bluuebunny/crossref_metadata_2025 huggingface dataset:
Pollen transfer patterns in a distylous plant with overlapping pollen‐size distributions
Authors: Pailler Thierry , Maurice Sandrine , Thompson John D.
Published: March 2003

In this study we quantify the relative rates and amounts of between‐morph (legitimate) cross‐pollination, within‐morph (illegitimate) cross‐pollination and self‐pollination in a natural population of distylous Gaertnera vaginata (Rubiaceae) endemic to the oceanic island of La Réunion in the Indian Ocean. Since pollen size distributions of each morph overlap we develop a method, based on chi‐squared comparisons of observed to expected pollen size distributions, to quantify pollen transfer proficiencies. This method, unlike previous methods, does not assume an equal chance of pollen transfer for pollen grains in the region of overlap. The probability that a single pollen grain from a short‐styled plant undergoes legitimate pollen transfer to long‐styled flowers (4.19×10−2) was more than twice that for illegitimate transfer to other short‐styled plants (1.56×10−2). In contrast, the probability that a single pollen grain from a long‐styled flower undergoes legitimate pollen transfer to short‐styled flowers (1.65×10−2) was no greater than that of illegitimate pollen transfer to other long‐styled plants (2.15×10−2). The transfer proficiencies of individual pollen grains are thus highly asymmetric. However, because long‐styled flowers produce significantly more pollen, the ratio of legitimate to illegitimate pollen on the stigmas of short‐styled flowers (1.85) markedly exceeded that for long‐styled flowers (1.11). Short‐styled flowers incurred significantly higher levels of self‐pollination than long‐styled plants, emphasizing the importance for self‐pollination to be removed before comparing stigmatic pollen loads. The method we propose could be applied to other distylous species with pollen size overlap between morphs and thus allow for a more widespread study of the pollination ecology of heterostylous species.
Lack of association between Streptococcus oralis and recurrent aphthous stomatitis
Authors: Riggio Marcello P. , Lennon Alan , Ghodratnama Fatemeh , Wray David
Published: March 2003

Abstract: In the present study, the potential involvement of Streptococcus oralis in the aetiology of recurrent aphthous stomatitis (RAS) was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. PCR was carried out using a primer pair that targets the D‐alanine:D‐alanine ligase gene and detects DNA from both S. oralis and the closely related species Streptococcus mitis. Discrimination between these two species was achieved by digestion of PCR products with the restriction endonucleases HaeIII and HindIII, which both give distinct restriction profiles for each species. S. oralis DNA was detected in 8 of 28 (29%) RAS samples, 10 of 20 (50%) OLP samples and 6 of 13 (46%) normal samples. These results suggest that S. oralis is not of primary aetiological significance in RAS.
Isolation of C. dubliniensis from insulin‐using diabetes mellitus patients
Authors: Willis A. M. , Coulter W. A. , Sullivan D. J. , Coleman D. C. , Hayes J. R. , Bell P. M. , Lamey P.‐J.
Published: March 2003

Abstract: The identification of the novel candidal species, C. dubliniensis, from oral swab studies of HIV‐seropositive and ‐seronegative individuals has led to speculation that such a species may also reside in the oral cavity of other patient groups. In this study involvement of the newly described species, C. dubliniensis, was investigated in oral carriage and disease in 414 insulin‐using diabetes mellitus patients. Seventy‐seven percent of the diabetic patients carried candidal species in the oral cavity. C. albicans was the most commonly identified candidal species. This was followed by C. dubliniensis, which was isolated on 64 occasions. Colonisation with multiple candidal species was common, and C. dubliniensis was present in both carriage and disease states. Seven patients without signs of oral disease had C. dubliniensis isolated as the sole candidal species, while the same species was associated with various forms of oral candidosis in six patients.
Raised tissue copper levels in oral submucous fibrosis
Authors: Trivedy C. R. , Warnakulasuriya K. A. A. S. , Peters T. J. , Senkus R. , Hazarey V. K. , Johnson N. W.
Published: March 2003

