Melanosomal Proteins – Role in Melanin Polymerization

Melanin, the major determinant of skin colour, is a tyrosine‐based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post‐tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin–protein interaction is not nonspecific.     

Confocal pH Topography in Plant Cells — Acidic Layers in the Peripheral Cytoplasm and the Apoplast

The distribution of intracellular pH was studied in cultured cells of Gossypium hirsutum by con-focal pH topography using the fluorescent probe car-boxy SNARF1 and a ratio imaging procedure. The resulting pH maps can visualize pH differences with an accuracy of 0.1 unit in the investigated range between 7.5 and 5.6. They reveal the following characteristic features of the Gossypium cells: – the pH of the cytoplasmic core regions ranges from near 7.4 in younger to near 6.0 in older cells; – vacuoles show the expected acidity with pH < 5.6; – the cell wall/apoplastic region is acidic with a pH near 5.6 or below, especially in young, growing cells; – interestingly, acidic areas appear also at the periphery of the cytoplasm, i.e. beneath the plasma membrane. They remain stable in the presence of 5/μmol/I of the protonophore CCCP. Acidic layers of peripheral cytoplasm were also detected in protoplasts of Penicillium cyclopium, i.e. eukaryotic cells of simpler structure, which served as a reference object. This ronfirms earlier findings obtained with classical fluorescence microscopy and another fluoroprobe (fluorescein diacetate). Though additional experimental support is needed, low pH regions at the cytoplasm/plasma membrane interface should be considered a real contribution to the pH control of plant and fungal cells, facilitating e.g. the maintenance of cytosolic pH in acidic environments.     

Two Acidic Proteins Associated with Brain Development in Chick Embryo

The developmental changes in protein composition of the chick optic tectum were analyzed by two-dimensional gel electrophoresis. Staining with Coomassie Brilliant Blue R revealed 54 major proteins, eight of which remarkably changed their abundance during development: Four of these proteins (S8, S14, S30, and S54) increased and two of them (S7 and S37) decreased in the course of the brain development. The other two proteins (S5 and S6) appeared at specific embryonic stages and were not detected in the adult. The abundance of S5 protein was highest at day 7, and that of S6 protein at days 9-18. The two proteins were present in other regions of the embryonic brain but were not detected in the embryonic liver. The proteins were purified from the soluble fraction of embryonic chick brains by pH 5.5 precipitation, DEAE-Sepharose column chromatography, ammonium sulfate precipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of S5 and S6 proteins were 95,000 and 100,000, respectively, and their isoelectric points were about 4.5. They were compared by peptide mapping using V8 protease and found to share 11 common peptides out of 17 distinct ones. This indicates a strong degree of structural homology between these two proteins.     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin     

Reversible inactivation of serpins at acidic pH

The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.     

The Effect of Sodium Iodate and Melanin on the Formation of Glyoxylate

Sodium iodate damages retinal pigment epithelium specifically, but the reason for this specificity is not well understood. The work reported here describes an effect of sodium iodate on melanin, a major component of the retinal pigment epithelium. Sodium iodate increases the ability of melanin to convert glycine to glyoxylate. Almost ten times as much glyoxylate is formed when sodium iodate is present compared to the amount formed with melanin alone, although iodate alone does not convert glycine to glyoxylate. A chemical reaction between sodium iodate and melanin is suggested as a partial explanation of the specificity of iodate toxicity towards retinal pigment epithelium.     

Recombinant Protein Production with Pichia pastoris in Continuous Fermentation – Kinetic Analysis of Growth and Product Formation

Continuous fermentation was applied to the production of recombinant human chymotrypsinogen B (hCTRB) by the methylotrophic yeast Pichia pastoris as a tool for the kinetic analysis of growth and product formation. Using methanol as the sole source of carbon, energy, and induction, cell growth could be described by a non‐competitive Monod approach. Maximum growth rate μ_max was determined to 0.084 h^‐‐1 and the K_M‐value for methanol to 0.22 g·L^‐‐1, respectively. With respect to product formation, a similar model was established exhibiting a methanol concentration of 0.13 g·L^‐‐1 as the K_M‐value and a maximum biomass‐specific product‐formation rate of π_max = 0.23 mg·g^‐‐1·h^‐‐1. The production of hCTRB was strictly growth‐coupled. The data provided covers the range of methanol concentrations between 0 and 4 g·L^‐‐1. Substrate concentrations exceeding this upper value led to a complete collapse of product formation. This change in phenotype turned out to be irreversible indicating a genetic instability of transformed Pichia pastoris caused by excess methanol.     

