The epithelial zinc transporter ZIP10 epigenetically regulates human epidermal homeostasis by modulating histone acetyltransferase activity.

BACKGROUND: The skin is the first organ that manifests changes in response to zinc deficiency. However, the molecular mechanism underlying how zinc is involved in skin homeostasis, especially its epigenetic regulation, is largely unknown. OBJECTIVES: In this study we demonstrate the importance of zinc levels and the zinc transporter ZIP10 in the epigenetic maintenance of human epidermal homeostasis. METHODS: Adult human skin, including skin appendages, were stained with anti-ZIP10 antibody. Histone acetyltransferase (HAT) activity was assessed after treating human keratinocytes with ZIP10 small interfering (si)RNAs or the zinc chelator TPEN. ZIP10- or HAT-regulated genes were analysed based on limma bioinformatics analysis for keratinocytes treated with ZIP10 siRNAs or a HAT inhibitor, or using a public database for transcription factors. A reconstituted human skin model was used to validate the role of ZIP10 in epidermal differentiation and the functional association between ZIP10 and HAT. RESULTS: ZIP10 is predominantly expressed in the interfollicular epidermis, epidermal appendages and hair follicles. ZIP10 depletion resulted in epidermal malformations in a reconstituted human skin model via downregulation of the activity of the epigenetic enzyme HAT. This decreased HAT activity, resulting from either ZIP10 depletion or treatment with the zinc chelator TPEN, was readily restored by zinc supplementation. Through bioinformatics analysis for gene sets regulated by knockdown of SLC39A10 (encoding ZIP10) and HAT inhibition, we demonstrated that ZIP10 and HATs were closely linked with the regulation of genes related to epidermal homeostasis, particularly filaggrin and metallothionein. CONCLUSIONS: Our study suggests that ZIP10-mediated zinc distribution is crucial for epidermal homeostasis via HATs. Therefore, zinc-dependent epigenetic regulation could provide alternatives to maintaining healthy skin or alleviating disorders with skin barrier defects.

PKCtheta II, a new isoform of protein kinase C specifically expressed in the seminiferous tubules of mouse testis.

Protein kinase C (PKC) theta, a Ca(2+)-independent isoform of PKC, has been known to be expressed in skeletal muscle and T cells. In the present study, we isolated and characterized a smaller transcript expressed in the mouse testis, the cDNA of which is referred hereafter as PKCthetaII and the original PKCtheta as PKCthetaI. The cDNA clone of PKCthetaII has 2184 base pairs and 464 amino acids in the possible open reading frame, consisting of the 5' unique sequence of 20 amino acids and the PKCthetaI sequence of 444 amino acids. Genomic DNA analysis revealed that transcription of PKCthetaII is initiated from the PKCthetaII-specific exon, which is located between exons 7 and 8 of the PKCtheta gene, indicating that alternative splicing is the mechanism by which PKCthetaII is generated. PKCthetaII is expressed exclusively in the testis in an age-dependent manner with sexual maturation. In situ hybridization and reverse transcription-polymerase chain reaction of microdissected tissues clearly demonstrated that PKCthetaII is expressed in the seminiferous tubules of the mouse testis. Consistent with its molecular structure lacking the C1 regulatory domain, PKCthetaII is constitutively active as determined by an in vitro kinase assay, being independent of PKC activators, e.g. phosphatidylserine and phorbol ester. PKCthetaII may play a crucial role in spermatogenesis or some related function of the testis.

[Experimental study on intraperitoneal and intravenous administration of anti-tumor agents for liver metastasis].

Intraperitoneal chemotherapy has been the treatment for peritoneal seedings. Most of the anti-tumor agent administered intraperitoneally is absorbed from visceral peritoneum, gets into the portal vein system and reaches the liver. Theoretically, intraperitoneal administration of anti-tumor agents must show equivalent effects on the liver metastasis to portal vein infusion. We compared the efficacy of intraperitoneal and intravenous administration of 5-FU, CDDP and CPT-11, using colon 26 mouse liver metastasis model. Intraperitoneal administration of 5-FU or CPT-11 was statistically superior to intravenous administration to diminish the liver metastatic deposits. CDDP experiment did not show a statistical difference, but the superiority intraperitoneal administration was recognized. Intraperitoneal administration of anti-tumor agents is more effective for not only peritoneal seedings but also liver metastases than intravenous administration. Intraperitoneal chemotherapy might be an effective adjuvant chemotherapy for gastrointestinal malignancies.

