bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell proliferation activity and the expression of cell cycle regulatory proteins in oral lichen planus
Authors: Hirota Makoto , Ito Takaaki , Okudela Koji , Kawabe Ryoichi , Yazawa Takuya , Hayashi Hiroyuki , Nakatani Yukio , Fujita Kiyohide , Kitamura Hitoshi
Published: March 2003

Background:  In oral lichen planus (OLP), destruction of the basal cell layer, which is one of the characteristic histological features, is seen and many changes in cell proliferation, cell repair and cell death occur in the injured mucosal epithelium.

Methods:  We studied mucosal tissues from 19 patients of OLP and 10 controls, with immunohistochemistry for Ki‐67, p53, cyclin dependent kinase inhibitors (CDKI) and cyclins. Mitotic count was calculated. TUNEL assay was also performed for evaluation of apoptotic cell death.

Results:  Mitotic count, Ki‐67 and cyclin D1 labeling indices in the basal and parabasal layers of OLP mucosa were elevated in comparison with those of controls. p53, p21Cip1 and TUNEL indices of OLP mucosa were also increased.

Conclusions:  These complex changes, which concomitantly occur in the injured mucosal epithelium, could contribute to the development and maintenance of characteristic mucosal epithelial architectures seen in OLP.
The Regulation of Normal Melanocyte Proliferation
Authors: Halaban Ruth
Published: March 2003

In vitro, normal human melanocytes require synergistic mitogens, in addition to the common growth factors present in serum, in order to proliferate. The peptide growth factors that confer stimulation are fibroblast growth factors (such as bFGF/FGF2), hepatocyte growth factor/scatter factor (HGF/SF), mast/stem cell factor (M/SCF), endothelins (such as ET‐1) and melanotropin (MSH). The proper function of these factors and their cognate receptors is likely to be important in vivo, as all five ligands are produced in the skin, and disruption of their normal function, by elimination due to deletions or mutations, or overproduction due to ectopic expression, disrupts the normal distribution of melanocytes. The synergistic growth factors activate intracellular signal transduction cascades and maintain the intermediate effectors at optimal levels and duration required for nuclear translocation and modification of transcription factors. The consequent induction of immediate‐early response genes, such as cyclins, and subsequent activation of cyclin‐dependent kinases (CDK4, CDK6 and CDK2) inactivates the retinoblastoma family of proteins (pRb, p107 and p130, together termed pocket proteins), and releases their suppressive association with E2F transcription factors. Molecular events that disrupt this tight control of pocket proteins and cause their inactivation, increase E2F transcriptional activity and confer autonomous growth on melanocytes.
Hermansky–Pudlak Syndrome and Pale Ear: Melanosome‐Making for the Millennium
Authors: Spritz Richard A.
Published: March 2003

Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized principally by oculocutaneous albinism, a bleeding tendency, and a ceroid‐lipofuscin lysosomal storage disease. These clinical manifestations of HPS are associated with defects of multiple cytoplasmic organelles – melanosomes, platelet granules, and lysosomes – suggesting that the HPS gene product is involved in some shared feature of the biogenesis or functions of these diverse organelles. The HPS gene has been cloned, and a number of pathologic mutations of the gene have been identified. Functional studies indicate that the HPS protein is part of a high‐molecular weight complex involved in the biogenesis of early melanosomes. Additional disorders with similarities to HPS have been identified in man, mouse, flies, and yeast, and it is rapidly becoming clear that understanding these disorders will shed new light on the mechanisms by which cells traffic newly synthesized proteins through the cytoplasm to assemble functional organelles.
Higher Plasma Catecholamine and Metabolite Levels in the Early Phase of Nonsegmental Vitiligo
Authors: Cucchi Maria Laura , Frattini Pietro , Santagostino Giuseppe , Orecchia Giovanni
Published: March 2003

The ethiology of vitiligo is still being debated, although neural factors seem to play a pivotal role in its pathogenesis. In our search for a link between vitiligo and the activity of monoaminergic systems, we used high‐pressure liquid chromatography and electrochemical detector (HPLC‐ED) methods to measure the plasma levels of the following substances in 35 healthy subjects and in 70 patients suffering from nonsegmental vitiligo at the different stages of the disease: catecholamines [norepinephrine (NE), epinephrine (E), and dopamine (DA)], their precursor 3,4‐dihydroxyphenylalanine (DOPA), their metabolites [3‐methoxy‐4‐hydroxyphenylglycol (MHPG), normetanephrine (NMN), metanephrine (MN), and homovanillic acid (HVA)], and 5‐hydroxyindolacetic acid (5‐HIAA) as the major metabolite of serotonin. We found that the levels of NE, E, NMN, MN, HVA, and 5‐HIAA were significantly higher in patients compared to controls. The patients at an active phase of the disease (n=49/70) showed significantly higher levels of NE, NMN, MHPG, and HVA than ones at a stable phase. The patients with progressive vitiligo and at its more recent onset (<1 year) showed significantly increased levels of E, NE, and MN in comparison with longer‐term sufferers. No significant differences were observed when the patients were subdivided according to the type of vitiligo or their age at its onset. The higher catecholamine and metabolite levels in the early phase of the disease may reflect increased activity by monoaminergic systems, probably due to stressful events, including the onset of vitiligo itself.
Probing the Building Blocks of Eumelanins Using Scanning Electron Microscopy
Authors: NOFSINGER J. BRIAN , FOREST SUSAN E. , EIBEST LESLIE M. , GOLD KATHRYN A. , SIMON JOHN D.
Published: March 2003