Abstract: Oral submucous fibrosis (OSF) is a well‐recognised‐potentially malignant condition of the oral cavity associated with areca nut chewing. Areca nut has been shown to have a high copper content compared to other commonly eaten nuts, and chewing areca nut for 5–30 min significantly increases soluble copper in whole mouth fluids. Our aims were to determine if tissue and serum concentrations of copper were raised in patients with OSF as a result of chewing areca nut. A panel of buccal mucosal biopsies from patients with OSF from Nagpur, India, was used to measure the tissue concentrations of copper by mass absorption spectrometry (MAS). By MAS, the mean tissue copper level was 5.5±2.9 μg/g in the OSF specimens (n=11) compared with 4±1.9 μg/g in the non‐areca chewing controls (n=7) (P=0.2). Energy dispersive x‐ray microanalysis (EDX) was used to identify the presence and distribution of the metal element. EDX showed distinct peaks corresponding to copper (Kα, 8.04 keV; Kβ, 8.91 keV) in the epithelium (21/23) and in the connective tissue (17/23) of the OSF specimens compared to spectra obtained from control oral biopsies from non‐areca chewing subjects (n=7). These findings were confirmed by secondary ion mass spectrometry (SIMS) analysis in a small number of samples. Serum copper (17.23±1.80 μmol/l), caeruloplasmin (0.32±0.04 g/l) levels and urinary copper (0.52±0.26 μmol/l) in OSF patients (n=14) were within the laboratory reference ranges. The finding of copper in OSF tissue supports the hypothesis of copper as an initiating factor in OSF, playing a role in stimulating fibrogenesis by the upregulation of lysyl oxidase activity.
Expression of matrix metalloproteinase‐2 in osteoarthritic fibrocartilage from human mandibular condyle
Authors: Tanaka Akira , Kawashiri Shuichi , Kumagai Shigehiro , Takatsuka Shigeyuki , Narinobou Masayoshi , Nakagawa Kiyomasa , Tanaka Shigenori
Published: March 2003

Abstract: Matrix metalloproteinase (MMP)‐2 is expressed in osteoarthritic cartilage and synovial fluid and is thought to be involved in the degradation of cartilage extracellular matrix. However, MMP‐2 expression and osteoarthritic changes in internal derangement of the temporomandibular joint are unknown. In the present study, we have examined the histological relationship between osteoarthritic changes on articular cartilage with or without articular disc perforation, and MMP‐2 expression, in 85 mandibular condyles from cadavers. The expression and tissue immunolocalization of MMP‐2 in fibrocartilages from these condyles was examined histochemically. The Mankin grade of histological criteria for specimens with disc perforation was significantly higher than that of specimens without perforation. MMP‐2 immunostaining was positive in the cytoplasm of chondrocytes and in their surrounding matrix. There was a linear correlation between MMP‐2‐positive cell rates and Mankin grade. Our data suggest that MMP‐2 plays an important role in fibrocartilage degradation in internal derangement of the temporomandibular joint.
The prevalence of oral mucosal lesions in a population in Ljubljana, Slovenia
Authors: Kovač‐Kavčič Marija , Skalerič Uroš
Published: March 2003

Abstract: The purpose of the present study was to evaluate the prevalence of oral mucosal lesions and conditions in a population in Ljubljana, capital of Slovenia. A total 1609 subjects represented the study population in the survey about the periodontal treatment needs in a population in Ljubljana, conducted from 1983 to 1987. Ten years later the same 1609 subjects were invited to the second examination. Altogether, 555 (34.5%) of the invited subjects in the age range 25–75 years came for an interview and clinical examination at the Department of Oral Medicine and Periodontology of the Dental Clinic in Ljubljana. Oral mucosal lesions and conditions were evaluated according to the WHO Guide to Epidemiology and Diagnosis of Oral Mucosal Diseases and Conditions. The results showed the presence of one or more mucosal lesions in 61.6% of the population. Fordyce’s condition was observed the most frequently (49.7%) followed by: fissured tongue (21.1%), varices (16.2%), history of herpes labialis (16.0%), history of recurrent aphthae (9.7%), denture stomatitis (4.3%), leukoplakia (3.1%), cheek biting (2.7%), lichen planus (2.3%), frictional keratosis (2.2%), geographic tongue (2.2%), geographic and fissured tongue together (1.1%), mucocele (0.9%), smoker’s palate (0.5%) and angular chelitis (0.4%). In the population examined, no oral malignancies were observed. Mucosal lesions like whitish lesions, denture related lesions, fissured tongue, varices and mucocele were more prevalent with increasing age. Tobacco‐related lesions (leukoplakia and smoker’s palate together) were significantly more prevalent among men than among women (P<0.05), while lichen planus, denture stomatitis and herpes labialis occured more frequently in the female population.
Immunohistochemical localization of type IV collagen and laminin (α1) in denture stomatitis
Authors: Le Bars Pierre , Piloquet Philippe , Daniel Alain , Giumelli Bernard
Published: March 2003

Abstract: Chronic clinical irritation of the palatal mucosa by dentures involves a series of histological changes in epithelial and connective tissues, inflammatory cells and the vasculature. No single change is pathognomic of this inflammatory process. The rupture of basement membrane associated with the development of denture stomatitis often marks an important stage. This study investigated modifications in basement membrane organisation, especially the distribution of type IV collagen and a specific laminin chain (α1), during denture stomatitis. Biopsies of palatal mucosa were obtained from 12 patients (8 with denture stomatitis and 4 with clinically healthy mucosa) who had worn removable dentures for more than 3 years. Immunohistochemical studies performed with specific antibodies to type IV collagen and a laminin (α1) revealed strong expression in the basement membrane of healthy palatal mucosa. In denture stomatitis, some discontinuities or disruptions in basement membrane were observed at the interface between connective tissue and epithelial cells. These findings suggest a relationship between the expression of laminin (α1) and type IV collagen and the development of denture stomatitis, a disorder involving modification of soft tissues in which initial inflammation of the palatal mucosa results from stress under the denture. These changes in basement membrane can be detected by histological studies.
Sequential immunohistochemical p53 expression in biopsies of oral lichen planus undergoing malignant evolution
Authors: Valente Guido , Pagano Marco , Carrozzo Marco , Carbone Mario , Bobba Virginio , Palestro Giorgio , Gandolfo Sergio
Published: March 2003