A test of Tyler’s Line – response of chironomids to a pH gradient in Tasmania and their potential as a proxy to infer past changes in pH

1. Tyler’s Line delimits two distinct limnological provinces that reflect differences in climate, geology and vegetation in Tasmania. Lakes west of Tyler’s Line are typically acidic and dystrophic with relatively shallow euphotic zones, whereas eastern lakes are circumneutral and oligotrophic or ultra‐oligotrophic, allowing deeper penetration of light. Consequently, Tyler’s Line defines a boundary where species assemblages change over a relatively short distance. 2. A survey of 48 Tasmanian lakes was undertaken to identify indicator taxa of the two limnological provinces and breakpoints along the pH gradient where shifts in taxa occur. Chironomidae (Diptera) were used because they are ideal candidates for lake classification. 3. Three independent methods (geographical position, piecewise linear regression, two‐way indicator species analysis) verified that chironomids accurately reflect the environmental variables defining Tyler’s Line at lake and catchment scales. Chironomid genera are often speciose, and members of the same genus can have markedly different responses to a given environmental variable. Although the types of taxa changed along the pH gradient, richness did not. This finding contrasts with many studies from the northern hemisphere but accords with other studies from Australia. 4. Models of pH, developed using both partial least squares and weighted averaging partial least squares, can be used to understand past natural variability of pH in Tasmania and to test hypotheses regarding the timing, magnitude and source of contamination in impacted aquatic ecosystems.     

Over‐expression of MSG1 Transcriptional Co‐activator Increases Melanin in B16 Melanoma Cells: A Possible Role for MSG1 in Melanogenesis

MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase.     

Production of pigment-free pullulan by swollen cell in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> NG which cell differentiation was affected by pH and nutrition

A black yeast strain “NG” was isolated from strawberry fruit and identified as Aureobasidium pullulans. Strain NG displayed yeast-like cell (YL), swollen cell (SC), septate swollen cell (SSC), meristematic structure (MS), and chlamydospore (CH) morphologies. pH was the key factor regulating cell morphogenesis of strain NG. Differentiation of YL controlled by extracellular pH had no relationship with nutrition level. YL was maintained at pH >6.0, but was transformed into SC at pH ∼4.5. SC, a stable cell type of A. pullulans, could bud, septate, or transform into MS or CH, in response to nutrition level and low pH. SC produced swollen cell blastospores (SCB) at pH 2.1 with abundant nutrition, and could transform into MS at lower pH (1.5). SC was induced to form CH by low level nutrition and pH <3, and this transition was suppressed by adjusting pH to ∼4.5. Crude polysaccharides without pigment (melanin) were produced by SC of strain NG. Pullulan content of the polysaccharides was very high (98.37%). Fourier-transform infrared spectroscopy confirmed that chemical structures of the polysaccharides and standard pullulan were identical. Swollen cells produced 2.08 mg/ml non-pigmented polysaccharides at 96 h in YPD medium. Controlling pH of fermentation is an effective and convenient method to harvest SC for melanin-free pullulan production.     

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch

Background

G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied.

Methods

Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation.

Results

G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle.

Conclusions

Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch.

General significance

These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates.     

Proteome of Acidic Phospholipid-binding Proteins: SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES*

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Whereas Coronin 1A co-localizes with PI(4,5)P₂ at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P₂ signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P₂-containing liposomes and that PI(4,5)P₂ antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.     

Inhibition of UVR‐Induced Tanning and Immunosuppression by Topical Applications of Vitamins C and E to the Skin of Hairless (hr/hr) Mice 1

Exposure of C3HBYB/Wq hairless (hr/hr) mice to ultra‐violet radiation (UVR) for 15 days induced intense tanning of their dorsal skin. Small, dark freckles appeared first, gradually enlarging and coalescing as treatment progressed yielding a uniform tan. Histologically, the gross changes in skin color were matched initially by the appearance of scattered epidermal melanocytes that subsequently proliferated to form discrete, progressively expanding and abutting populations resulting in a uniform melanocyte network throughout the basal layer of the interfollicular epidermis. In contrast, when applied topically before each daily exposure to UVR, a cream or lotion vehicle containing both vitamins C and E (Vits C/E) inhibited UVR‐induced erythema and tanning. Application of Vits C/E, both before and after irradiation, was no more effective in providing photoprotection than pre‐treatment only. At the tissue level, UVR‐induced proliferation and melanogenesis of melanocytes were reduced compared with irradiated controls. The density of individual melanocyte populations was reduced, as was the number of melanocyte populations achieving merger (confluence) with others. Confluence grades and cell counts, estimating the maximum density of melanocyte populations in UVR–Vits C/E‐treated mice, were approximately two thirds those of UVR–vehicle‐treated controls. However, tanning was only one fifth that of UVR–vehicle‐treated controls, suggesting that melanogenesis was also inhibited. In addition to its inhibitory actions on irradiated melanocytes, Vits C/E also inhibited UVR‐induced suppression of contact hypersensitivity (CHS) in haired (Hr/hr) and hr/hr mice of the C3HBYB/Wq strain. The common denominators for most, if not all, of the influences of topically‐applied Vits C/E in muting the responses of the melanocyte and immune systems to UVR may stem from the vitamins’ combined ability to suppress UVR‐stimulated inflammation and its associated cascade of mediators.     