Scanning electron microscopy (SEM) is used to examine the structure of natural and synthetic melanins. Eumelanin from Sepia officinalis and synthetic eumelanin are found to be structurally dissimilar. The natural sample has a significant structural order with subunits that have a lateral dimension of ∼15 nm. The synthetic samples, on the other hand, appear to be amorphous solids. These results lend support for the existence of fundamental structural units proposed from the analyses of wide‐angle X‐ray diffraction measurements and previous mass‐spectrometry results. These findings also provide insight into the disparate photophysical behavior of Sepia and synthetic eumelanin.
A Novel Approach to Gene Therapy of Albino Hair In Histoculture with a Retroviral Streptomyces Tyrosinase Gene
Authors: ZHAO MING , SAITO NORIMITSU , LI LINGNA , BARANOV EUGENE , KONDOH HIROFUMI , MISHIMA YUTAKA , SUGIYAMA MASANORI , KATSUOKA KENSEI , HOFFMAN ROBERT M.
Published: March 2003

In order to induce melanin production in mammalian cells with pigment disorders such as albino hair, a recombinant retrovirus containing the mel locus of Streptomyces antibioticus was constructed. The S. antibioticusmel locus, which consists of the open reading frame (ORF)‐438 and the tyrosinase gene, was specifically derived by polymerase chain reaction (PCR) from Streptomyces plasmid pIJ702. The ORF‐438 is required for the transfer of copper to apotyrosinase, which is essential for tyrosinase enzymatic activity. The tyrosinase gene was inserted into the XhoI/BamHI cloning site of the pLXSN retroviral vector to obtain pLtyrSN. An internal ribosome entry site (IRES) suitable for mammalian cell expression was obtained from the pLXIN retroviral vector by PCR. The ORF‐438 and IRES DNA fragments were inserted into the pLtyrSN vector to obtain the tyrosinase‐expression retroviral vector pLmelSN. The expression vector was amplified in murine PT67 packaging cells, where the ORF‐438 and tyrosinase genes were also co‐expressed as determined by reverse transcription‐PCR. In order to evaluate the vector's ability to restore pigment production in cells with a pigment disorder, albino ‐mouse skins were histocultured and then infected with the pLmelSN retrovirus. Six days after infection, melanin granules were observed in ∼60% of albino‐mouse hair follicles in the histocultured skin. These results demonstrated that the S. antibioticusmel operon could express an active tyrosinase and produce melanin in the albino‐mouse hair follicles. This novel gene therapy approach, using a small and simple tyrosinase operon in a high‐expression vector, has a potentially wide application for therapy of pigment disorders in hair follicles.
The Mouse p (pink‐eyed dilution) and Human P Genes, Oculocutaneous Albinism Type 2 (OCA2), and Melanosomal pH
Authors: H. BRILLIANT MURRAY
Published: March 2003

Recessive mutations of the mouse p (pink‐eyed dilution) gene lead to hypopigmentation of the eyes, skin, and fur. Mice lacking a functional p protein have pink eyes and light gray fur (if non‐agouti) or cream‐colored fur (if agouti). The human orthologue is the P protein. Humans lacking a functional P protein have oculocutaneous albinism type 2 (OCA2). Melanocytes from p‐deficient mice or OCA2 individuals contain small, minimally pigmented melanosomes. The mouse and human proteins are predicted to have 12 membrane spanning domains and possess significant sequence homology to a number of membrane transport proteins, some of which are involved in the transport of anions. The p protein has been localized to the melanosome membrane. Recently, it has been shown that melanosomes from p protein‐deficient melanocytes have an abnormal pH. Melanosomes in cultured melanocytes derived from wild‐type mice are typically acidic, whereas melanosomes from p protein‐deficient mice are non‐acidic. Melanosomes and related endosome‐derived organelles (i.e., lysosomes) are thought to have an adenosine triphosphate (ATP)‐driven proton pump that helps to generate an acidic lumen. To compensate for the charge of these protons, anions must also be transported to the lumen of the melanosome. In light of these observations, a model of p protein function is presented in which the p protein, together with the ATP‐driven proton pump, regulates the pH of the melanosome.
Ceramide Inhibits Cell Proliferation through Akt/PKB Inactivation and Decreases Melanin Synthesis in Mel‐Ab Cells
Authors: KIM DONG‐SEOK , KIM SOOK‐YOUNG , MOON SEONG‐JOON , CHUNG JIN‐HO , KIM KYU‐HAN , CHO KWANG‐HYUN , PARK KYOUNG‐CHAN
Published: March 2003

