N-Methyl-D-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric Oxide/cGMP signal transduction pathway. A novel possible role for glutamate as physiologic activator of melanogenesis

The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefish Sepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time that l-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via the N-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l-glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO-stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3, 5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway.     

The melanogenic system of the liver pigmented macrophages of Rana esculenta L.--tyrosinase activity

The enzyme system responsible for Amphibian Kupffer Cell (KC) melanogenesis has not been entirely elucidated. This research demonstrates that the KC melanosomes of Rana esculenta L. possess a tyrosine-hydroxylase (TH) activity, showing that a tyrosinase is the enzyme involved in the melanogenesis. The TH reaction depends on catalytic Dopa as a cofactor and is not affected by catalase or H2O2, showing that it is catalysed by the tyrosinase and not by the peroxidase present in the melanosomes. The TH reaction is activated by Cu2+ ions but not by other tyrosinase activators such as limited proteolysis, protein ageing, and Sodium Dodecyl Sulphate (SDS). SDS inhibited the KC TH activity even below the critical micelle concentration. All these results suggest that the KC-tyrosinase differs in structure from other known tyrosinases. Using anti-KC-tyrosinase antobodies, we observed that the sites of the tyrosinase location within the cell are the same as those described in the melanocytes. In the immunoblots, the anti-KC-tyrosinase antibodies also recognised two protein bands, at the higher molecular weight ranges, in the protein electrophoretic pattern. Moreover, the tyrosinase activity was limited to the highest molecular weight band of about 260 kDa, suggesting that the enzyme activity could depend on a molecular aggregate. The melanin produced in the liver was found to be a 5,6-dihydroxyindole-rich eumelanin similar to the Sepia melanin.     

Transient Expression of High Molecular Weight,Heat Sensitive,Trypsin-Resistant Form of Tyrosinase in B-16 Melanoma Cells

High molecular weight forms of tyrosinase have been found to be expressed during spontaneous remelanization of the amelanotic B-16 melanoma cells in culture as well as in melanotic tumors formed from amelanotic melanoma cells grown in C57BL/6J mice. Overnight extraction of the crude melanosomal fractions from such tumors and cultured melanoma cells reveal the presence of an additional DOPA-MBTH positive band well below the stacking gel. This band has been found to be α-PEP7 (antibody specific for tyrosinase) positive and α-PEPl (antibody specific for TRP-1) negative on Western blot analysis. Heat treatment at 60°C for 60 min results in the loss of this band and considerable loss of activity of the melanosomal extract. Trypsin treatment of these melanosomal extracts resulted in a minor change in the mobility of the high molecular weight band. SDS-PAGE under reduced conditions followed by Western blotting revealed that the high molecular weight band was lost and not detected by α-PEP7 or α-PEPl. These findings indicate that high molecular weight, heat sensitive and trypsin resistant forms of tyrosinase are transiently expressed in B-16 melanoma cells and tumors that are initiating remelanization following phenotypic drift towards the amelanotic state.     

Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus

A toxic protein, called bolevenine, was isolated from the toxic mushroom Boletus venenatus based on its lethal effects on mice. On SDS-PAGE, in either the presence or absence of 2-mercaptoethanol, this protein showed a single band of approximately 12 kDa. In contrast, based on gel filtration and MALDI-TOFMS, its relative molecular mass was estimated to be approximately 30 kDa and approximately 33 kDa, respectively, indicating that the protein consists of three identical subunits. This toxin exhibited its lethal activity following injection at 10mg/kg into mice. The N-terminal amino acid sequence was determined up to 18, and found to be similar to the previously reported bolesatine, a toxic compound isolated from Boletus satanas.     

Lectins from bulbs of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae).

The isolation of three lectins with similar N-terminal amino acid sequences from the bulbs of the Chinese daffodil Narcissus tazetta was achieved. The isolation protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on mannose-agarose, and fast protein liquid chromatography-gel filtration on Superose 12. The lectins were all adsorbed on mannose-agarose and demonstrated a single band with a molecular weight of 13 kDa in SDS-polyacrylamide gel electrophoresis and a single 26 kDa peak in gel filtration, indicating that they were mannose-binding, dimeric proteins. The lectins differed in hemagglutinating activity, with the magnitude of the activity correlating with the ionic strength of the buffer required to elute the lectin from the DEAE-cellulose column. The bulb lectin did not exert potent cytotoxicity against cancer cell lines or fetal bovine lung cells but inhibited syncytium formation in, and reinstated viability of, fetal bovine lung cells infected with bovine immunodeficiency virus.     