Abstract: Transformation in squamous cell carcinoma (SCC) may occur in a small percentage of patients affected by oral lichen planus (OLP), but the pathogenesis remains to be elucidated. Overexpression of p53 protein was investigated immunohistochemically in 28 cases of OLP, followed up by sequential biopsies for up to 96 months. In 15 cases (Group 1), no dysplastic changes or neoplastic transformation occurred during the follow‐up period; in 7 cases, OLP and SCC were synchronously observed (Group 2), whereas in another 6 cases (Group 3) SCC developed several months or years after diagnosis of OLP. The percentage of p53‐positive epithelial cells at first diagnosis was significantly higher in the cases of Groups 2 and 3 than in those of Group 1. In contrast, evaluation of growth fraction by MIB‐1 monoclonal antibody did not show any statistical differences among the three groups. Although no conclusions can be drawn about the molecular pathway leading to neoplastic transformation of OLP, or about the role of p53, the results indicate that immunohistochemical evaluation of p53 expression may be a practical tool to select cases of OLP with a high risk of neoplastic transformation.
Detection of cell cycle‐related factors in ameloblastomas
Authors: Kumamoto Hiroyuki , Kimi Kenji , Ooya Kiyoshi
Published: March 2003

Abstract: To determine whether cell cycle regulation or alteration plays a role in oncogenesis and cytodifferentiation of odontogenic epithelium, cell cycle‐related factors, including cyclin D1, p16INK4a, p21WAF1/Cip1 and p27Kip1 proteins, DNA topoisomerase IIα and histone H3 mRNA, were examined in 8 tooth germs and 31 ameloblastomas. Cyclin D1 was expressed in epithelial cells near the basement membrane in tooth germs and ameloblastomas, suggesting that this protein participates in cell proliferation in odontogenic epithelium. Immunoreactivity for p16 protein was observed in most epithelial cells in tooth germs and ameloblastomas. Expression of p21 protein was detected in most epithelial cells in tooth germs and ameloblastomas, but not in keratinizing or granular cells in variants of ameloblastomas. Expression of p27 protein was chiefly found in central polyhedral cells and keratinizing cells in tooth germs and ameloblastomas. These cyclin‐dependent kinase inhibitors were well preserved in ameloblastomas as compared with tooth germs, suggesting that the odontogenic epithelium is strictly regulated by these factors. The cell cycle phase/cellular proliferation markers, DNA topoisomerase IIα and histone H3 mRNA, were localized in scattered epithelial cells attached to the basement membrane in tooth germs and ameloblastomas.
PTCH gene altered in dentigerous cysts
Authors: Pavelić B. , Levanat S. , Crnić I. , Kobler P. , Anić I. , Manojlović S. , Šutalo J.
Published: March 2003

Abstract: Motivated by the evidence that odontogenic keratocysts are associated with genetic alterations, we examined the possibility that development of other odontogenic cysts can be attributed to gene malfunctioning, in particular to the PTCH gene. Cyst epithelium was examined for polymorphism on chromosome 9q22.3, the region that contains the PTCH gene. Loss of heterozygosity (LOH) for the D9S287 marker and/or D9S180 marker was observed in about 50% of dentigerous cysts, whereas radicular cysts gave no indication of lesions in the PTCH region. As a more direct argument for PTCH involvement in cystic growth, we report evidence of PTCH expression in dentigerous cyst lining, which indicates malfunctioning of the relevant signaling pathway. While we found no reason to believe that PTCH should be associated with radicular cysts, other genes may be implicated in their development. We performed immunohistochemical comparisons of keratocysts, dentigerous and radicular cysts for the nonmetastatic marker Nm23. A graded response placed radicular cysts in between the other two types, suggesting a similar neoplastic character for their epithelial proliferation.
Oral administration of milk from cows immunized with human intestinal bacteria leads to significant improvements of symptoms and signs in patients with oral submucous fibrosis
Authors: Tai Y. S. , Liu B. Y. , Wang J. T. , Sun A. , Kwan H. W. , Chiang C. P.
Published: March 2003

Background: Previous studies have shown that the local and systemic upregulation of fibrogenic cytokines and downregulation of antifibrotic cytokine are central to the pathogenesis of oral submucous fibrosis (OSF). The milk from cows immunized with human intestinal bacteria (immune milk) contains an anti‐inflammatory component that may suppress the inflammatory reaction and modulate cytokine production. Therefore, it was decided to test whether immune milk may have some beneficial effects on controlling the symptoms and signs in OSF patients.

