Ultrastructural localization of calcitonin in C-cells of dog thyroid; an immunocytochemical study

Summary Light microscopic observations of normal rat peritoneal mast cells and ultrastructural observations of human mast cells from lesions of nodular mastocytosis indicated that structural damage results in a pronounced increase in percentage of cells with a positive dopa reaction. Recent studies have indicated that the dopa reaction in mast cells is peroxidase-dependent. Enhancement of the dopa reaction by structural damage (latency) is probably related to increased substrate-enzyme interaction. Ultrastructural localization of dopa melanin to mast cell granules and the high percentage of mast cells showing a positive dopa reaction after structural damage is evidence against the possibility that dopa melanin is formed in mast cells by phagocytized enzyme.Supported by USPHS Grant Tl Am 5220, and by The General Research Support Fund, Boston City Hospital.     

Histochemical differentiation of peroxidase-mediated from tyrosinase-mediated melanin formation in mammalian tissues

Summary Preincubation with the copper-chelator, sodium diethyldithiocarbamate (DDC) and the presence of catalase in the incubation media allowed an accurate and reproducible differentiation of the role of tyrosinase from that of peroxidase in the oxidation of tyrosine and dopa in melanocytes, mast cells and eosinophils. These studies indicated that mammalian peroxidase in melanocytes, mast cells and eosinophils can mediate the conversion of tyrosine to melanin in the presence of dopa co-factor, as well as the conversion of dopa to melanin. With the methods employed, there was no evidence that tyrosinase in the preparations studied had significant ability to mediate the oxidation of tyrosine to melanin (even in the presence of dopa co-factor), although there was abundant evidence that it can mediate the conversion of dopa to melanin. Mammalian peroxidase may have roles in initiating melanin synthesis and catechol amine synthesis in vivo.Supported by USPHS Grant T1 AM 5,220, The General Research Support Fund, Boston City Hospital, and The Pathology Research Fund, St. Vincent Hospital.     

Continuous culture of normal adult mammalian hepatocytes exhibiting parenchymal functions

Summary Past in vitro studies of liver-cell functions have been performed on nonproliferating primary cells or serially propagated hepatic monolayers of neoplastic or fetal origin. We optimized conditions for the selective culture of adult rabbit and canine liver parenchymal cells and presently have four differentiated proliferating monolayer strains. At the 30th passage level these hepatic cultures still display the specific liver parenchymal functions of albumin and fibrinogen synthesis as well as tyrosine aminotransferase activity. Optimization of the conditions for hepatocyte culture was monitored by [³H]thymidine incorporation. Albumin and fibrinogen synthesis were measured by bioradioimmunoassay and tyrosine transaminase activity by a modification of Diamondstone's assay. Albumin and fibrinogen synthesis were correlated with hepatocyte growth kinetics. Supported by the Medical Staff Research and Education Fund, Wayne County General Hospital, Eloise, Michigan 48132, and American Cancer Society Institutional Grant No. 40M, The University of Michigan, Ann Arbor, Michigan 48109.     

Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL-1

The human eosinophilic leukemia cell line, EoL-1, differentiated with butyrate as an eosinophilic cellular model was evaluated for peroxidase-dependent tyrosine nitration. Butyrate suppressed cell growth and induced eosinophilic granules in EoL-1 cells after 9 days of culture. Peroxidase activity was detected biochemically and histochemically from 3-day cultures and it increased in a time dependent manner. This peroxidase activity was inhibited by cyanide. Nitrotyrosine formation catalysed by peroxidase using hydrogen peroxide and nitrite was detected at a high level similar to that of mature eosinophils. However, no expression of eosinophil peroxidase (EPO) was detected by RT-PCR or immunocytochemistry. In contrast, the induction of myeloperoxidase (MPO) by butyrate was clearly detected by RT-PCR, Northern blot, and immunocytochemical staining. These results suggest that butyrate induces MPO rather than EPO in EoL-1 cells and that the formation of nitrotyrosine in butyrate-induced cells is dependent on MPO.     

