bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical and genetic analysis of mandibular cysts in heterozygous ptc knockout mice
Authors: Ohki Kousuke , Kumamoto Hiroyuki , Kondo Mari , Taniguchi Yoshihiro , Tanigami Akira , Ooya Kiyoshi
Published: March 2003

Background:  Alterations of human patched (ptc) homolog have been proven to be responsible for basal cell nevus syndrome (BCNS). Mandibular cysts in heterozygous ptc knockout mouse (ptc+/− mouse) were microradiologically, histologically, immunohistochemically, and genetically examined to investigate the possible role of the ptc gene and its associates in the jaw cysts.

Methods:  The mandibular bones were prepared from 63 ptc+/− mice and 6 ptc+/+ mice. Soft X‐ray radiographs and histological sections were examined for detection of the presence of mandibular cysts. The mandibular cysts were immunohistochemically investigated using anti‐ptc, shh, and smo antibodies. PCR analysis of loss of heterozygosity (LOH) of ptc was performed in genomic DNA from the mandibular cysts.

Results:  Six ptc+/+ mice showed no pathologic change in any examinations. Microradiologically, ptc+/− mice did not show any apparent lesion. Mandibular cysts were often multiple, and were histologically detected in the alveolar bones or periodontal ligaments of the molars in 16 (25.4%) ptc+/− mice. The mandibular cysts were lined by thin parakeratotic stratified squamous epithelium and contained keratinized materials. Immunohistochemical examination showed sonic hedgehog (shh) protein mainly in cyst lining epithelium, and ptc and smoothened (smo) proteins in cyst lining epithelium, and surrounding fibrous connective tissue. Expression of ptc protein in the cyst lining epithelium tended to be weak as compared with incisor enamel organs and gingival stratified squamous epithelium. LOH of the ptc gene couldn't be found in lining epithelium of mandibular cysts in any ptc+/− mice.

Conclusions:  Ptc
+/− mouse is a useful model of BCNS from the standpoint of occurrence of jaw cysts, and downregulation of ptc protein in cyst lining epithelium caused by gene targeting would be associated with formation of jaw cysts in ptc+/− mice.
Inhibition of UVR‐Induced Tanning and Immunosuppression by Topical Applications of Vitamins C and E to the Skin of Hairless (hr/hr) Mice	1
Authors: QUEVEDO JR. WALTER C. , HOLSTEIN THOMAS J. , DYCKMAN JACOB , MCDONALD CHARLES J. , ISAACSON ERNEST L.
Published: March 2003

Exposure of C3HBYB/Wq hairless (hr/hr) mice to ultra‐violet radiation (UVR) for 15 days induced intense tanning of their dorsal skin. Small, dark freckles appeared first, gradually enlarging and coalescing as treatment progressed yielding a uniform tan. Histologically, the gross changes in skin color were matched initially by the appearance of scattered epidermal melanocytes that subsequently proliferated to form discrete, progressively expanding and abutting populations resulting in a uniform melanocyte network throughout the basal layer of the interfollicular epidermis. In contrast, when applied topically before each daily exposure to UVR, a cream or lotion vehicle containing both vitamins C and E (Vits C/E) inhibited UVR‐induced erythema and tanning. Application of Vits C/E, both before and after irradiation, was no more effective in providing photoprotection than pre‐treatment only. At the tissue level, UVR‐induced proliferation and melanogenesis of melanocytes were reduced compared with irradiated controls. The density of individual melanocyte populations was reduced, as was the number of melanocyte populations achieving merger (confluence) with others. Confluence grades and cell counts, estimating the maximum density of melanocyte populations in UVR–Vits C/E‐treated mice, were approximately two thirds those of UVR–vehicle‐treated controls. However, tanning was only one fifth that of UVR–vehicle‐treated controls, suggesting that melanogenesis was also inhibited. In addition to its inhibitory actions on irradiated melanocytes, Vits C/E also inhibited UVR‐induced suppression of contact hypersensitivity (CHS) in haired (Hr/hr) and hr/hr mice of the C3HBYB/Wq strain. The common denominators for most, if not all, of the influences of topically‐applied Vits C/E in muting the responses of the melanocyte and immune systems to UVR may stem from the vitamins’ combined ability to suppress UVR‐stimulated inflammation and its associated cascade of mediators.
Up‐regulation of Tyrosinase Gene by Nitric Oxide in Human Melanocytes
Authors: SASAKI MINORU , HORIKOSHI TOSHIO , UCHIWA HIDEYO , MIYACHI YOSHIKI
Published: March 2003