Human cerebral acetylcholinesterase activity measured with positron emission tomography: procedure, normal values and effect of age.

The regional cerebral metabolic rate of [11C]N-methyl-4-piperidyl acetate, which is nearly proportional to regional cerebral acetylcholinesterase (AChE) activity, was measured by dynamic positron emission tomography in 20 healthy subjects with a wide age range (24-89 years). Quantitative measurement was achieved using a kinetic model which consisted of arterial plasma and cerebral tissue compartments. The plasma input function was obtained using thin-layer chromatography and an imaging phosphor plate system at frequent sampling intervals to catch the rapid metabolism of the tracer in the blood. The distribution of the rate constant k3, an index of AChE activity, agreed well with reported post-mortem AChE distribution in the cerebral cortex (0.067-0.097 min-1) and thalamus (0.268 min-1), where AChE activity was low to moderate. The k3 values in the striatum and cerebellum, where AChE activity was very high, did not respond linearly to AChE activity because of increased flow dependency. No significant effect of age was found on AChE activity of the cerebral cortex, suggesting that the ascending central cholinergic system is preserved in normal aging. This study has shown that quantitative measurement of enzyme activity in the living brain is possible through appropriate modelling of tracer kinetics and accurate measurement of the input function. The method should be applicable to patients with Alzheimer's disease and those with other kinds of dementia whose central cholinergic system has been reported to be disturbed.

[Hemodynamics at hepatoarterial infusion of 5-FU in a chronic renal failure patient maintained by hemodialysis].

The authors studied the hemodynamics of 5-FU hepatoarterial infusion in a colorectal cancer patient with multiple liver metastases, who had chronic renal failure maintained by hemodialysis. Under weekly high dose 5-FU hepatoarterial infusion (1,000 mg/m2, 5 hours), on a non-dialysis day, serum 5-FU concentration was 1,090 ng/ml just after the 5 h infusion, 391 ng/ml at 15 min, 217 ng/ml at 30 min, 47 ng/ml at 60 min, and < 4 ng/ml at 120 min after infusion. On a dialysis day it was 1,500 ng/ml just after infusion, 41 ng/ml at 15 min, 5 ng/ml at 30 min, and < 4 ng/ml at 60 and 120 min after infusion. A control group (n = 4), who had liver metastases from colorectal cancers and normal renal functions, showed 5-FU serum concentration of 987 +/- 384 ng/ml just after infusion, 226 +/- 117 ng/ml at 15 min, 18.5 +/- 5.8 ng/ml at 30 min, < 4 ng/ml at 60 and 120 min after infusion. The serum 5-FU concentration of the patient was maintained higher on non-dialysis days, while it decreased more rapidly on dialysis days than that of the control group. There were no clinical complications due to the weekly high dose 5-FU hepatoarterial infusion. Under the treatment of continuous 5-FU hepatoarterial infusion (500 mg/day), the serum 5-FU concentration of this patient was kept under 115 ng/ml. After hemodialysis, the concentration decreased. The serum 5-FU concentration of the control group (n = 4) was under 66 ng/ml. There were no side effects under the protocol of continuous 5-FU hepatoarterial infusion. 5-FU hepatoarterial infusion for liver metastasis was a safe treatment for a renal failure patient with hemodialysis.

The metabolic pathway of visual pigment chromophore formation in Drosophila melanogaster--all-trans (3S)-3-hydroxyretinal is formed from all-trans retinal via (3R)-3-hydroxyretinal in the dark.