Isolation and purification of a Campylobacter upsaliensis autolysin.

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS)-PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.     

Nitric oxide synthase in the nervous system and ink gland of the cuttlefish Sepia officinalis: molecular cloning and expression

Nitric oxide (NO) signaling is involved in numerous physiological processes in mollusks, e.g., learning and memory, feeding behavior, neural development, and defence response. We report the first molecular cloning of NOS mRNA from a cephalopod, the cuttlefish Sepia officinalis (SoNOS). SoNOS was cloned using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with RACE procedure. Two splicing variants of SoNOS, differing by 18 nucleotides, were found in the nervous system and the ink gland of Sepia. In situ hybridization shows that SoNOS is expressed in the immature and mature cells of the ink gland and in the regions of the nervous system that are related to the ink defence system.     

Tyrosine hydroxylase purification from rat PC12 cells.

Tyrosine hydroxylase was purified in high yield from rat PC12 cells. This three-day procedure consisted of differential ammonium sulfate precipitation, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. It yielded an average of 15 mg of purified protein from 100 flasks of PC12 cells, with greater than 40% recovery of tyrosine hydroxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single protein band with a molecular weight of approximately 60,000. The protein had a specific activity of 670 nmol/min/mg and had a Km for its reducing cofactor tetrahydrobiopterin of 1.8 mM. The purified protein can be phosphorylated and activated by cyclic AMP-dependent protein kinase.     

Melanogenesis in the ink gland of Sepia officinalis

Among the various melanin-producing systems, the ink gland of the cuttlefish (Sepia officinalis) has traditionally been regarded as a most convenient model system for the studies of melanogenesis. The ink gland is a highly specialized organ with immature cells in the inner portion, from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to particulate melanosomes. When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand. Biochemical studies carried out over the past two decades have shown that the ink gland contains a variety of melanogenic enzymes, including tyrosinase, a peculiar dopachrome rearranging enzyme (which catalyses the rearrangement of dopachrome to 5,6-dihydroxyindole) and a peroxidase (presumably involved in the later stages of melanin biosynthesis). These enzymes are functionally interactive in close subcellular compartments of ink gland cells and appear to act in a concerted fashion during the process of melanogenesis in the mature portion of the gland. More recent studies have revealed that ink production and ejection are affected and modulated by the N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP (cGMP) signalling pathway. Glutamate NMDA receptor and NO synthase, the enzyme responsible for the synthesis of NO, have been detected by biochemical and immunohistochemical techniques in immature ink gland cells. Stimulation of NMDA receptors caused a marked elevation of cGMP levels, activation of tyrosinase and increased melanin synthesis in the mature portion of the gland, via the NO-guanylyl cyclase interaction. This signalling is also present in different regions of the nervous system in Sepia and in certain neural pathways controlling contraction of the ink sac sphincters and wall muscle in the ejection mechanism. Overall, these and other findings allowed elaboration of an improved model of melanin formation in Sepia, which underscores the complex interplay of melanogenic enzymes and regulatory factors, highlighting both the similarities and the differences with melanogenesis in mammals.     