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell‐permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel‐Ab. Our results show that C2‐ceramide inhibits DNA synthesis in Mel‐Ab cells and G361 human melanoma cells in a dose‐dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C2‐ceramide is able to influence extracellular signal‐regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C2‐ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C2‐ceramide‐induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel‐Ab cells. Furthermore, we assessed the effects of C2‐ceramide on the pigmentation of Mel‐Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C2‐ceramide at concentrations in the range of 1–10 μM, and that the pigmentation‐inhibiting effect of C2‐ceramide is much greater than that of kojic acid at 1–100 μM. In addition, we found that the activity of tyrosinase is reduced by C2‐ceramide treatment. Our results demonstrate that C2‐ceramide reduces the pigmentation of Mel‐Ab cells by inhibiting tyrosinase activity.
Cyclic Oscillations in Melanin Composition within Hairs of Baboons
Authors: Ito Shosuke , Wakamatsu Kazumasa , Matsunaga Naoko , Hearing Vincent J. , Carey K. Dee , Anderson Sydney , Dooley Thomas P.
Published: March 2003

The wild‐type agouti‐banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of α‐melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such ‘agouti’‐banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non‐pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana‐Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.
Inhibition of Melanosome Transfer from Melanocytes to Keratinocytes by Lectins and Neoglycoproteins in an In Vitro Model System
Authors: Minwalla Ljiljana , Zhao Yang , Cornelius James , Babcock George F. , Wickett R. Randall , Le Poole I. Caroline , Boissy Raymond E.
Published: March 2003

We propose that some of the critical molecules involved in the transfer of melanosomes from melanocytes to keratinocytes include plasma membrane lectins and their glycoconjugates. To investigate this mechanism, co‐cultures of human melanocytes and keratinocytes derived from neonatal foreskins were established. The process of melanosome transfer was assessed by two experimental procedures. The first involved labeling melanocyte cultures with the fluorochrome CFDA. Labeled melanocytes were subsequently co‐cultured with keratinocytes, and the transfer of fluorochrome assessed visually by confocal microscopy and quantitatively by flow cytometry. The second investigative approach involved co‐culturing melanocytes with keratinocytes, and processing the co‐cultures after 3 days for electron microscopy to quantitate the numbers of melanosomes in keratinocytes. Results from these experimental approaches indicate significant transfer of dye or melanosomes from melanocytes to keratinocytes that increased with time of co‐culturing. Using these model systems, we subsequently tested a battery of lectins and neoglycoproteins for their effect in melanosome transfer. Addition of these selected molecules to co‐cultures inhibited transfer of fluorochrome by approximately 15–44% as assessed by flow cytometry, and of melanosomes by 67–93% as assessed by electron microscopy. Therefore, our results suggest the roles of selected lectins and glycoproteins in melanosome transfer to keratinocytes in the skin.
Distinct Melanogenic Response of Human Melanocytes in Mono‐culture, in Co‐Culture with Keratinocytes and in Reconstructed Epidermis, to UV Exposure
Authors: DUVAL CHRISTINE , RÉGNIER MARCELLE , SCHMIDT RAINER
Published: March 2003

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.
Molecular Anatomy of Tyrosinase and its Related Proteins: Beyond the Histidine‐Bound Metal Catalytic Center
Authors: García‐Borrón José C. , Solano Francisco
Published: March 2003

The structure of tyrosinase (Tyr) is reviewed from a double point of view. On the one hand, by comparison of all Tyr found throughout nature, from prokaryotic organisms to mammals and on the other, by comparison with the tyrosinase related proteins (Tyrps) that appeared late in evolution, and are only found in higher animals. Their structures are reviewed as a whole rather than focused on the histidine (His)‐bound metal active site, which is the part of the molecule common to all these proteins. The availability of crystallographic data of hemocyanins and recently of sweet potato catechol oxidase has improved the model of the three‐dimensional structure of the Tyr family. Accordingly, Tyr has a higher structural disorder than hemocyanins, particularly at the CuA site. The active site seems to be characterized by the formation of a hydrophobic pocket with a number of conserved aromatic residues sited close to the well‐known His. Other regions specific of the mammalian enzymes, such as the cytosolic C‐terminal tail, the cysteine clusters, and the N‐glycosylation sequons, are also discussed. The complete understanding of the Tyr copper‐binding domain and the characterization of the residues determinant of the relative substrate affinities of the Tyrps will improve the design of targeted mutagenesis experiments to understand the different catalytic capabilities of Tyr and Tyrps. This may assist future aims, from the design of more efficient bacterial Tyr for biotechnological applications to the design of inhibitors of undesirable fruit browning in vegetables or of color skin modulators in animals.
Influence of Tyrosinase Levels on Pigment Accumulation in the Retinal Pigment Epithelium and on the Uncrossed Retinal Projection
Authors: Rachel Rivka A. , Mason Carol A. , Beermann Friedrich
Published: March 2003