Methods: In this preliminary study, 26 OSF patients who received immune milk treatment (45 g of immune milk powder twice a day) for 3 months and oral habit intervention were included in the experimental group. Another 20 OSF patients who received only oral habit intervention served as the control group.

Results: We found that the interincisor distance was significantly improved (≧3 mm of the baseline measurement) in 18 of the 26 (69.2%) OSF patients in the experimental group at exit. However, in the control group none of the OSF patients had an increase in interincisor distance greater than 2 mm. In addition, disappearance or significant improvement of symptoms at exit was observed in 80% (16/20) of the patients with intolerance to spicy foods (P<0.001) and 72.2% (13/18) of the patients with xerostomia (P<0.005) in the experimental group, compared with 17.6% (3/17) of the patients with improvement of intolerance to spicy foods and 15.4% (2/13) of the patients with improvement of xerostomia in the control group. Partial regression of concomitant oral leukoplakia or erythroplakia (judged from the size reduction of the lesions) at exit was noted in 71.4% (5/7) of the patients in the experimental group (P<0.05), compared with none (0/5) of the patients with improvement in the control group.

Conclusion: We conclude that oral administration of immune milk leads to significant improvements of symptoms and signs in OSF patients.
Detection of TP53 mutation in ameloblastoma by the use of a yeast functional assay
Authors: Shibata Toshiyuki , Nakata Daichi , Chiba Itsuo , Yamashita Tetsuro , Abiko Yoshihiro , Tada Mitsuhiro , Moriuchi Tetsuya
Published: March 2003

Background:   Mutations in TP53 have been observed in a variety of tumors including oral lesions, though there are no reports in ameloblastomas. The purpose of the study was to examine the TP53 status of ameloblastomas using newly developed yeast functional assay whose accuracy and sensitivity has been proven to be higher than those of the previous DNA structure‐based methods such as single strand conformation polymorphism (SSCP) analysis.

Methods:   TP53 status was analyzed by yeast functional assay and DNA sequencing in 12 cases of ameloblastoma which were diagnosed histologically and represented the clinical features of a benign tumor. After the extraction of RNA from the frozen tissue samples without microdissection, reverse transcription (RT)–PCR was carried out and these samples were used. The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA‐binding activity of the protein and reveal them as red colonies.

Results:   One case of 47‐year‐old male gave 17% red colonies of yeast and the other 11 cases gave 5.4% (mean; range, 3–8%). To confirm this result, we obtained other nine samples from the case of 17% red colonies, which contained or did not contain tumor tissues, and analyzed them by the assay. Seven samples that were histologically negative for tumor cells gave 4.7% red colonies (mean; range, 1–7%). Two samples that were histologically positive for tumor cells gave 20 or 46% of red colonies. The p53 plasmids were recovered from the red colonies of these three samples showing high red colony ratios and were subjected to sequencing analysis after purification. In all these samples, the same clonal mutation of TGT (Cys) 238 TAT (Tyr) was demonstrated.

Conclusions:  These results suggest that TP53 mutation may be involved in molecular pathogenesis in a subset of ameloblastomas, though it is infrequent.
Oral manifestations of an HIV positive cohort in the era of highly active anti‐retroviral therapy (HAART) in South London
Authors: Eyeson J. D. , Tenant‐Flowers M. , Cooper D. J. , Johnson N. W. , Warnakulasuriya K. A. A. S.
Published: March 2003

Background: Human Immunodeficiency Virus (HIV) infection is associated with oral manifestations of diagnostic and prognostic importance. With the advent of Highly Active Anti‐retroviral Therapy (HAART) there is anecdotal evidence to suggest that the prevalence of oral lesions has declined. The number of prevalence studies, carried out in the era of HAART is, however, meagre. Our aim was to study the prevalence of the oral manifestations of HIV in a population, predominantly on HAART, attending a Genito‐Urinary Medicine Centre in South London.

Methods: This cross sectional study included 203 adult volunteers, comprising 76% males and 24% females. One third of the subjects were from the predominantly African or Afro‐ Caribbean ethnic minority groups resident in London. The relationship between the prevalence of oral lesions and demographic variables, therapeutic regimes, viral load and CD4 counts were evaluated.

Results: One hundred (49%) of the patients had no detectable oral lesions. Oral lesions detected most frequently included oral hairy leukoplakia (9.9%), HIV associated periodontal diseases (9.9%) and oral candidiasis (4.9%). Three subjects had multiple papillomatous growths. Most cases (n = 17/20) of oral hairy leukoplakia were in individuals with a detectable (> 400 copies/ml) plasma RNA viral load. The majority (n = 8/10) of our patients with oral candidiasis had a plasma RNA viral load > 10,000 copies/ml and half (n = 5/10) had a CD4 count < 200 cells/mm3. Logistic regression analysis suggested that the presence of an oral lesion was not associated with any demographic features except for periodontal diseases which were associated with tobacco smoking (P = 0.023).