Tarantula (Eurypelma californicum) venom, a multicomponent system

The venom of the tarantula Eurypelma californicum was analysed biochemically, the components were isolated and characterized. The pH value of the crude venom is 5.3 +/- 0.3. After dilution with distilled water, UV-absorption spectra showed a single maximum at 258 nm (pH ca. 7.0). A second maximum at 328 nm emerged above pH 8.0. Protein concentration of the venom is ca. 65 mg/ml. After Coomassie staining SDS-PAGE patterns show three major bands with apparent molecular masses around 40 kDa, 4.3 kDa and 1.3 kDa besides some weak high molecular protein bands. The following low-molecular mass constituents were determined in the crude venom: ATP, ADP, AMP, glutamic acid, aspartic acid, gamma-aminobutyric acid, glucose and the ions potassium, sodium, calcium, magnesium and chloride; the osmolality was 361 micro0smol/ml. The LD50 value for female cockroaches was 0.15 microliters venom per g body weight and for male cockroaches 0.4 microliters venom per g body weight. Separation of the crude venom by gel chromatography yielded four elution peaks. Peak I contains the enzyme hyaluronidase. The activity is 200-900 U/microliters. Peak II contains a mixture of toxic peptides. Peak III contains the 1.3-kDa components of SDS-PAGE and peak IV mainly contains ATP. Venom proteins including the enzyme hyaluronidase were precipitated by 5% trichloroacetic acid. The supernatant was separated by HPLC into 13 fractions. Fraction 1 contains glutamic acid, aspartic acid, gamma-aminobutyric acid and ATP; fraction 2 contains ATP, ADP and AMP as well as a component 2' visible in SDS-PAGE as 1.3-kDa band and consisting of spermine and tryptophan; fraction 3 contains ATP and an unknown component 3'; fractions 4-6 also show a 1.3-kDa band in SDS-PAGE, fraction 4 being tyrosylspermine and fractions 5 and 6 containing compounds of spermine and aromatic molecules; fraction 7 contains a peptide which lacks aromatic amino acids, it was sequenced from the N-terminus; fractions 8-13 contain very similar toxic peptides. The peptides in fractions 11 and 12, labeled ESTX for Eurypelma spider toxin, were cleaved with different enzymes and sequenced. They differ in one amino acid in position 26. Homologies with scorpion toxins and with a toxin of the spider Segestria florentina were found.     

Melanogenesis inhibitory effect of low molecular weight fucoidan from <Emphasis Type="Italic">Undaria pinnatifida</Emphasis>

In this study, fucoidans with different molecular weight that were isolated from the brown alga Undaria pinnatifida (Phaeophyceae, Laminariales) were investigated for their ability to inhibit melanogenesis and scavenge superoxide and hydroxyl radicals. Fucoidan samples with low molecular weights of 89, 35, 17, and 6 kDa were prepared by radiation-degradation of a 378 kDa fucoidan isolated from U. pinnatifida. The inhibitory activity of fucoidan against melanin biosynthesis in B16BL6 melanoma cells was enhanced for low molecular weight samples. To investigate the increase in melanogenesis inhibition exhibited by the low molecular weight fucoidan, tyrosinase inhibition activity and radical scavenging activities were measured. There was an increase in the tyrosinase inhibition activity for low molecular weight samples. Additionally, the radical scavenging activity was increased for lower molecular weight fucoidans. These results suggest that low molecular weight fucoidans from seaweeds may have beneficial biological properties.     

中国林蛙卵核糖核酸酶的分离纯化及其抗肿瘤作用

以中国林蛙卵为原料,采用丙酮分级沉淀、SP-Trisacryl阳离子交换色谱、Sephadex G-75凝胶过滤色谱、C8反相色谱等纯化方法,得到一种具有核糖核酸酶活性的蛋白质,采用SDS-PAGE电泳对该蛋白质进行了相对分子质量和纯度测定。结果表明：纯化的中国林蛙卵核糖核酸酶为相对分子质量13kDa的单一成分。该酶最适反应温度为65℃,最适反应pH为5.5~6.0,米氏常数为4.11μmol/L,最大反应速率为2.82 pmol/s。在体外细胞毒性实验中,对人三种肿瘤细胞HeLa、K562、MCF-7具有抑制作用,其IC50分别为0.6μmol/L、0.8μmol/L和4μmol/L,而对于正常人成纤维细胞在酶浓度达到8μmol/L时仍未见明显细胞毒性。这种从中国林蛙卵中分离纯化出的具有选择性细胞毒性的小分子量核糖核酸酶,为肿瘤的治疗提供了新的候选蛋白分子。     

Low molecular weight phosphotyrosine protein phosphatase from PC12 cells. Purification,some properties and expression during neurogenesis in vitro and in vivo