Carotenoid-depleted fruit flies, Drosophila melanogaster, were reared on yeast/glucose medium containing lipid-depleted white corn grits and cholesterol. After rearing for more than a year, the yield of flies remained constant and the content of 3-hydroxyretinal in a head was three logarithmic units less than that of normal flies reared on medium containing yellow corn grits. When all-trans retinal was supplied as the sole source of retinoids, the flies formed and accumulated all-trans 3-hydroxyretinal in the dark. To examine the metabolic pathway to produce (3S)-3-hydroxyretinal in Drosophila, all-trans retinal was supplemented for two hours to carotenoid-depleted flies in the dark, and the subsequent changes in the composition of 3-hydroxyretinal enantiomers were analyzed using a chiral column on HPLC. The results indicated initial formation of (3R)-3-hydroxyretinal followed by isomerization into the 3S enantiomer. In another set of experiments, the membrane fraction was obtained from the head homogenate of retinoid-depleted flies and an in vitro assay of 3-hydroxyretinal formation from retinal was performed. The 3-hydroxyretinal produced was the 3R enantiomer, supporting the result obtained from the in vivo experiment whereby (3S)-3-hydroxyretinal is produced from retinal via (3R)-3-hydroxyretinal. Addition of NADPH enhanced 3-hydroxyretinal formation and the presence of carbon monoxide inhibited it, suggesting that hydroxylation at the C3 position of retinal occurred via the monooxygenase activity of cytochrome P-450.

Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells.

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl- concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K+ with valinomycin. Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl- permeability, but neither by the increased K+ conductance with valinomycin nor by the increased Ca2+ conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A. These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells; the K+ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl- channel opening depends on calyculin A-sensitive protein phosphatase activity.

Cloning and characterization of the gene encoding the iron-sulfur protein of succinate dehydrogenase from Pleurotus ostreatus.

Genomic and cDNA fragments encoding the iron-sulfur protein (Ip) subunit of dehydrogenase (EC 1.3.99.1) have been cloned from the edible basidiomycetous fungus, Pleurotus ostreatus. The gene is interrupted by five introns and is predicted to encode a polypeptide of 268 amino acid residues. Sequence comparison with the Ip subunit from other species identified three conserved cysteine-rich clusters. One of these contains a critical histidine residue implicated in carboxin sensitivity in the heterobasidiomycete Ustilago maydis.

Time courses of changes in cerebral blood flow and blood-brain barrier integrity by focal proton radiation in the rat.

In order to know the pathophysiological mechanisms underlying radiation brain injury, cerebral blood flow and blood-brain barrier integrity were studied using N-isopropyl-p-[123l]iodoamphetamine (IMP) and [14C]-alpha-aminoisobutyric acid (AIB), respectively, in the rat focal proton radiation model (a single dose of 30 or 60 Gy radiation with 70 MeV proton beams). One, 2, 4, and 5.5 months after irradiation, [123l]IMP and [14C]AIB were intravenously injected and uptake of IMP and AIB in the cerebral cortex, striatum, hippocampus, and thalamus was measured. Significant decreases in IMP uptake were observed in the cerebral cortex and thalamus of the irradiated side at 4 and 5.5 months after 60 Gy irradiation; the effects at 5.5 months were more prominent than those at 4 months. AIB uptake markedly increased in all the brain regions of the irradiated side at 5.5 months after 60 Gy irradiation, and at 4 months, only in the hippocampus. The results suggest that there are dose- and time-dependent responses in radiation effects and regional differences in tissue vulnerability to radiation. Proton focal radiation model appears to be a useful model for studies of radiation brain injury in small animals such as rats.

Structures of genomic and complementary DNAs coding for Pleurotus ostreatus manganese (II) peroxidase.

To study the mechanism of regulation and structure/function relationship of the Pleurotus ostreatus manganese (II) peroxidase (MnP), we amplified the full-length genomic and complementary DNAs for the major isozyme of the MnP mainly by the cassette-primer PCR technique and then sequenced them. The cDNA contained an open reading frame of 1083 bp encoding for a polypeptide of 361 amino-acid residues, including the suggested signal peptide of 29 amino-acid residues with a prepro structure. The predicted amino-acid sequence of the protein shared several common characteristics with those of fungal lignin and manganese (II) peroxidases. We could find a suggested metal response element and two heat-shock element-like sequences in the 5'-flanking region of the structural gene. The structural gene contained 15 introns, many of which lie identical to those in lignin peroxidase genes rather than to those in the known MnP genes.

In vivo measurement of acetylcholinesterase activity in the brain with a radioactive acetylcholine analog.

A novel method for visualization of brain acetylcholinesterase (AChE) in vivo has been developed. Following intravenous administration of a radiolabelled acetylcholine analog, N-methyl-3-piperidyl acetate, there was very good agreement between the distribution of radioactivity and AChE activity in the brain of rat and monkey. The method would be applicable for in vivo studies of human brain AChE activity in disorders of central cholinergic systems such as Alzheimer's disease.