粗糙脉孢霉酸性蛋白酶基因的克隆与鉴定

以粗糙脉胞霉CICIM F00021染色体DNA为模板,通过PCR扩增得到粗糙脉胞霉酸性蛋白酶结构基因。对其序列进行了测定和分析,表明扩增得到的片段为酸性蛋白酶基因。将扩增出的酸性蛋白酶基因克隆入酵母表达载体中获得重组质粒pPIC9K-ap。将其转化入毕赤氏酵母获得重组菌NA3。重组酸性蛋白酶在pH4．0和45℃下表现出最高活性。     

Conformational transition and mass transfer in extraction of proteins by AOT–alcohol–isooctane reverse micellar systems

We examined quantitatively the effect of alcohols on protein and reverse micellar structure. We used circular dichroism (CD) to compare the effects of various alcohols on the protein structure, and percolation phenomena to evaluate the effects of various alcohols on reverse micellar structure. Upon the addition of alcohols to the bulk aqueous phase, proteins were denatured significantly, depending on the alcohol species and concentration, suggesting that use of alcohol directly to the stripping solution is not effective in back-extraction processes of proteins. In the present study, a new method, a small amount of alcohol is added to the surfactant–organic solution to improve the back-extraction behaviors of proteins. Practically, in the back-extraction process, the alcohols suppressing the cluster formation of reverse micelles (high value of β_t), remarkably improved the back-extraction behavior of proteins. In addition, the same alcohol molecules showed a positive effect on the rate and fraction of protein back-extraction. From a result of the CD measurement of the back-extracted proteins, it was known that the alcohols added to reverse micellar solution allowed the proteins to back-extract safely without causing structural changes. These results show that the values of β_t, defined by the variation of percolation processes, and the back-extraction behaviors of proteins have a good relationship, suggesting that the back-extraction processes were controlled by the micellar–micellar and protein–micellar interactions.     

State-dependent Inhibition of TRPM2 Channel by Acidic pH

Transient receptor potential melastatin 2 (TRPM2) channel fulfills an important role in oxidative stress signaling in immune and other cells, to which local extracellular acidosis is known to occur under physiological or pathological conditions and impose significant effects on their functions. Here, we investigated whether the ADP-ribose-activated TRPM2 channel is a target for modulation by extracellular acidic pH by patch clamp recording of HEK293 cells expressing hTRPM2 channel. Induced whole cell or single channel currents were rapidly inhibited upon subsequent exposure to acidic pH. The inhibition in the steady state was complete, voltage-independent, and pH-independent in the range of pH 4.0–6.0. The inhibition was irreversible upon returning to pH 7.3, suggesting channel inactivation. In contrast, exposure of closed channels to acidic pH reduced the subsequent channel activation in a pH-dependent manner with an IC₅₀ for H⁺ of 20 μm (pH 4.7) and rendered subsequent current inhibition largely reversible, indicating differential or state-dependent inhibition and inactivation. Alanine substitution of residues in the outer vestibule of the pore including Lys⁹⁵² and Asp¹⁰⁰² significantly slowed down or reduced acidic pH-induced inhibition and prevented inactivation. The results suggest that acidic pH acts as a negative feedback mechanism where protons bind to the outer vestibule of the TRPM2 channel pore and inhibit the TRPM2 channels in a state-dependent manner.     

Depigmenting Action of Phenylhydroquinone,an o‐Phenylphenol Metabolite,on the Skin of JY‐4 Black Guinea‐Pigs

The effects of o‐phenylphenol (OPP) and its metabolite, phenylhydroquinone (PHQ) on the skin of JY‐4 black guinea‐pigs were studied. Topical application of 1 or 5% PHQ on the black skin of the back caused marked depigmentation and hypopigmentation of the skin after 5 weeks, whereas OPP applied at the same concentrations had little effect. Depigmented skin had an increased L* (lightness) value in the CIE‐L*a*b* color system. This corresponded with a decreased number of melanocytes and melanosomes in the melanocytes and keratinocytes, the disruption of melanosomes in the melanocytes, and destruction of the membranous organelles of the melanocytes. These morphological and numerical changes in epidermal melanocytes indicate that selective melanocyte toxicity occurred. Furthermore, application of PHQ to the skin of white guinea‐pigs caused skin irritation, as shown by a colorimetric increase in a* value (redness) and by histological observation of inflammation. This study confirmed that OPP, which is a reported depigmenter, has little depigmenting action, while its metabolite, PHQ, is a potent depigmenter preferentially affecting melanocytes.