The purification and partial characterization of low molecular weight phosphotyrosine phosphatase (LMW-PTP) was reported for the first time in PC12 cells. In addition, the expression levels during neuronal phenotype induction by nerve growth factor (NGF) and during neurogenesis in chick embryos were investigated. LMW-PTP was purified to homogeneity and showed a single band of about 18 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. A native molecular mass of 20.1 kDa was determined by gel filtration on Sephadex G-75 column. The LMW-PTP from PC12 cells displays structural and biochemical characteristics similar to the enzyme isolated for normal tissues. It was specifically immunoprecipitated by an affinity purified antibody directed against the bovine liver enzyme. The enzyme is present in the cytosolic and cytoskeletal cell compartment where is tyrosine phosphorylated. Time course expression of LMW-PTP in PC12 cells was investigated after NGF treatment and showed an increase of about 30% in the basal level of LMW-PTP from 0 to 72 h. These changes were related to the appearance in PC12 cells of neuronal processes and to a decrease in cell proliferation. An increase of the LMW-PTP expression was also observed in vivo during chick embryo neurogenesis from 8-day-old embryos to adult chicks. The protein level, assayed by immunoblotting, increases from 14-day-old embryos to the hatched chicks reaching the adult levels within the first week after birth. These data indicate that the neurogenesis process is accompanied by a physiological increment of LMW-PTP expression in vitro and in vivo.     

Seasonal variations of Rana esculenta L. skin tyrosinase

Various enzymes are known to be involved in melanin biosynthesis, but the key role appertains to tyrosinase. In amphibians this enzyme displays peculiar characteristics: i) it requires an activation process; ii) its level of enzymatic activity in the animal skin changes depending on the season. In this work, by using chymotrypsin, subtilisin and SDS as putative activators, we studied the activation process of the skin pro-tyrosinase of Rana esculenta L. in different seasons over a period of two years. We found that chymotrypsin and subtilisin were able to yield an active enzyme, but not SDS. The maximum levels of tyrosinase activity were recorded in winter and the minimum in summer. We detected tyrosinase activity in the melanosomal fraction, where the enzyme form was least sensitive to proteolytic activation, probably corresponding to a "mature" tyrosinase. The enzyme forms found in the microsomal and soluble fractions were more sensitive to proteolytic activation, probably corresponding to "immature" tyrosinase. On SDS-PAGE, the tyrosinase activity assays showed a dopa-positive band at 200 kDa and a second aggregated band with a still higher molecular mass. The significance of these results in frog melanogenesis regulation is discussed.     

Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production of reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular weight of toxin was estimated by chromatography and SDS-PAGE. Two protean peaks with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 13.3 kDa). The relative amounts between the peaks (P1/P2 = 0.36) changed when 2-mercaptoethanol was employed (P1/P2 = 0.59). When Sephadex G-200 chromatography was performed, a protean peak of Ve = 113 mL (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa. SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented three bands of trimer, dimer, and monomer. An increase of casein hydrolysate and albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin.     

Effect of an abundant human skin melanosomal 66 kDa protein (MP 66) on m urine tyrosinase: Its physiological implications on melanogenesis

A single polypeptide protein (MP 66) of molecular weight 66 kDa purified to homogeneity from melanosomes of normal human skin epidermal melanocytes, was partially characterized. The isoelectric point of MP 66 is in the range of 7.3 to 7.6. This protein, which was shown to inhibit partially purified human skin tyrosinase activity at pH 6.8, also inhibits murine tyrosinase at pH 6.8. However, at pH 5.0, it stimulates murine tyrosinase activity. The physiological implications of these results are discussed.     

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.     

Evidence of the presence of GTPase inhibiting proteins for a low M(r) GTP-binding protein, ram p25, in rat spleen and pheochromocytoma PC12 cells.

1. GTPase inhibiting activity for a low M(r) GTP-binding protein, ram p25, was detected in the cytosolic fractions of rat spleen and PC12 cells. 2. The inhibitors were heat-labile and trypsin-sensitive, indicating that they were of proteinous nature. 3. The molecular mass of the inhibitor in the spleen appeared to be about 65 kDa on the elution profile from the gel filtration column.     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Cytotoxic component(s) of Klebsiella oxytoca on HEp-2 cells

A cytotoxic component(s) was detected in culture filtrates of Klebsiella oxytoca isolated from patients with antibiotic-associated hemorrhagic colitis. Eleven of 12 isolates exhibited cytotoxicity on HEp-2 cells. The cytotoxic activity of K. oxytoca strain MH43-1 was stable for the treatment of 60 C for 30 min, but inactivated by the treatment of 100 C for 15 min. This cytotoxicity was not destroyed by the treatment with trypsin or pronase, and the component was filtrable through a membrane filter which cut off molecular weight 5,000.