Conclusions: The prevalence of so called ‘strongly associated’ oral lesions of HIV is low in this South London HIV‐infected population on HAART, and the relative frequency is different from that cited in the literature from the pre‐HAART era. The oral lesions detected were found mostly in people with low CD4 counts and high HIV‐1 RNA viral loads, suggesting they were very immunocompromised, not on, or declining therapy, or that their therapy was failing.
Invasive front grading: reliability and usefulness in the management of oral squamous cell carcinoma
Authors: Sawair Faleh A. , Irwin Christopher R. , Gordon Derek J. , Leonard Alan G. , Stephenson Mike , Napier Séamus S.
Published: March 2003

Background:  The value of histological grading was examined with emphasis on reliability of assessment in 102 cases of intraoral squamous cell carcinoma from Northern Ireland with known outcome.

Methods:  Two pathologists independently graded the invasive tumour front blinded to the stage and outcome.

Results:  Intraobserver agreement was acceptable but interobserver agreement was not satisfactory. The degree of keratinisation was assessed most consistently while nuclear polymorphism was the least reliable feature. Multivariate survival analysis showed that the total grading score was associated with overall survival while the pattern of tumour invasion was the most valuable feature in estimating regional lymph node involvement. The number of positive lymph nodes was strongly associated with regional relapse, while the treatment modality and status of the surgical margins correlated with local relapse.

Conclusions:  Grading of selected features in OSCC is reliable and can facilitate treatment planning.
Cyclic AMP a Key Messenger in the Regulation of Skin Pigmentation
Authors: BUSCÀ ROSER , BALLOTTI ROBERT
Published: March 2003

Compelling evidence has been gathered indicating that pro‐opiomelanocortin peptides, α‐melanocyte stimulating hormone (α‐MSH) and adrenocorticotropic hormone (ACTH), through the cyclic AMP pathway, play a pivotal role in melanocyte differentiation and in the regulation of melanogenesis. Recently, the molecular events linking cAMP to melanogenesis up‐regulation have been elucidated. This cascade involves the activation of protein kinase A and CREB transcription factor, leading to the up‐regulation of the expression of Microphthalmia associated transcription factor (MITF). MITF has been found mutated in patients with Waardenburg syndrome 2A, and plays a crucial role in melanocyte development. MITF binds and activates melanogenic gene promoters, thereby increasing their expression which results in an increased melanin synthesis. Beyond this simplified scheme, It appears that melanogenic gene expression is controlled by a complex network of regulation involving other transcription factors such as Brn2, TBX2, PAX3 and SOX10. Further studies are required to better understand the respective roles of these factors in the regulation of melanin synthesis. In addition, other intracellular signaling pathways, like the phosphatidyl inositol 3‐kinase pathway, as well as the molecular cascade of events governed by the small GTP‐binding protein Rho, seem to be involved in the regulation of melanogenesis and melanocyte dendricity. Finally, it should be mentioned that cAMP activates a melanocyte‐specific pathway leading to MAP kinase activation. MAP kinase, ERK2, phosphorylates MITF, thereby targeting the transcription factor to proteasomes for degradation. Thus, in addition to the complex transcriptional regulation, melanogenesis is also subjected to a post‐translational regulation that controls MITF or tyrosinase function. Taken together, these complex molecular processes would finally allow a fine tuning of melanocyte differentiation leading to melanin synthesis.
The Melanocortin 1 Receptor (MC1R): More Than Just Red Hair
Authors: REES JONATHAN L.
Published: March 2003

The melanocortin 1 receptor, a seven pass transmembrane G protein coupled receptor, is a key control point in melanogenesis. Loss‐of‐function mutations at the MC1R are associated with a switch from eumelanin to phaeomelanin production, resulting in a red or yellow coat colour. Activating mutations, in animals at least, lead to enhanced eumelanin synthesis. In man, a number of loss‐of‐function mutations in the MC1R have been described. The majority of red‐heads (red‐haired persons) are compound heterozygotes or homozygotes for up to five frequent loss‐of‐function mutations. A minority of red‐heads are, however, only heterozygote. The MC1R is, therefore, a major determinant of sun sensitivity and a genetic risk factor for melanoma and non‐melanoma skin cancer. Recent work suggests that the MC1R also shows a clear heterozygote effect on skin type, with up to 30% of the population harbouring loss‐of‐function mutations. Activating mutations of the MC1R in man have not been described. The MC1R is particularly informative and a tractable gene for studies of human evolution and migration. In particular, study of the MC1R may provide insights into the lightening of skin colour observed in most European populations. The world wide pattern of MC1R diversity is compatible with functional constraint operating in Africa, whereas the greater allelic diversity seen in non‐African populations is consistent with neutral predictions rather than selection. Whether this conclusion is as a result of weakness in the statistical testing procedures applied, or whether it will be seen in other pigment genes will be of great interest for studies of human skin colour evolution.
Depigmenting Effects of Calcium d‐Pantetheine‐S‐Sulfonate on Human Melanocytes
Authors: FRANCHI JOCELYNE , COUTADEUR MARIE CHRISTINE , MARTEAU CLARISSE , MERSEL MARCEL , KUPFERBERG ALEXANDRE
Published: March 2003