Ultraviolet light (UV) radiation causes skin‐tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO‐induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S‐nitroso‐N‐acetyl‐
l‐arginine. An increase of tyrosinase activity was also detected time‐dependently within the 24‐hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO‐induced melanogenesis.
The Tyrosinase Gene and Oculocutaneous Albinism Type 1 (OCA1): A Model for Understanding the Molecular Biology of Melanin Formation
Authors: OETTING WILLIAM S.
Published: March 2003

Through the last century there has been a steady progression in our understanding of the biology of melanin biosynthesis. Much of this work includes the analysis of coat color mutations of the mouse and albinism in man. Our understanding has been greatly enhanced in the last 10 years, as the molecular pathogenesis of albinism has been better understood. Different mutations of the tyrosinase gene (TYR) , and their association with oculocutaneous albinism type 1 (OCA1) has provided insight into the biology of tyrosinase, including protein trafficking and structure/function analysis. Several questions still remain, including cryptic mutations that affect tyrosinase activity and the minimum amount of pigment required for normal optic development. The next 10 years should prove just as exciting as the last.
EDNRB/EDN3 and Hirschsprung Disease Type II
Authors: Mccallion Andrew S. , Chakravarti Aravinda
Published: March 2003

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1–4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah‐Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type‐B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest‐derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest‐derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB‐mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3‐induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post‐coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3 – a receptor‐mediated pathway with pleiotropic effects.
Agouti: from Mouse to Man, from Skin to Fat
Authors: Voisey Joanne , Van Daal Angela
Published: March 2003

The agouti protein regulates pigmentation in the mouse hair follicle producing a black hair with a subapical yellow band. Its effect on pigmentation is achieved by antagonizing the binding of α‐melanocyte stimulating hormone (α‐MSH) to melanocortin 1 receptor (Mc1r), switching melanin synthesis from eumelanin (black/brown) to phaeomelanin (red/yellow). Dominant mutations in the non‐coding region of mouse agouti cause yellow coat colour and ectopic expression also results in obesity, type II diabetes, increased somatic growth and tumourigenesis. At least some of these pleiotropic effects can be explained by antagonism of other members of the melanocortin receptor family by agouti protein. The yellow coat colour is the result of agouti chronically antagonizing the binding of α‐MSH to Mc1r and the obese phenotype results from agouti protein antagonizing the binding of α‐MSH to Mc3r and/or Mc4r. Despite the existence of a highly homologous agouti protein in humans, agouti signal protein (ASIP), its role has yet to be defined. However it is known that human ASIP is expressed at highest levels in adipose tissue where it may antagonize one of the melanocortin receptors. The conserved nature of the agouti protein combined with the diverse phenotypic effects of agouti mutations in mouse and the different expression patterns of human and mouse agouti, suggest ASIP may play a role in human energy homeostasis and possibly human pigmentation.
Transcription of Prostanoid Receptor Genes and Cyclooxygenase Enzyme Genes in Cultivated Human Iridial Melanocytes from Eyes of Different Colours
Authors: Wentzel Parri , Bergh Kerstin , Wallin Örjan , Niemelä Pekka , Stjernschantz Johan
Published: March 2003

Several prostaglandin analogues used for glaucoma treatment have been shown to cause increased iridial pigmentation as side‐effect. In the present study we identified the types of prostanoid receptors and cyclooxygenase (COX) enzymes that are expressed in human iridial melanocytes isolated from eyes of different colours. Iris specimens were obtained during trabeculectomy surgery, or from enucleated eyes, and the iridial melanocytes were isolated and cultivated. The transcription of the DP, EP1, EP2, EP3, EP4, FP, IP and TP prostanoid receptor genes as well as the COX‐1 and COX‐2 enzyme genes was investigated using reverse transcriptase‐polymerase chain reaction (RT‐PCR). Of the prostanoid receptors the FP receptor gene was found to be most consistently transcribed in the melanocytes isolated from both blue‐ and hazel‐coloured eyes. No RNA of the DP, EP2 and TP receptor genes could be detected, whereas the EP1, EP3, EP4 and IP receptor genes were found to be transcribed in melanocytes from some eyes. The COX‐2 gene was found to be transcribed, but the COX‐1 gene less consistently. There was no difference in gene transcription pattern between melanocytes originating from eyes treated with latanoprost, and eyes not previously treated with the prostaglandin. These results indicate that the FP prostanoid receptor gene is transcribed in cultivated human iridial melanocytes of both blue and hazel eyes, whereas the other prostanoid receptor genes seem to be transcribed much less frequently, or not at all. Surprisingly, the COX‐2 rather than the COX‐1 gene, was found to be transcribed in the melanocytes.
Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans
Authors: Nonaka Koji , Ishisaki Akira , Okahashi Nobuo , Koseki Takeyoshi , Kato Satsuki , Muro Miyuki , Nakashima Keisuke , Nishihara Tatsuji , Kowashi Yusuke
Published: March 2003