To study the relationship among tyrosinase activity, melanin production, and the routing of retinal ganglion cell (RGC) axons at the optic chiasm, we analysed mice with varying doses of the tyrosinase gene. These include the dark‐eyed albino (Tyrc44H), a radiation‐induced hypomorphic allele of tyrosinase; and transgenic mice carrying 1 or 2 alleles of a tyrosinase minigene on both wild‐type (Tyr+) and albino (Tyrc) backgrounds. Melanization of the retinal pigment epithelium (RPE) occurred gradually even at <2% wild‐type tyrosinase activity and was sensitive to tyrosinase activity up to <35% of wild‐type levels, beyond which melanin synthesis appeared to be saturated. Overexpression of tyrosinase led to tyrosinase activity above wild type level, but did not increase melanin production. Although a loss of melanin because of a mutation in tyrosinase is associated with a decrease in the number of uncrossed fibers, elevating tyrosinase levels does not appear to cause an increase in the size of the uncrossed retinal projection. Our results suggest that replacing less than 35% of wild‐type tyrosinase activity is sufficient to restore normal pigmentation of the RPE, and potentially, to allay visual defects.
Artificial Chromosome Transgenesis in Pigmentary Research
Authors: Giraldo Patricia , Montoliu Lluís
Published: March 2003

Pigmentary genes were among the first mammalian genes to be studied, mostly because of the obvious phenotypes associated with their mutations. In 1990, tyrosinase, encoding the limiting enzyme in the melanin synthesis pathway, was eventually assigned to the c (albino) locus by classical rescue experiments driven by functional constructs in transgenic mice. These pioneer reports triggered the study of the regulation of endogenous tyrosinase gene expression by combining different amounts of upstream regulatory and promoter regions and testing their function in vivo in transgenic animals. However, faithful and reproducible transgenic expression was not achieved until the entire tyrosinase expression domain was transferred to the germ‐line of mice using artificial‐chromosome‐type transgenes. The use of these large tyrosinase transgenic constructs and the ease with which they could be manipulated in vitro enabled the discovery of previously unknown but fundamental regulatory regions, such as the tyrosinase locus control region (LCR), whose presence was required in order to guarantee position‐independent and copy‐number‐dependent expression of tyrosinase transgenes, with an expression level, per copy, comparable to that of an endogenous wild‐type allele. Subsequently, functional dissection of elements present within this LCR through the generation of new artificial‐chromosome type tyrosinase transgenes has revealed the existence of different regulatory activities. The existence of some of these units had been suggested previously by standard‐type transgenic analyses. In this review, we will discuss both independent approaches and conclude that optimal tyrosinase transgene expression requires the use of its complete expression domain.
Expression of Cre Recombinase in Pigment Cells
Authors: Guyonneau Laurence , Rossier Anita , Richard Christelle , Hummler Edith , Beermann Friedrich
Published: March 2003

Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre‐mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell‐specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell‐specific Cre/loxP‐mediated recombination.
Skin Responses to Ultraviolet Radiation: Effects of Constitutive Pigmentation, Sex, and Ancestry
Authors: Wagner Jennifer K. , Parra Esteban J. , L. Norton Heather , Jovel Celina , Shriver Mark D.
Published: March 2003