The effects of calcium d‐pantetheine‐S‐sulfonate (PaSSO3Ca) on human pigmentation were examined by in vitro assays using two types of human melanocytes: normal adult melanocytes (HNM) and M4Be melanoma cells. The compound, when added to a culture medium at doses indicating no cytotoxicity, causes a visually recognizable, reversible loss of pigment in both types of cells. Determination of melanin content, incorporation of

C‐DOPA into melanins and tyrosinase activities demonstrated that treatment of these cells with PaSSO3Ca resulted in a marked decrease in all three areas. When homogenates of these cells were assayed with lectins, the glycosylation pattern was modified, as tyrosinase activities were reduced in the cells treated with the compound. Immunoprecipitation of tyrosinase and tyrosinase‐related protein 1 (Tyrp1 or TRP1) in cells incubated with radioactive glucosamine disclosed that glucosamine uptake by these enzymes was apparently increased, suggesting structural alterations in their sugar moieties. It is also noted that PaSSO3Ca is analogous in its chemical structure to Coenzyme A (CoA), which plays an important role in the intracellular transport of proteins. Based on these findings, it is likely that the compound exerts its depigmenting effects in human pigment cells through the modification of glycosylation of tyrosinase and TRP1, which are key enzymes for melanogenesis.
Melanocytes Respond to Mechanical Stretch by Activation of Mitogen‐Activated Protein Kinases (MAPK)
Authors: KIPPENBERGER STEFAN , LOITSCH STEFAN , MÜLLER JUTTA , GUSCHEL MAIKE , RAMIREZ‐BOSCA ANA , KAUFMANN ROLAND , BERND AUGUST
Published: March 2003

Cells of human epidermis are permanently targeted by mechanical stimuli. Besides mechanical forces from external sources the body itself generates mechanical forces via muscle contractions and growth processes. Recently, it was demonstrated that mechanical stretch is connected to enhanced proliferation in epidermal cells. The underlying biochemical events are still a matter of debate. Here we show that mechanical stretch leads to activation of both ERK1/2 and SAPK/JNK in human melanocytes and keratinocytes. In response to a 5 min single stretch ERK1/2 becomes moderately induced in melanocytes and peaked 30 min after the stimulus. In keratinocytes strong activation of ERK1/2 is present directly after the stimulus. SAPK/JNK shows the same activation pattern in both cell species – a slow but steady activation. The different kinetics of both MAPK suggest that different signalling cascades were activated. Future studies should evaluate the relevance of stretch‐dependent MAPK activation in triggering the cell proliferation.
Melanosynthesis, Differentiation, and Apoptosis in Kupffer Cells from Rana esculenta
Authors: PURRELLO MICHELE , SCALIA MARINA , CORSARO CONCETTA , DI PIETRO CINZIA , PIRO SALVATORE , SICHEL GIOVANNI
Published: March 2003

It is well known that amphibian Kupffer cells (KCs) contain eumelanins. In this paper, we demonstrate through a molecular analysis that Rana esculenta KCs synthesize high levels of mRNA for tyrosinase and through cytochemistry that they possess dopa oxidase activity: both these data prove that frog KCs are capable of autonomously synthesizing eumelanins. On the other hand, by using a highly sensitive reverse transcription‐polymerase chain reaction assay we clearly show that in mammalian KCs the tyrosinase gene is not expressed. Quite unexpectedly, we have detected tyrosinase mRNA in Rana esculenta spleen, lung, and heart; to explain this finding, we suggest that it could be due to the presence of pigmented macrophages within the spleen, that probably behave as KCs, and of melanophores in lung and heart. It also may be hypothesized that the Rana esculenta tyrosinase gene, as opposed to its mammalian counterpart, is expressed in many cell types because its promoter contains sequences that are recognized by widely synthesized transcription factors. Our experiments also demonstrate that there is an inverse correlation between the amount of tyrosinase mRNA and melanin content, and that populations of terminally differentiated KCs are characterized by a high degree of apoptosis. Based on these data, we propose that differentiating KCs start accumulating eumelanins, as a result of previous expression of high levels of tyrosinase and of dopa oxidase activity, acquire the full KC phenotype (characterized by both phagocytic and melanosynthetic ability), and then undergo apoptosis. Accordingly, we propose that these cells could represent an interesting model to study, at the molecular level, the relationship between differentiation, specific gene expression, and programmed cell death in higher eukaryotes.
Regulation of Tyrosinase‐related Protein‐2 (TYRP2) in Human Melanocytes: Relationship to Growth and Morphology
Authors: FANG DONG , KUTE TIM , SETALURI VIJAYASARADHI
Published: March 2003