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans‐infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl‐Val‐Ala‐Asp (OMe)‐fluoromethyl ketone (Z‐VAD‐FMK) and benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp (OMe)‐fluoromethyl ketone (Z‐DEVD‐FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin‐1β(IL‐1β) was detected in the culture supernatants of J774.1 cells. IL‐1β secretion was blocked by the caspase‐1 inhibitor, Z‐VAD‐FMK, indicating that caspase‐1 is involved in not only the induction of apoptosis but also the IL‐1β secretion from A. actinomycetemcomitans‐infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase‐3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans‐infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase‐3 inhibitor, Z‐DEVD‐FMK. Furthermore, caspase‐3‐like activity was markedly increased in J774.1 cells infected with A. actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase‐3 inhibitor, Z‐DEVD‐FMK. These findings indicate that caspase‐3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase‐1 and caspase‐3 are involved in the induction of apoptosis in A. actinomycetemcomitans‐infected macrophages.
Induction of collagenase‐2 (matrix metalloproteinase‐8) gene expression by interleukin‐1β in human gingival fibroblasts
Authors: Abe M. , Kawamoto K. , Okamoto H. , Horiuchi N.
Published: March 2003

Collagenase‐2 (matrix metalloproteinase‐8 or MMP‐8) is synthesized mainly by polymorphonuclear neutrophils and plays a crucial role in inflammatory periodontal tissue destruction. We tested the effect of interleukin(IL)‐1β, a proinflammatory cytokine, on collagenase‐2 gene expression in cultured human gingival fibroblasts and also compared this effect with IL‐1β‐induced changes in collagenase‐1 and ‐3 gene expression. By a combination of reverse transcription‐polymerase chain reaction and Southern analysis, IL‐1β was found to dose‐dependently induce gene expression for collagenase‐1, ‐2, and ‐3 in gingival fibroblasts. Although collagenase‐2 mRNA was the least abundant among the three collagenase mRNAs tested in the cultured fibroblast system, addition of 1 ng/ml IL‐1β significantly increased collagenase‐2 gene transcription within 6 h, and maximal stimulation was maintained for 12 to 48 h. Significant mRNA induction was observed with as little as 0.1 ng/ml IL‐1β. IL‐1β was also found to increase the stability of collagenase‐2 mRNAs after transcription arrest was induced by an RNA polymerase inhibitor. Stimulation of collagenase‐2 mRNA expression by IL‐1β was prevented by pretreatment with cycloheximide, an inhibitor of protein synthesis. These results indicate that IL‐1β increased mRNA expression for collagenases including collagenase‐2 in gingival fibroblasts. The findings also suggest that enhancement of collagenase‐2 mRNA expression by IL‐1β involves both protein synthesis and suppression of mRNA degradation.
Crevicular fluid elastase levels in relation to periodontitis and metabolic control of diabetes
Authors: Alpagot Tamer , Silverman Sol , Lundergan William , Bell Colin , Chambers David W.
Published: March 2003