Constitutive skin pigmentation and skin responses to ultraviolet radiation were measured on a sample of volunteers (n=250) living in State College, PA, USA. The sample was composed of individuals of European American (n=190), Hispanic (n=45), and East Asian ancestry (n=15). Constitutive pigmentation was measured using the Adjusted Melanin Index (AMI), Erythemal Dose Response (EDR) was measured using the slope of a* at 24 h (Δa*), and Melanogenic Dose–Response (MDR) was measured using ΔAM, the slope of AMI at 7 d. The relationships between constitutive skin pigmentation, EDR, MDR, sex, age, and ancestry were investigated. European Americans showed a lower constitutive pigmentation, had a significantly higher burn response (EDR), and had a significantly lower tanning response (MDR) than Hispanics and East Asians. No significant difference is seen between Hispanics and East Asians for either constitutive pigmentation or EDR. Constitutive pigmentation in females was slightly lower than in males in all three samples, but the difference was not significant. While no differences were observed in MDR between sexes, males had a stronger EDR than females regardless of population or constitutive pigmentation level, and this difference was significant in European Americans and Hispanics. We observed no age‐related differences in any of the populations or measures investigated. We evaluated the relationship between constitutive pigmentation, EDR and MDR. There was a strong inverse correlation between constitutive pigmentation and EDR in the three samples (European Americans, R2=0.176, P < 0.001; Hispanics, R2=0.204, P=0.009; East Asians, R2=0.223, P=0.098) and a strong direct correlation between constitutive pigmentation and MDR in European Americans and Hispanics (European Americans, R2=0.094, P < 0.001; Hispanics, R2=0.164, P=0.012). In other words, persons with lower constitutive pigmentation both burn more and tan less than persons with higher pigmentation. However, after controlling for constitutive pigmentation, EDR and MDR were significantly correlated in European Americans (R2=0.041 P=0.006). Thus, the general observation that persons who burn more tan less is probable because of the common link that these two phenotypes have with constitutive skin pigmentation and, in fact, once pigmentation has been adjusted for, there is a positive correlation between tanning response and burning response in European Americans.
Catecholamines Increase in the Urine of Non‐Segmental Vitiligo Especially During Its Active Phase
Authors: Cucchi Maria Laura , Frattini Pietro , Santagostino Giuseppe , Preda Stefania , Orecchia Giovanni
Published: March 2003

Neural factors appear to play a major role in the pathogenesis of vitiligo. To investigate the possible correlation between vitiligo and peripheral monoaminergic system activity, we used high‐pressure liquid chromatography and electrochemical detector methods to evaluate the basal urine excretion values of catecholamines [norepinephrine (NE), epinephrine and dopamine (DA)], their relative metabolites [3‐methoxy‐4‐hydroxyphenylglycol (MHPG), normetanephrine (NMN), metanephrine (MN), vanilmandelic acid (VMA) and homovanillic acid], as well as 5‐hydroxyindoleacetic acid (5‐HIAA), in 35 healthy subjects and in 70 patients, suffering from non‐segmental vitiligo at different stages of the disease. Levels of NE, DA, NMN, MN, MHPG, VMA and 5‐HIAA were found to be significantly higher in patients than in controls. The patients with progressive vitiligo (n = 56) presented increased urinary excretion values for all parameters (in particular, NE levels) than other patients. Interestingly, in patients at its more recent vitiligo onset (<1 yr), NE values were different to those of subjects affected from 1 to 5 yr and from 6 to 10 yr. This result was confirmed by the significant negative relationship detected between NE excretion values and disease duration. In both vitiligo and control groups, significant correlations were found between monoamines as well as between these monoamines and their metabolites. The increase in catecholamine turnover, mainly occurring at the onset of the disease, is probably due to the stress associated with the appearance of lesions. Moreover, considering that these compounds readily produce toxic free‐radicals and that vitiliginous subjects have a defective free radical defence mechanism, they may also contribute to the disappearance of melanocytes in the early phases of vitiligo.
Accumulation of Tyrosinase in the Endolysosomal Compartment is Induced by U18666A
Authors: Hall Andrea M. , Krishnamoorthy Lalitha , Orlow Seth J.
Published: March 2003

The 3β‐(2‐diethylaminoethoxy)‐androstenone HCl (U18666A), progesterone and several cationic amphiphilic drugs have been shown to alter the trafficking of a number of intracellular membrane proteins including CD63/Lamp‐3, insulin growth factor 2/mannose 6‐phosphate receptor (IGF2/MPR), and the Niemann‐Pick C1 gene product (NPC1) as well as ganglioside GM1. We have examined the effects of these compounds on cultured melanocytes at concentrations that have been shown to effectively alter intracellular trafficking. Treatment of melanocytes with U18666A (2.5 µM) or progesterone (15 µM) for 96 h decreased melanin content an average of 67% as compared with control without lowering the total cellular tyrosinase activity. Steroidal alkaloids that preferentially act on the Sonic Hedgehog signaling pathway showed no related specificity in their ability to decrease pigmentation. In melanocytes treated with U18666A, tyrosinase accumulates in a compartment that contains both lysosome‐associated membrane protein‐1 (Lamp 1) and MPR, and stains with filipin, consistent with cholesterol‐laden late endosomes/lysosomes. Our results suggest that tyrosinase, like the NPC1 gene product, traverses a U18666A‐sensitive trafficking pathway.
Effect of periodontal treatment on the activity of chitinase in whole saliva of periodontitis patients
Authors: Van Steijn G. J. , Nieuw Amerongen A. V. , Veerman E. C. I. , Kasanmoentalib S. , Overdijk B.
Published: March 2003