Treatment of human melanoma cells with the differentiation‐inducing agent hexamethylene bisacetamide (HMBA) results in reciprocal changes in expression of melanocyte‐specific genes tyrosinase‐related proteins‐1 and ‐2 (TYRP1 and TYRP2). In this study, we investigated the effects of HMBA on cultured neonatal human cutaneous melanocytes. Flow cytometric analysis showed that HMBA inhibited 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐dependent growth of melanocytes by reducing the population of cells entering the DNA synthesis phase of cell cycle. Melanocyte growth inhibition was accompanied by an increase in the number of cells exhibiting polydendritic morphology. This morphologic change was less pronounced when HMBA was added to melanocytes in the absence of TPA. Northern blot analyses of total cellular RNA showed that expression of microphthalmia‐associated transcription factor (MITF), tyrosinase (TYR), TYRP1, Silver (SILV/Pmel17) gene was down‐regulated by HMBA, while TYRP2 mRNA was up‐regulated (>10‐fold). When the inducer was added to cells in the absence of TPA, there was >50‐fold increase in TYRP2 mRNA with a moderate increase in MITF, tyrosinase and SILV gene mRNAs and complete repression of TYRP1 gene. Studies using inhibitors for protein kinases involved in cell signaling pathways suggested that stress‐activated kinase p38 and mitogen‐activated protein kinase kinase MEK are involved in TPA‐independent regulation of TYRP2 expression in melanocytes. These data show that treatment of proliferating melanocytes with the differentiation inducer HMBA results in a distinct change in morphology and up‐regulation of TYRP2, while quiescent melanocytes respond by a dramatic increase in expression of TYRP2 without change in morphology. These results suggest an inverse relationship of TYRP2 gene regulatory mechanisms to melanocyte growth regulatory pathways.
Tissue Factor Expression and Serum Level in Patients with Melanoma does not Correlate with Disease Progression
Authors: Kageshita Toshiro , Funasaka Yoko , Ichihashi Masamitsu , Wakamatsu Kazumasa , Ito Shosuke , Ono Tomomichi
Published: March 2003

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.
Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation, Differentiation and Migration in Cultured Mouse Neural Crest Cells
Authors: Takano Nagako , Kawakami Tamihiro , Kawa Yoko , Asano Mari , Watabe Hidenori , Ito Masaru , Soma Yoshinao , Kubota Yasuo , Mizoguchi Masako
Published: March 2003

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.
Animal Models of Melanoma: An HGF/SF Transgenic Mouse Model May Facilitate Experimental Access to UV Initiating Events
Authors: Noonan Frances P. , Dudek James , Merlino Glenn , De Fabo Edward C.
Published: March 2003

Cutaneous malignant melanoma, the most lethal of the skin cancers, known for its intractability to current therapies, continues to increase in incidence, providing a significant public health challenge. There is a consensus that skin cancer is initiated by sunlight exposure. For non‐melanoma skin cancer there is substantial evidence that chronic exposure to the ultraviolet B radiation (UVB) (280–320 nm) portion of the sunlight spectrum is responsible. Experimentally, UVB is mutagenic and chronic UVB exposure can cause non‐melanoma skin cancer in laboratory animals. Non‐melanoma tumors in animals and in humans show characteristic UVB signature lesions in the tumor suppressor p53 and/or in the patched (PTCH) gene. An action spectrum or wavelength dependence for squamous cell carcinoma in the mouse shows a major peak of efficacy in the UVB. For malignant melanoma, however, the situation is unclear and the critical direct target(s) of sunlight in initiating melanoma and even the wavelengths responsible are as yet unidentified. This lack of information is in major part a result of a paucity of animal models for melanoma which recapitulate the role of sunlight in initiating this disease. The epidemiology of melanoma differs significantly from non‐melanoma skin cancer. Intense sporadic sunlight exposure in childhood, probably exacerbated by additional adult exposure, is associated with elevated melanoma risk. Melanoma is also a disease of gene–environment interactions with underlying genetic factors playing a significant role. These major differences indicate that extrapolation from information for non‐melanoma skin cancer to melanoma is unlikely to be useful. We summarize in this review the experimental information available on the role of UV radiation in melanoma and give an overview of animal melanoma models. A new model derived by neonatal UV irradiation of hepatocyte growth factor/scatter factor (HGF/SF) transgenic mice is described which recapitulates the etiology, the histopathology and molecular pathogenesis of human disease. It is anticipated that the HGF/SF transgenic model will provide a means to access the mechanism(s) by which sunlight initiates this lethal disease and provide an appropriate vehicle for derivation of appropriate therapeutic and preventive strategies.
Expression of fibrillins and tropoelastin by human gingival and jreiodontal ligament fibroblasts in vitro
Authors: Tsuruga Eichi , Irie Kazuharu , Sakakura Yasunori , Yajima Toshihiko
Published: March 2003

The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In jreiodontal tissues, oxytalan fibers are known to be distributed in the jreiodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and jreiodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since fibrillin‐1 and fibrillin‐2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed fibrillin‐1 and fibrillin‐2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the jreinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the exjreimental jreiod, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and jreiodontal ligament in vivo.
Different distribution of immunocompetent cells in the dentogingival junction during root formation in rat molars
Authors: Tamura Hiroshi , Nakakura‐Ohshima Kuniko , Maeda Takeyasu , Ohshima Hayato
Published: March 2003