The purpose of this study was to investigate the associations between gingival crevicular fluid (GCF) elastase levels, clinical measures of periodontal status, and metabolic control of diabetes in insulin dependent (type 1) diabetes (IDDM) and non‐insulin dependent (type 2) diabetes (NIDDM) patients. Sixty patients were recruited from the Diabetes Center at the University of California in San Francisco. Thirty subjects were type 1 diabetics and 30 subjects were type 2 diabetics. Metabolic control was evaluated by glycosylated hemoglobin (HbA1c) levels. Demographic information was obtained using a structured interview with the subjects. Clinical measurements and GCF samples were taken from the mesio‐buccal surfaces of 2 premolars and 2 molars from the most diseased sextant. GCF elastase was determined by measurement of p‐Nitroanalide resulting from hydrolysis of elastase specific peptide. Crevicular fluid elastase levels were significantly correlated with gingival index, bleeding index, probing depth and attachment level in both type 1 and type 2 diabetes groups (0.01<p<0.001). HbA1c levels were not correlated with clinical measurements and GCF elastase. The results suggest that GCF elastase, age and smoking are risk indicators for periodontitis in patients with diabetes mellitus, and periodontal status is not associated with the duration and metabolic control of diabetes.
Characterization of cellular infiltrate, detection of chemokine receptor CCR5 and interleukin‐8 and RANTES chemokines in adult periodontitis
Authors: Gamonal J. , Acevedo A. , Bascones A. , Jorge O. , Silva A.
Published: March 2003

Leukocyte migration is essential for immune surveillance of tissues by focusing immune cells to sites of antigenic challenge. The control of leukocyte migration depends on the combined actions of adhesion molecules and a vast array of chemokines and their receptors. The purpose of the present study was to investigate the involvement of Interleukin‐8 (IL‐8), RANTES, the associated infiltrating cells and expression of CCR5 chemokine receptors in periodontitis; furthermore, the effect of periodontal therapy on these parameters was evaluated. Patients included in the study had moderate to advanced periodontal disease with at least 5–6 teeth with probing depth >6 mm, attachment loss ≥3 mm and extensive radiographic bone loss. The inflammatory infiltrate was analyzed by immunohistochemistry in gingival biopsies obtained from subjects at the beginning of the study and 2 months after periodontal treatment. Gingival crevicular fluid (GCF) was collected for 30 seconds using periopaper strips, and chemokines were quantified by ELISA. The cellular components of the inflammatory infiltrate included B (CD19) and T (CD3, CD4+ and CD8+) lymphocytes and monocytes/macrophages (CD11c). CCR5 chemokine receptor expressing cells were exclusively found in periodontitis gingiva. IL‐8 and RANTES were detected in the periodontitis group, obtaining a total amount of 212.5 pg and 42.0 pg, respectively. However, IL‐8 was also detectable in the GCF of the healthy group (total amount of 44.8 pg). Periodontal therapy reduced the cell number in the infiltrate and the levels of IL‐8 and RANTES, suggesting a relationship between these chemokines and periodontal status. We propose that the presence of these chemokines and the expression of chemokine receptors may represent a marker of lymphocyte subsets with the ability to migrate to inflammatory sites.
Bacteroides forsythus prtH genotype in periodontitis patients: occurrence and association with periodontal disease
Authors: Tan Kai Soo , Song Keang‐Peng , Ong Grace
Published: March 2003

Bacteroides forsythus has been described as a periodontopathogen and its presence in the subgingival plaque can lead to periodontal disease. Recently, a cysteine protease designated as prtH was isolated and characterized from B. forsythus ATCC 43037. The purpose of this study was to determine the prevalence and the association of the prtH gene of B. forsythus with periodontal disease. A total of 160 subgingival plaque samples were assayed with the polymerase chain reaction method using oligonucleotide primers targeting the prtH and the 16S rDNA genes of B. forsythus. Primers targeting the 16S rDNA gene of B. forsythus were used to determine the occurrence of the bacteria in the subgingival plaque samples at baseline. At baseline, B. forsythus was detected in 78 out of 86 (91%) diseased sites and 33 out of 74 (45%) healthy sites studied. Among the 86 diseased sites examined, 73 sites (85%) were colonized by the bacteria with the prtH genotype. In sites of the periodontally healthy, 7 out of 73 (10%) possessed B. forsythus with the prtH genotype. The results obtained suggested strong association of the prtH gene of B. forsythus with adult periodontitis. Although this bacterial species was detected from about half of the periodontally healthy samples, only a fraction of these subjects possess the bacteria strain with the prtH genetic subtype. We propose the use of the prtH gene as an alternative to the more widely used 16S rDNA gene of B. forsythus, for a more accurate determination of the prevalence of periodontal health and disease in epidemiological studies and clinical screening.
Initial effect of controlled release chlorhexidine on subgingival microorganisms
Authors: Daneshmand Nazanin , Jorgensen Michael G. , Nowzari Hessam , Morrison John L. , Slots Jørgen
Published: March 2003

Background:  Little or no data exist on the ability of subgingival application of PerioChip® (2.5 mg chlorhexidine gluconate in a biodegradable chip; Astra Pharmaceuticals, Westborough, MA, USA) to suppress periodontopathic microorganisms. The present study compared the subgingival microbiota of periodontitis sites receiving the chlorhexidine chip plus scaling and root planing (Sc/Rp) or Sc/Rp alone.