Human salivary chitinase could play a role in the defence against chitin‐containing oral pathogens. The activity levels of chitinase in the whole saliva of periodontitis patients were significantly higher than those in saliva from controls. Periodontal treatment for a period of 5–6 months resulted in a three‐ to fourfold decrease in this enzyme activity. The activity of β‐N‐acetylhexosaminidase, which is another enzyme that hydrolyses glycosidic linkages, also decreased as a result of treatment, although to a lesser extent. The decrease in chitinase activity upon treatment of the disease did not correlate with the decrease that was seen in clinical attachment loss and bleeding on probing, and only a weak correlation was observed with the changes in probing pocket depth and plaque index. No correlations were found between the above clinical parameters and the decrease in β‐N‐acetylhexosaminidase activity.
Hepatocyte growth factor (HGF) in jreiodontal disease: detection of HGF in gingival crevicular fluid
Authors: Ohshima Mitsuhiro , Sakai Akihiko , Ito Koichi , Otsuka Kichibee
Published: March 2003

Hepatocyte growth factor (HGF) acts as a mitogen, motogen, morphogen, anti‐apoptotic factor, and scatter factor for various kinds of epithelial cells. It is a protein secreted by mesenchymal cells such as fibroblasts, and promotes motility and matrix invasion of epithelial cells. To clarify whether HGF is involved in jreiodontal disease, this study was conducted to determine whether HGF is present in gingival crevicular fluid (GCF) and to investigate the relationship between levels of HGF and the clinical parameters of jreiodontal disease, probing depth (PD), gingival index (GI) and bleeding on probing (BOP). We examined and collected GCF samples from 80 sites in 38 subjects with jreiodontal or other oral diseases. The concentrations of HGF, IL‐1β and PGE2 were determined by ELISA, and active collagenase activity was determined by functional assay. The HGF level correlated positively with PD and GI, and was significantly higher in specimens from BOP‐positive sites and those where PD exceeded 4 mm compared with those from sites that were BOP‐negative or with a PD less than 3 mm. There was a significant positive correlation between the concentrations of HGF and IL‐1β. These results indicate that the HGF level in GCF correlates well with clinical parameters of jreiodontal disease, and suggest that HGF may be involved in epithelial invasion through its role as a scatter factor.
Oral mucosa alterations induced by cyclosporin in mice: morphological features
Authors: Meller A. T. , Rumjanek V. M. , Sansone C. , Allodi S.
Published: March 2003

Background and objective:  The mechanisms involved in the pathogenesis of cyclosporin A‐induced gingival hyperplasia are not well understood. The present work aimed at developing a mouse model with the characteristics of the human process, i.e. time of appearance, dose dependency and the capacity of developing in a variety of genetic backgrounds. This model would present the advantages of using a very well known animal species, small and easy to handle, with a number of experimental reagents (antibodies, etc.) already available against its products.

Methods:  Three different strains of mice were used: CBA, F1(C57Bl × DBA), Balb/c. Groups of mice received different concentrations of cyclosporin A (CSA) (10 mg/kg, 25 mg/kg and 40 mg/kg body weight) intraperitoneally five times a week. Anatomical and histological alterations were recorded at various time intervals.

Results:  All strains of mice presented gingival hyperplasia after 8 weeks of CSA treatment. A dose‐dependency was observed with regard to the time of first appearance of alterations. Increased redness was seen in all animals at the sixth week, independent of the dosage used. Histologic examination exhibited increased vascularization, epithelial and connective tissue thickening, edema and a mononuclear infiltrate.

Conclusions:  It was possible to develop CSA‐induced gingival hyperplasia in mice with the characteristics described in humans and other species. The use of this animal model may help in the elucidation of the process involved in CSA‐induced gingival overgrowth.
Expression of receptor activator of NF‐kappa B ligand and osteoprotegerin in culture of human periodontal ligament cells
Authors: Hasegawa Tomokazu , Yoshimura Yoshitaka , Kikuiri Takashi , Yawaka Yasutaka , Takeyama Sadaaki , Matsumoto Akira , Oguchi Haruhisa , Shirakawa Tetsuo
Published: March 2003

The receptor activator of NF‐kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins implicated in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament (PDL) cells and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down‐regulated remarkably by application of 10−8 m one‐alpha, 25‐dihydroxyvitamin D3[1,25‐(OH)2D3] and 10−7 m dexamethasone (Dex). In contrast, RANKL mRNA was up‐regulated by the same treatment. Western blotting demonstrated decrease of OPG by the application of 1,25‐(OH)2D3 and Dex. Tartrate‐resistant acid phosphatase‐positive multinuclear cells were markedly induced when the PDL cells were cocultured with mouse bone marrow cells in the presence of an anti‐OPG antibody together with 1,25‐(OH)2D3 and Dex. These results indicate that PDL cells synthesize both RANKL and OPG and that inactivation of OPG may play a key role in the differentiation of osteoclasts.
Differential expression and distribution of syndecan‐1 and ‐2 in periodontal wound healing of the rat
Authors: Worapamorn W. , Xiao Y. , Li H. , Young W. G. , Bartold P. M.
Published: March 2003