The distribution of immunocompetent cells in the dentogingival junction of rat molars during root formation was investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6‐antibody) and monocyte/macrophage lineage cells (ED1‐antibody) as well as by histochemical reaction for periodic acid–Schiff (PAS). Two portions (the junctional epithelium in the mesial gingiva of the first molar, and the interdental gingiva between the first and second molars) were selected for observations. At the eruption stage of the first molar (16–18 days after birth), OX6‐positive cells, dendritic or oval in shape, were abundantly distributed in the connective tissue between the oral epithelium and tooth germ. Positive cells with slender cell processes were also found beneath the ameloblast layer. At the commencement stage of the first molar occlusion (24–28 days after birth), numerous OX6‐positive cells displaying a dendritic fashion existed preferentially in the mesial gingiva, but were fewer in the interdental gingiva. In contrast, the interdental gingiva showed a denser distribution of ED1‐positive cells and PAS‐reactive polymorphonuclear leukocytes (PMLs) than the mesial gingiva. At the completion stage of root formation (100–120 days after birth), the OX6‐immunopositive cells invaded the deeper position of the mesial gingiva with the downgrowth of the epithelium; they had a considerably higher cell density compared with those in the interdental gingiva where PAS‐reactive PMLs persisted. These findings indicated that the immunocompetent cells showed a region‐specific distribution and cell density by their roles in immune response.
Immunohistochemical characteristics of epithelial cell rests of Malassez during cementum repair
Authors: Hasegawa Naohiko , Kawaguchi Hiroyuki , Ogawa Tetsuji , Uchida Takashi , Kurihara Hidemi
Published: March 2003

To clarify the roles of epithelial cell rests of Malassez (ECRM) during periodontal repair, experimental root resorption was induced in rats and then the ECRM that existed in periodontal ligament during cementum repair was investigated using morphological and immunohistochemical approaches. At day 7, after mechanical injury, root resorption was observed and ECRM were present adjacent to the site of resorption lacunae. They were observed in periodontal ligament adjacent to site of the resorption lacunae. These ECRM were immunoreactive for bone morphogenetic protein‐2. During the stage of early cementum repair, the ECRM were immunoreactive for osteopontin and ameloblastin. They strongly reacted to proliferating cell nuclear antigen. In uninjured control sections, ECRM located in the periodontal ligament adjacent to cementum were not immunoreactive for any antibodies. These findings suggested that ECRM may be related to cementum repair by activating their potential to secrete matrix proteins which have been expressed in tooth development.
Effects of rhIGF‐I and Insulin‐Induced Hypoglycaemia on Cardiovascular Parameters Recorded with Telemetry in the Conscious Dog
Authors: Ilbäck Nils‐Gunnar , Gunnarsson Kjell , Stålhandske Torbjörn
Published: March 2003

Abstract: Using telemetry, the effects on cardiovascular parameters after single intravenous administration (0.5 mg/kg) of recombinant human insulin‐like growth factor‐I (rhIGF‐I) were studied in conscious and unrestrained dogs. Insulin (1.1 IU/kg) was used as a reference compound. Telemetry transmitters were implanted subcutaneously with a pressure catheter in the femoral artery and electrodes for ECG subcutaneously. Cardiovascular effects and changes in blood glucose levels induced with rhIGF‐I were similar to those induced with insulin. Heart rate increased continuously for about 90 min. after treatment, regardless of compound. Thereafter, heart rate slowly decreased but did not fully reach predose values 4 hr after treatment. Both systolic and diastolic blood pressure decreased continuously for about 90 min. and remained low for up to 4 hr after treatment. Treatment with rhIGF‐I or insulin did not influence dp/dt values. Treatment with glucose intravenously to abolish the rhIGF‐I‐induced hypoglycaemia reduced the heart rate, but caused a substantial increase in dp/dt and a slight increase in blood pressure. RhIGF‐I and insulin induced an almost identical onset and degree of hypoglycaemia. Blood glucose reached a minimum level 1 hr after treatment and was almost returned to normal 4 hr after treatment. There was an increase in the amplitude of the T‐waves, though this effect occurred earlier and was longer lasting with insulin than with rhIGF‐I. After 4 hr, the T‐wave amplitude was normal with rhIGF‐I but remained high with insulin. These T‐wave effects were probably due to an increase in the transport of potassium or calcium since they decreased in plasma. This suggests that the effects of rhIGF‐I and insulin are highly comparable and that the cardiovascular changes induced by rhIGF‐I are likely caused by its insulin‐like activity. As shown by the recorded cardiovascular responses induced by rhIGF‐1 and insulin, the telemetric recording system makes it possible to evaluate the effects of different drugs in a continuous way that is not possible with conventional techniques. This new telemetric technique can be of significant importance in the process of future drug development.