Methods: Seven males and six females, mean age 49 years, with moderate to advanced periodontitis participated in the study. In each patient, two bilateral pockets probing 6–7 mm were randomly assigned to treatment by chlorhexidine chip + Sc/Rp, or by Sc/Rp alone. Subgingival placement of chlorhexidine chips was carried out according to the manufacturer's instructions. Sc/Rp was performed with hand instruments for at least 10 min in each study tooth. Subgingival samples were collected by paper‐points at baseline, at 2 weeks and at 4 weeks post‐treatment. Anaerobic culture methods were used for microbial isolation and identification. The microbiologic examination was carried out blindly. Microbiological data were evaluated by a repeated measures analysis of variance.

Results: No statistical difference was found in total colony counts between subgingival sites treated with chlorhexidine chip + Sc/Rp and those treated with Sc/Rp alone. Also, the percentage of major periodontal pathogens (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Bacteroides forsythus) and the percentage of total periodontal pathogens (A. actinomycetemcomitans, P. gingivalis, B. forsythus, Prevotella intermedia‐group, Fusobacterium, Eubacterium, Campylobacter rectus, Peptostreptococcus micros, Eikenella corrodens, enteric rods) were not significantly different between the chlorhexidine chip + Sc/Rp group and the Sc/Rp group. At baseline, A. actinomycetemcomitans was recovered from 4 chlorhexidine chip + Sc/Rp sites and 2 Sc/Rp sites, P. gingivalis from 5 chlorhexidine chip + Sc/Rp sites and 4 Sc/Rp sites, and B. forsythus from 9 chlorhexidine chip + Sc/Rp and 7 Sc/Rp sites. At 4 weeks, A. actinomycetemcomitans was detected in 2 chlorhexidine chip + Sc/Rp sites but not in any site receiving Sc/Rp, P. gingivalis in 2 chlorhexidine chip + Sc/Rp sites but not in any Sc/Rp site, and B. forsythus in 1 chlorhexidine chip + Sc/Rp and in 2 Sc/Rp sites.

Conclusion: The present data obtained from bilateral periodontitis lesions of 13 adults suggest that chlorhexidine chip treatment of adult periodontitis lesions provides little or no additional antimicrobial benefits compared to thorough Sc/Rp alone.
Initial attachment of osteoblasts to various guided bone regeneration membranes: an in vitro study
Authors: Wang H.‐L. , Miyauchi M. , Takata T.
Published: March 2003

Objective and background:  Guided bone regeneration (GBR) has proved to be a suitable and somehow predictable technique for promoting bone regeneration. Avariety of synthetic and naturally derived GBR barriers have been used in clinics to facilitate bone regeneration. These barriers may differ in composition and structure and these may affect the outcomes of GBR. Therefore, the present study was undertaken to evaluate the in vitro ability of osteoblasts (MC3T3‐E1) to attach to various GBR membranes.

Materials and methods:  Six GBR/GTR (guided tissue regeneration) membranes [BioMend® (BM), Resolut® (RL), Guidor® (GD), EpiGuide® (EG), Gore‐Tex® (GT) and Millipore filter® (MP)] were tested. For controls, cells were directly plated on culture dishes (CD). Each test membrane was secured to the bottom of a culture dish with a double‐sided adhesive tape. All samples were triplicate. At 1.5 and 24 h after plating of 2 ml (5 × 104 cells/ml) of MC3T3‐E1 (passage 7) cells, the specimens were rinsed with phosphate‐buffered saline to wash out any unattached cells and then fixed with a 10% buffered formalin solution for 1 d. After washing with distilled water, the cells were stained with hematoxylin. The number of attached cells was counted under a light microscope equipped with an ocular‐micrometer in a unit area of 0.25 mm2 (five areas on each membrane). In addition, cell morphology attached to the membranes was evaluated under scanning electron microscope.