Immunohistochemical localization of a specificileal peptide in the pig

Summary The tissue distribution of a polypeptide purified from pig ileal mucosa tentatively called porcine ileal polypeptide (PIP) and known to have potent acid secretagogue activity has been studied with immunohistochemical methods together with extraction of different tissues followed by radioimmunoassay for PIP content. Histochemically the peptide is found in superficial epithelial cells in the mucosa of the distal 20% of the small intestine and to some extent in the mucosa of the urinary tract. There is no staining of goblet cells or crypt cells. The staining in the urinary tract mucosa is due to antigenic peptides with Mr identical to PIP. While the presence of PIP in the ileum is compatible with a function as an enterooxyntin, it is not possible at present to explain the physiologic role of PIP entirely as a hormone regulating acid secretion in light of the immunohistochemical distribution.Supported in part by a grant from the NIH AM-27077 and the Sinai Hospital General Research Fund     

DOPA compared with dihydroxyfumarate as co-factor in peroxidase-mediated oxidation of tyrosine to melanin

Summary Dihydroxyfumarate was used as a co-factor in the histochemical demonstration of peroxidase-mediated oxidation of tyrosine to melanin in eosinophils, mast cells, melanoma cells and neurons. The use of dihydroxyfumarate as co-factor provides a direct method for demonstrating peroxidase-mediated oxidation of tyrosine to melanin in tissues, and allows for the ultrastructural localization of this pigment.Supported by USPHS Grant T1 AM 5220 and by the St. Vincent Hospital Research Foundation.     

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.     

Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing

Peroxidase activity in isolated thylakoids from degreening canola ( Brassica napus cv. Westar) seeds was demonstrated. The enzyme catalyzes the degradation of thylakoid-bound pigments in the presence of H₂O₂ and 2,4-dichlorophenol. Peroxidase activity is related to degreening, with periods of rapid degreening associated with high enzyme activity. Both de novo synthesis and substrate availability appear to control enzyme activity. Peroxidase is initially inhibited and then stimulated by sublethal freezing. Therefore, inhibition of peroxidase activity following sublethal freezing may be responsible, in part, for a failure of the seed to degreen.     

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.     

抑制黑色素合成的乳酸菌胞外多糖的筛选和性质研究

【目的】筛选可抑制黑色素合成的乳酸菌胞外多糖。【方法】通过观察凝乳拉丝外观筛选产胞外多糖的乳酸菌菌株,测量胞外多糖对B16黑色素瘤细胞黑色素合成和细胞活力的影响。对胞外多糖进行纯化,并通过PMP衍生-HPLC、红外光谱、抑制酪氨酸酶活性、抗氧化能力对其单糖组成和结构、作用机制进行研究。【结果】筛选到一株乳酸菌Lactobacillus rhamnosus HLAB122,发酵产生的胞外多糖在5 g/L浓度下可使B16细胞黑色素产量下降至空白对照的32.7%,且在96 h内对细胞活力无影响。纯化后的多糖由鼠李糖、葡萄糖、半乳糖构成,各单糖摩尔比为1?5.44?5.37。该胞外多糖不抑制酪氨酸酶活力且抗氧化性微弱。【结论】L.rhamnosus HLAB122产生的胞外多糖在个人护理产品中有潜在应用价值。     

Electron microscope autoradiographic studies of RNA metabolism in Trillium erectum microspores

3H-Uridine has been used to investigate the sites of RNA synthesis in the post-meiotic G₁ phase of Trillium microspores using electron microscope autoradiography. The dilute, non-condensed component of the nucleus has been found to be the site of synthesis. When the labelled cells were further incubated in non-radioactive medium the label was found to shift towards the condensed chromatin regions within the nucleus. Two hypotheses to explain the observations are considered, one involving migration of the RNA from the relaxed to condensed regions, the other involving a change in state of the nuclear regions involved in the synthesis. The data are interpreted as favoring the latter possibility.This study supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and from the Swiss National Fund, Grant Nr. 3202.Fellow of the Jane Coffin Childs Fund for Medical Research.     

Temperature and species differences in susceptibility of kidney cell cultures to mercury toxicity

Summary The effect of temperature on inorganic mercury toxicity was investigated using kidney tissue culture systems. The relative susceptibility of rabbit (homeothermic) kidney to mercury intoxication was compared to that of Coho salmon (poikilothermic) kidney over temperature ranges consistent with the habitat of each of the two species. It was demonstrated that susceptibility to mercury toxicity is species dependent; that is, the rabbit kidney cells tolerated higher mercury concentrations in the medium than did the fish-derived cells. Within a given species, susceptibility to mercury toxicity was temperature dependent. Decreasing the temperature increased the toxicity of mercury to cultures of rabbit kidney cells, whereas decreasing temperatures decreased the effect of mercury toxicity on the salmon kidney cells. As a consequence, fish taken from arctic waters are liable to be more toxic when introduced into mammalian food chains. Albumin was shown to act as a protective agent in vitro against inorganic mercury toxicity. Research was supported in part by the University of Victoria Faculty Grant No. 08-869 and a Medical Staff Research and Education Fund Grant from Wayne County General Hospital, Eloise, Michigan.     