Cell‐surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell–cell and cell–matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan‐1 and ‐2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan‐1, or ‐2 core proteins. Both syndecan‐1 and ‐2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan‐1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast‐like cells and newly formed blood vessels. Syndecan‐1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan‐1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan‐2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan‐2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan‐2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan‐1 and ‐2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan‐1 may involve both cell–cell and cell–matrix interactions, while syndecan‐2 showed a predilection to associate with cell–matrix interactions during hard tissue formation.
Emdogain® regulation of cellular differentiation in wounded rat periodontium
Authors: Chano Laura , Tenenbaum Howard C. , Lekic P. Charles , Sodek Jaro , McCulloch Christopher A.
Published: March 2003

Emdogain® is an enamel matrix derivative that may promote periodontal regeneration by recapitulating critical events in tooth morphogenesis. We hypothesized that Emdogain® enhances periodontal regeneration by promoting the differentiation of cells required for the synthesis of periodontal ligament, bone and cementum. Cell differentiation was examined in rat periodontal window wounds in which there is no microbial biofilm or epithelial downgrowth, thereby simplifying the model system. Defects were filled with vehicle control or Emdogain® (3 mg/ml or 30 mg/ml). Rats were sacrificed at 7, 14 and 21 d after wounding. Specimens of periodontium were immunostained for osteopontin, bone sialoprotein, osteocalcin as markers of osteogenic differentiation and for α‐smooth muscle actin, a myofibroblastic marker. Morphometry and 3H‐proline radioautography were used for assessment of tissue homeostasis and matrix production. Rats treated with Emdogain® (only at 30 mg/ml) showed widening of the periodontal ligament at 7 d; by 14 and 21 d, periodontal ligament width was restored to normal values for all groups. Emdogain® exerted no effect on cementum thickness, bone volume, osteoid deposition rates, or extracellular staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein was unaffected by Emdogain®. Staining for α‐smooth muscle actin was abundant in the repopulating wound but was also unaffected by Emdogain®. In conclusion, Emdogain® does not apparently affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium. Therefore the effect of Emdogain® on wound healing in the periodontium may be independent of differentiation in the cell populations examined in this model.
Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease
Authors: Garlet Gustavo P. , Martins Walter , Ferreira Beatriz R. , Milanezi Cristiane M. , Silva João S.
Published: March 2003

Current knowledge states that periodontal diseases are chronic inflammatory reactions raised in response to periodontopathogens. Many cell types and mediators, including Th1 and Th2 lymphocytes, cytokines and chemokines, appear to be involved in the immunopathogenesis of periodontal diseases. Chemokines, a family of chemotactic cytokines, bind to specific receptors and selectively attract different cell subsets to the inflammatory site. They can also interact with classical cytokines and modulate the local immune response. In order to study the role of chemokines in periodontal diseases, we examined the expression of chemokines, chemokine receptors and cytokines by means of reverse transcription‐polymerase chain reaction (RT‐PCR) techniques. Characteristic patterns of such factors' expression were found in gingival biopsies from patients presenting with aggressive periodontitis and chronic periodontitis. The expression of the chemokines macrophage inflammatory protein‐1 alpha (MIP‐1α) and interferon‐gamma inducible protein 10 (IP‐10) and of their respective receptors, CCR5 and CXCR3, were more prevalent and higher in aggressive periodontitis, and associated with higher interferon‐gamma (IFN‐γ) expression and lower interleukin‐10 (IL‐10) expression. In contrast, chronic periodontitis patients exhibited a more frequent and higher expression of monocyte chemoattractant protein‐1 (MCP‐1) and its receptor CCR4, and higher expression of IL‐10. It is possible that chemokines, in addition to the classical cytokines, are involved in the immunopathogenesis of periodontal disease, driving the migration and the maintenance of several inflammatory cell types such as polymorphonuclear leukocytes, dendritic cells (DCs), natural killer cells, macrophages, and subsets of lymphocytes in the gingival tissues. These cells are thought to participate in the inflammatory and immune reaction that takes place in periodontal disease, killing pathogens, presenting antigens, and producing cytokines. The selective recruitment of polarized lymphocyte subsets could result in differential cytokine production at the site of response, which is supposed to determine the stable or progressive nature of the lesion. Besides, the role of chemokines as activators and chemoattracts of osteclasts may be involved in the determination of disease severity.
Ambroxol Inhibits Peroxynitrite‐Induced Damage of α1‐Antiproteinase and Free Radical Production in Activated Phagocytic Cells
Authors: Lee Chung Soo , Jang Yoon Young , Song Joon Sup , Song Jin Ho , Han Eun Sook
Published: March 2003