Results:  Data were presented as mean ± standard error and analyzed for statistical difference using a generalized Wilcoxon's test. Cell attachment at 1.5 h was as follows: MP (27.5 ± 2.1) > RL (17.0 ± 1.4) ≈ BM (14.5 ± 1.4) ≈ EG (11.4 ± 1.0) > GD (5.2 ± 0.8) ≈ GT (3.1 ± 0.6); and at 24 h was: MP (67.6 ± 3.6) > RL (35.8 ± 1.8) > BM (15.4 ± 0.9) ≈ EG (13.3 ± 1.3) > GD (5.9 ± 0.7) ≈ GT (5.6 ± 1.3). At 24 h, the scanning electron microscope finding revealed that cells attached on MP, RL, BM and EG were flatter in shape, like cells on CD, than cells on GD and GT, where cells were rather round.

Conclusions:  Results from this study suggested that MP, BM, RL and EG enhanced the early osteoblast attachment. However, the true benefit of this observation in clinic remains to be determined.
Effects of CD14 receptors on tissue reactions induced by local injection of two gram‐negative bacterial lipopolysaccharides
Authors: Chiang Cheng‐Yang , Fu Earl , Shen E‐Chin , Chiu Hsien‐Chung
Published: March 2003

Lipopolysaccharide (LPS) was recognized by CD14, which may be an important mediator in the deleterious effects of LPS on the periodontal destruction. To investigate the roles of CD14 molecules on LPS‐induced soft tissue inflammation and bone destruction, the tissues of CD14‐deficient mice were examined histopathologically following a local injection of either Salmonella minnesota or Porphyromonas gingivalis LPS. In the first group, 12 mice received a local injection of 500 µg of purified P. gingivalis LPS and six mice were injected with saline to the calvaria as controls. In the second group 13 mice were injected subcutaneously on the laterally abdominal skin with 50 µg of S. minnesota LPS and three mice were injected with PBS. Mice were sacrificed at day 5. After histological preparation, the tissue sections of calvaria and soft tissue specimen were stained with tartrate‐resistant acid phosphatase (TRAP) marker for osteoclast and macrophage. The soft tissue sections were also stained with hematoxylin & eosin (H&E). Resorption surface and osteoclast index were measured to quantify bone resorption. Necrotic area and inflammatory cell numbers were estimated to assess the situation of local inflammation. Our results indicated that LPS‐induced bone resorption is inhibited in CD14‐deficient mice. An increase in the number of total inflammatory cells was noticed in both CD14‐deficient mice and wild‐type mice; however, the cell numbers were less in CD14‐deficient mice than those in wild‐type mice (two‐ to three‐fold decrease). Therefore, we conclude that the LPS‐stimulated bone resorption is mainly via CD14 receptor but the LPS‐induced soft tissue inflammation appears to be partially dependent on the receptor.
Centrally mediated reflex vasodilation in the gingiva induced by painful tooth‐pulp stimulation in sympathectomized human subjects
Authors: Satoh‐Kuriwada S. , Sasano T. , Date H. , Karita K. , Izumi H. , Shoji N. , Hashimoto K.
Published: March 2003

This study was designed to determine whether painful electrical stimulation of the tooth pulp induces centrally mediated reflex vasomotor changes in human gingiva and whether the sympathetic nervous system is involved in the vasomotor responses. Dynamic changes in maxillary gingival blood flow (GBF) following painful electrical stimulation of the mandibular lateral incisor were investigated, by means of laser‐Doppler flowmetry, in both healthy volunteers and patients undergoing sympathetic blockade for hyperhidrosis. Increases in GBF were observed in both healthy volunteers and patients on the ipsilateral side without an increase in systemic blood pressure, but the evoked GBF increase disappeared when pain sensation was abolished by local anesthetization with 2% xylocaine solution. The vasodilator responses did not differ in amplitude between before and after the sympathectomy. These results suggest that painful tooth stimulation evokes centrally mediated reflex vasodilation, presumably via parasympathetic efferent fibers, in the human gingiva and that sympathetic vasomotor mechanisms are not involved in these responses.
Evaluation of Dextromethorphan N‐Demethylation Activity as a Biomarker for Cytochrome P450 3A Activity in Man
Authors: Kawashima Yoshiaki , Hagiwara Mieko , Inoue Yoshimasa , Someya Toshiyuki
Published: March 2003

