-ACS6 promoter/-glucuronidase (GUS) fusion gene were generated. Strong GUS expression occurred by auxin treatment of leaves of T0 transformants and hypocotyls of T1 etiolated seedlings. Magnitude of the response to auxin was dose-dependent, and the increased GUS activity was detected at 0.1 microM and higher concentrations of IAA. Other plant hormones did not induce GUS activity, but greatly modified the response to auxin. Cytokinin enhanced the IAA-induced expression of GUS reporter gene, whereas ABA and ethylene suppressed the expression. These characteristics of VR-ACS6 promoter activity in transgenic tobacco are in good accordance with the expression patterns of the gene in mungbean hypocotyls. Histochemical staining showed that GUS activity was evident in both etiolated and light grown seedings treated with IAA. Cytokinin enhanced the intensity of auxin-induced GUS stain and also expanded the stained area, whereas ABA and ethylene reduced both intensity and area of the stain.

VR-ACS6 is an auxin-inducible 1-aminocyclopropane-1-carboxylate synthase gene in mungbean (Vigna radiata).

We have isolated four cDNA clones of ACC synthase from etiolated mungbean seedlings treated with auxin. pVR-ACS2, pVR-ACS3 and pVR-ACS6 contained the same sequences as the previously reported DNA fragments, pMAC2, pMAC3 (Botella et al. 1992b) and pMBA1 (Kim et al. 1992), respectively. pVR-ACS1 was identical with pAIM-1 (Botella et al. 1992a). VR-ACS6 was specifically induced in response to the auxin signal. The IAA-induction of VR-ACS6 was very rapid (within 30 min) and insensitive to cycloheximide treatment at concentrations up to 100 microM. Significant accumulation of VR-ACS6 mRNA was detected at 1 microM IAA. The IAA-induced expression of VR-ACS6 was suppressed by ABA and ethylene, but enhanced by BA. These characteristics of VR-ACS6 expression were well correlated with the physiological data of auxin-induced ethylene production in mungbean hypocotyls. VR-ACS1 was strongly induced by cycloheximide, but was found to be not auxin-specific. Inhibitors of either ethylene biosynthesis (AOA) or action (NBD) increased the basal level of VR-ACS1 mRNA.