Ribosomal proteins of mouse kidney: Normal status and during compensatory renal hypertrophy

Summary Ribosomes were isolated from normal and growing kidney and the protein complement was examined by a two-dimensional gel electrophoretic procedure. Proteins were resolved in the first dimension on the basis of charge and, in the second dimension, on the basis of molecular weight. 60S and 40S ribosomal subunits from normal kidney contained respectively 42 and 31 proteins. 80S ribosomes contained 23 proteins not found with either sub-unit. Nineteen of these proteins were removed from the ribosomes when isolated ribosomes were washed in a high salt buffer. Six proteins of the 80S ribosome corresponded to proteins associated with both sub-units. 80S ribosomal proteins were also studied during compensatory renal hypertrophy after 4-96 h of induced growth. The protein complement displayed by electrophoresis was identical to the pattern seen from normal renal cells.Abbreviations Bis-Tris [bis(2-Hydroxyethyl)imino-tris (Hydroxymethyl)methane] - MES 2(N-morpholino)ethane sulfonic acid Supported by NIH Grants AM-12769 and RR-05486 and the Damon Runyon-Walter Winchell Fund. Dr. Irwin is a fellow of the Damon Runyon-Walter Winchell Fund (DRG-51-F). Dr.Northrup is a Research Fellow in Developmental Medicine (HD00362) at Massachusetts General Hospital.     

D‐tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity

Although L‐tyrosine is well known for its melanogenic effect, the contribution of D‐tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L‐tyrosine, D‐tyrosine dose‐dependently reduced the melanin contents of human MNT‐1 melanoma cells and primary human melanocytes. In addition, 500 μM of D‐tyrosine completely inhibited 10 μM L‐tyrosine‐induced melanogenesis, and both in vitro assays and L‐DOPA staining MNT‐1 cells showed that tyrosinase activity is reduced by D‐tyrosine treatment. Thus, D‐tyrosine appears to inhibit L‐tyrosine‐mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D‐tyrosine inhibited melanogenesis induced by α‐MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D‐tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D‐tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte‐derived cells.     

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

孕烷X受体配体结合结构域蛋白晶体的X线衍射结构解析

目的:利用X线衍射技术解析孕烷X受体(PXR)配体结合结构域(LBD)蛋白晶体的3维结构。方法:对PXR蛋白LBD(130~434氨基酸残基)序列进行密码子优化并化学合成后克隆至pRSFDuet-1表达载体,再将载体导入大肠杆菌BL21(DE3),对PXR-LBD蛋白进行原核表达与分离纯化;采用晶体筛选试剂盒筛选蛋白结晶条件,采用悬滴法获得目标蛋白的晶体;对获得的蛋白晶体进行X线晶体衍射检测,并收集相关数据建立PXR-LBD的三维结构。结果:获得了PXR-LBD的高质量晶体并利用X线衍射解析了该蛋白质晶体的结构数据,使用Phenix.refine软件和COOT软件等对结构进行修正,最终获得了高分辨率的3维结构数据。结论:完成了孕烷X受体配体结合结构域蛋白晶体的X线衍射结构解析,为研究和开发PXR相关药物奠定了基础。     

Influence of low dozes of ionizing radiation on accumulation of melanin pigments and catalase and superoxidedismutase activities in Cladosporium cladosporioides

Influence of low dozes of ionizing radiation on melanin pigments synthesis and activity of antioxidant enzymes catalase and superoxidedismutase of two strains of Cladosporium cladosporioides 4, (isolated from radioactive soil) and 396 (control) were investigated. It was shown, that in C. cladosporioides 4 under the exposure of ionizing radiation an increase of melanin synthesis in a stationary growth phase and increase of superoxidedismutase activity in a logarithmic phase were observed; in the control strain C. cladosporioides 396 activation of melanin synthesis and superoxide dismutase activity in both growth phases was revealed. It was established that in C. cladosporioides 4 the endocellular catalase activity in a logarithmic phase is 3.2 times higher, than in control strain. Under the action of ionizing radiation a 2-fold increase of this enzyme activity unlike the control strain in which the activity inhibition was revealed. The obtained results testify to the complex response of antioxidant systems and melanin to the action of low dozes of radiation which depends on the growth phase and presence of radioadaptation properties in the investigated fungi.     

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine.

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and beta-1, 3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and beta-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conclusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.