Analysis of the UV-Induced Melanogenesis and Growth Arrest of Human Melanocytes

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm² UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G₂-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 μM α-melanocyte stimulating hormone (α-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of α-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.     

The Presence of Tyrosinase and Related Proteins in Human Epidermis and Their Relationship to Melanin Type

The present study was carried out to investigate the abundance of tyrosinase and related proteins (TRP-1 and TRP-2) in human epidermis and their relationship to melanin type. Positive immunocytochemical staining was seen for all three proteins in epidermal melanocytes. For each protein the numbers of positively stained melanocytes were similar in all subjects studied irrespective of skin type. Following 5 daily suberythemal doses of UVB the melanocytes were larger, more dendritic, and increased in number. With TRP-1 and TRP-2 the increase in number in response to UVB was unrelated to skin type and, hence, with melanin type but with tyrosinase there was a much greater increase in skin types III and IV than in skin type I and II. The enhanced numbers of tyrosinase-positive melanocytes were accompanied by increased staining intensity, suggesting a greater expression of tyrosinase in the melanocytes from skin types III and IV compared with skin types I and II. This increase in tyrosinase could be related to the greater levels of eumelanin found in skin types III and IV, and this is in keeping with the view that higher levels of tyrosinase are associated with the production of eumelanin than phaeomelanin.     

Coordinated mRNA and Protein Expression of Human LAMP-1 in Induction of Melanogenesis After UV-B Exposure and Co-Transfection of Human Tyrosinase and TRP-1 cDNAs

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome-associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV -B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase-related protein-1 (TRP-1). Repeated exposure of UV B (2.5–5.0 mJ/cm²) caused not only upregulation of tyrosinase and TRP-1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp-1 (lysosome-associated membrane protein-1). When COS-7 kidney cells and amelanotic melanoma (C32 and SKMEL-24) and melanotic melanoma (G361 and SK-MEL-23) cells were exposed to cotransfection of human tyrosinase and TRP-1 cDNAs, there was also an increased expression of Lamp-1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP-1 cDNA or cotransfectants of human tyrosinase and TRP-1 cDNAs, indicating that TRP-1 prevented, along with Lamp-1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP-1 and Lamp-1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp-1 gene, showing the decreased expression of TRP-1 as identified by three different types of anti-TRP-1 monoclonal antibodies. We propose therefore that human tyrosinase and TRP-l, when activated or expressed together, will coordinate to upregulate the mRNA expression and protein synthesis of Lamp-1. The Lamp-1 molecules will, in turn, cover the inner surface of melanosomal membrane, together with TRP-1 molecules, thus protecting the melanosomal membrane from toxic melanin intermediates generated during melanogenesis in the presence of active tyrosinase. In contrast, the expression of other lysosome-related proteins, e.g., β-galactosidase and CD63 is not stimulated in new melanogenesis.     

Human Melanoma Cell Lines Show Little Relationship Between Expression of Pigmentation Genes and Pigmentary Behaviour In Vitro

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with α-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by α-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; α-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or α-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinaserelated genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study

The human hair cycle is characterized by successive phases of growth and involution that imply tissue regression and regeneration. As a consequence, the hair melanin unit has to be renewed in a cyclic manner. Actually, the behavior of human hair follicle melanocytes throughout the hair cycle has been poorly studied. Thus, the origin of melanocytes present in the bulb after human hair regeneration is still not clarified, and neither are the events that control the melanin biosynthesis activity in the human hair bulb. In this study, we showed at the cellular level that in human pigmented hair follicles, the expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) was detectable during the anagen phases III/IV through VI, only in those melanocytes which were located in the bulb. During the catagen phase, the two evaluated melanogenic enzymes were detectable no more, although melanocytes were still present in the preceding bulbar area. The epithelial column of catagen follicles and the capsule of telogen follicles also contained inactive melanocytes as evidenced by pMel-17 labeling. At the induction of a new anagen hair follicle, some melanocytes were committed to cell division, but only when located in the nascent bulb close to the dermal papilla. Our results emphasize the close relationship between melanogenesis and the hair cycle and suggest that in humans, melanogenesis is restricted to anagen hair follicles not because of the regulation of tyrosinase activity, but because of melanogenic enzyme expression, e.g., tyrosinase and TRP-1. Furthermore, the fact that in the newly developing anagen hair follicles, cell-division commitment and tyrosinase and TRP-1 expression were observed in melanocytes only when located in the nascent bulb suggests a highly regio-specific melanocyte stimulation in early the anagen phase.     

AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.     

Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Biological Role of Tyrosinase Related Protein and its Biosynthesis and Transport From TGN to Stage I Melanosome,Late Endosome,Through Gene Transfection Study

Tyrosinase-related protein (TRP)-1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP-1 and its transport from TGN (trans-Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP-1 cDNA co-transfection studies indicated that human TRP-1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP-1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome-associated membrane protein)-1 that would directly prevent the tyrosinase-mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP-1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP-1 will be transported from TGN to the stage I melanosome. At this stage, TRP-1 will have its own target signal, in particular, tyrosine-rich leucine residues in cytoplasmic tail. Our TRP-1 cDNA transfection and immunoelectron microscopy study shows that TRP-1 will be transported through small vesicles, probably non-clathrin-coated type, to large vacuoles, identical to the MPR (mannose-6-phosphate receptor)-positive, late endosomes. In this transport process, a low molecular weight G-protein, rab-7, was isolated from the purified melanosomal protein on 2D-PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co-localization of rab-7 and TRP-1 in the melanosomes with early stages of maturation (I-III). Furthermore, this process will also be regulated by phosphatidylinositol 3-kinase (PI-3 kinase).     

lpa-miR-nov-66通过调控cAMP路径抑制α-MSH介导的羊驼黑色素生成

α-黑素细胞刺激素(α-MSH)和lpa-miR-nov-66在羊驼黑色素细胞产生黑色素过程中均起重要的调控作用,但二者之间的关系尚未报道.本研究在体外培养的羊驼黑色素细胞中通过转染lpamiR-nov-66和添加α-MSH处理,用实时定量PCR和Western印迹检测黑色素细胞内基因表达水平,ELISA法检测c AMP和cGMP的产量,RTCA实时无标记细胞功能分析黑色素细胞增殖以及紫外分光光度法检测黑色素产量,证实二者在调控羊驼黑色素细胞产生黑色素颗粒过程中的关系.结果显示,与单纯α-MSH处理相比,lpa-miR-nov-66转染结合α-MSH处理组中,小眼转录因子(MITF)和酪氨酸酶(TYR)在转录水平和翻译水平的表达均降低,而酪氨酸酶相关蛋白2(TYRP2)在转录和翻译水平的表达均升高;cGMP的产量升高,cAMP的产量下降;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量下降.与单纯转染lpa-miR-nov-66相比,lpa-miR-nov-66转染结合α-MSH处理组中,MITF、TYR和TYRP2在转录水平和翻译水平的表达均升高;cGMP的产量下降,cAMP的产量升高;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量升高.上述结果证明,lpa-miR-nov-66通过调控羊驼黑色素细胞中毛色形成的c AMP路径,抑制α-MSH对黑色素细胞产生黑色素的促进作用.     

Synthesis of caffeic acid ester morpholines and their activation effects on tyrosinase

Two new caffeic acid ester derivatives, caffeic acid-4-(2-hydroxyethyl) morpholine ester (Z-1) and caffeic acid-4-(3-hydroxypropyl) morpholine ester (Z-2), were synthesized, and their structures were characterized by LC–MS, IR, and ¹H NMR. The activation effects of the two compounds on tyrosinase activity were evaluated. Compounds Z-1 and Z-2 showed potent activation effects, and their EC₅₀ values were determined to be 0.075 and 0.0375 mmol/L, respectively. Moreover, the activation mechanism was determined to be of noncompetitive activation type. Interactions of the two compounds with tyrosinase were further analyzed by fluorescence quenching and molecular simulation assays. The results indicated that the effects on the tyrosinase activity were relative to the length of the carbon chain of the compounds. In addition, human M14 melanoma cells showed increased intracellular tyrosinase activity after treatment with the two compounds for 24 h. The expressions of TYR, TRP-1, TRP-2, and α-MSH proteins were upregulated in a dose-dependent manner during a 24-h treatment. These results suggested that compounds Z-1 and Z-2 might represent a novel approach for an effective therapy for diseases associate with tyrosinase dysfunction, such as vitiligo and hair graying.     

Antecedent short-term central nervous system administration of estrogen and progesterone alters counterregulatory responses to hypoglycemia in conscious male rats

The aim of this study was to test the hypothesis that antecedent short-term administration of estradiol or progesterone into the central nervous system (CNS) reduces levels of neuroendocrine counterregulatory hormones during subsequent hypoglycemia. Conscious unrestrained male Sprague-Dawley rats were studied during randomized 2-day experiments. Day 1 consisted of an 8-h lateral ventricle infusion of estradiol (1 mug/mul; n = 9), progesterone (1 mug/mul; n = 9), or saline (0.2 mul/min; n = 10). On day 2, a 2-h hyperinsulinemic (30 pmol.kg(-1).min(-1)) hypoglycemic (2.9 +/- 0.2 mM) clamp was performed on all rats. Central administration of estradiol on day 1 resulted in significantly lower plasma epinephrine levels during hypoglycemia compared with saline, whereas central administration of progesterone resulted in increased levels of plasma norepinephrine and decreased levels of corticosterone both at baseline and during hypoglycemia. Glucagon responses during hypoglycemia were unaffected by prior administration of estradiol or progesterone. Endogenous glucose production following day 1 estradiol was significantly lower during day 2 hypoglycemia, and consequently, the glucose infusion rate to maintain the glycemia was significantly greater after estradiol administration compared with saline. These data suggest that 1) CNS administration of both female reproductive hormones can have rapid effects in modulating levels of counterregulatory hormones during subsequent hypoglycemia in conscious male rats, 2) forebrain administration of reproductive hormones can significantly reduce pituitary adrenal and sympathetic nervous system drive during hypoglycemia, 3) reproductive steroid hormones produce differential effects on sympathetic nervous system activity during hypoglycemia, and 4) reduction of epinephrine resulted in significantly blunted metabolic counterregulatory responses during hypoglycemia.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKCα had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKCα in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1. © 1996 Wiley-Liss, Inc.     

Human TRP-1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of ³H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.