Abstract: The present study examined the effect of ambroxol on toxic action of peroxynitrite and the respiratory burst in activated phagocytic cells. Ambroxol decreased the inactivation or destruction of α1‐antiproteinase induced by peroxynitrite (ONOO−) or hypochlorous acid (HOCl), which was similar to penicillamine and glutathione and was greater than diclofenac sodium and naproxen sodium. Ambroxol significantly decreased ONOO−–mediated tyrosine nitration and iron plus EDTA‐mediated degradation of 2‐deoxy‐D‐ribose. Ambroxol significantly attenuated the production of superoxide, hydrogen peroxide, HOCl, and nitric oxide in fMLP– or IL‐1‐activated phagocytic cells, while the inhibitory effects of antiinflammatory and thiol compounds were only observed in HOCl production. Ambroxol and antiinflammatory drugs did not show a cytotoxic effect on macrophages. The results suggest that ambroxol protects tissue components against oxidative damage by an action different from antiinflammatory drugs. Ambroxol may interfere with oxidative damage of α1‐antiproteinase through a scavenging action on ONOO− and HOCl and inhibition of the respiratory burst of phagocytic cells.
In vitro Tests for Predicting Drug‐Drug Interactions: The Need for Validated Procedures
Authors: Kremers Pierre
Published: March 2003

Abstract: Over the past decade, the prediction of drug‐drug interactions from in vitro studies has become a rapidly expanding field of research. Numerous papers and excellent review articles (Bertz & Granneman 1997; Ito et al. 1998a & b; Lin 2000; Bachmann & Ghosh 2001; Ekins & Wrighton 2001; Weaver 2001) have been published in this area. Yet like any new and fast‐growing subject, this one has been developing with some confusion and without any real, efficient organisation. Depending on the drug tested, the models and extrapolation parameters used, etc., results and conclusions may vary widely from study to study (von Moltke et al. 1998; Weaver 2001). Several authors have called for validation of these procedures (Rodrigues et al. 2001; Kummar & Surapaneni 2001; Pelkonen et al. 2001a & b; Kremers 2002), and regulatory authorities intend to require better traceability and reliability (FDA & EMEA guidelines). A systematic and reliable approach is needed also to allow such protocols to be incorporated into early screening for potential drugs and new chemical entities. There is certainly a great need to standardise these studies and to verify their conclusions, but is true validation possible in this field? The main purpose of the present paper is to discuss this issue and to examine what is possible and what is needed to improve the quality of predictions made from in vitro experiments.
The in vitro Effects of Galactose and its Derivatives on Rat Brain Mg2+‐ATPase Activity
Authors: Tsakiris Stylianos , Marinou Kyriakoula , Schulpis Kleopatra H.
Published: March 2003

Abstract: Galactosaemia is an inborn error of metabolism characterized by irreversible damage to neural tissue. To evaluate whether galactose metabolic disorders, (e.g. classical galactosaemia, galactokinase deficiency galactosaemia), is implicated for alterations of brain Mg2+‐ATPase activity, various concentrations (1–16 mM) of galactose, galactose‐1‐phosphate, galactitol, glucose‐1‐phosphate or glucose were preincubated with whole brain homogenates of suckling rats at 37 ° for 1 hr. Mg2+‐ATPase activities were determined according to Bowler & Tirri's (1974). Galactose‐1‐phosphate or glucose‐1‐phosphate excessively activated the brain Mg2+‐ATPase in a concentration‐dependent way. Additionally, galactitol, galactose or glucose stimulated the enzyme up to 35–45% (P<0.001) at concentrations >4 mM. A mixture of galactose‐1‐phosphate (2 mM), glactitol (2 mM) and galactose (4 mM), concentrations commonly found in blood and brain of untreated patients with classical galactosaemia, resulted in a 500% enzyme activation (P<0.001) as compared to control. Moreover, a mixture of galactitol (2 mM) and galactose (1 mM), concentrations measured in patients with galactokinase deficiency, caused an enzyme stimulation (35%, P<0.001). These findings suggest: a) The great Mg2+‐ATPase activation by galactose‐1‐phosphate or glucose‐1‐phosphate may be due to the epimer of galactose and the presence of phosphorus. b) The brain Mg2+‐ATPase stimulation by galactose and its derivatives could be toxic by modulating the Mg2+ concentration, the ATP availability, the activity of other ATP‐ and Mg2+‐dependent enzymes as well as the rates of protein synthesis and cell growth.