Abstract: The present study evaluates the usefulness of dextromethorphan N‐demethylation activity indices to reflect cytochrome P450 (CYP) 3A activity in man. Indices of dextromethorphan N‐demethylation activity were categorized as N1=3‐methoxymorphinan/dextromethorphan, N2=3‐hydroxymorphinan/dextrorphan, N3=(3‐methoxymorphinan + 3‐hydroxymorphinan)/(dextromethorphan + dextrorphan). Two mg of midazolam were administered orally to 22 Japanese male volunteers, and midazolam clearance determined. Thirty mg of dextromethorphan were also orally administered to these volunteers and N1, N2 and N3 indices determined by 12 hr urine collection. Results showed N2 and N3 were highly correlated (r>0.99, P<0.001), and significantly correlated to oral midazolam clearance (r=0.45, P<0.05); suggesting that N2 and N3 are more suitable than N1 when using dextromethorphan as an index of individual CYP3A activity.
Antioxidant/Restorative Effects of Calcined Gold Preparations Used in Indian Systems of Medicine against Global and Focal Models of Ischaemia
Authors: Shah Zahoor Ahmad , Vohora Shashi Bharat
Published: March 2003

Abstract:Stroke, or ischaemic brain damage, is of great geriatric importance being the third most common cause of death after cancer and heart diseases in developed countries. Despite such high frequency, its management has received inadequate attention. Many studies have shown the role of free radicals in the pathogenesis of ischaemic brain damage. Search for safe and effective antioxidant and free radial scavenger agents, therefore, appear to be a promising approach for stroke therapy. Gold, widely used in modern medicine for the treatment of rheumatoid arthritis, is highly valued for various medicinal uses in Indian systems of medicine. Traditional gold preparations are attributed with tonic/rejuvenating and antioxidant properties. Our earlier studies revealed interesting analgesic, immunostimulant, adaptogenic and glycogen sparing properties in these preparations, but their effects in cerebral ischaemia have not been investigated. This prompted us to initiate the present study using global and focal models of ischaemia in albino rats. Enzymatic parameters (lipid peroxidase, reduced glutathione, catalase, glutathione reductase, glutathione‐S‐transferase, glutatione peroxidase, superoxide dismutase, and glucose‐6‐phosphate dehydrogenase) were employed to assess ischaemic brain damage and its modulation. Significant restoration of altered values to near normal levels by AyurvedicSwarna Bhasmaand UnaniKushta Tila Kalan(25 mg/kg, orally for 10 days), suggest potentials for gold preparations in cerebrovascular diseases. The preparations deserve more scientific attention for possible therapeutic exploitation.
Receptor‐Mediated Hepatocarcinogenesis: Role of Hepatocyte Proliferation and Apoptosis
Authors: Oliver Jason D. , Roberts Ruth A.
Published: March 2003

Abstract: The rodent liver is a target organ for the action of several non‐genotoxic carcinogens. These include dioxins, polychlorinated biphenyls, phenobarbital, peroxisome proliferators and organochlorine pesticides. These chemicals disrupt the homeostasis of the liver by perturbing hepatocyte cell death and proliferation, causing hyperplasia leading to tumour formation. Significant progress has been made towards elucidating the mechanisms of action of these toxicants since the discovery of receptors that bind specific classes of xenobiotics. Dioxins and polychlorinated biphenyls bind to the aryl hydrocarbon receptor, phenobarbital binds to the constitutive androstane receptor and peroxisome proliferators act via the their activated receptor alpha. These three receptors have ligand‐dependent transcription activities and therefore mediate changes in gene expression in response to toxicant exposure. The development of transgenic mouse strains where the genes for these receptors are disrupted has demonstrated that receptor activity is essential for the toxicity of these carcinogens. This implies that changes in the expression of key target genes control proliferation and apoptosis in the xenobiotic‐induced hepatocyte phenotype.
Oestrogen Modulates Cholecystokinin: Opioid Interactions in the Nervous System
Authors: Micevych Paul , Chaban Victor , Quesada Arnulfo , Sinchak Kevin
Published: March 2003

Abstract: Responses of the nervous system to introceptive and extroceptive inputs depend upon the state of the brain. Oestrogen has the ability to modulate brain state and dramatically alter interactions among neural circuits to influence an organism's responses to given stimuli. Cholecystokinin (CCK) and endogenous opioid peptides (EOP) have a wide and parallel distribution in the nervous system. Their reciprocal interactions regulate a diverse physiology including reproduction, cortical function and nociception. The actions of CCK and EOP are diametrically opposed, in many regions. For example, when opioids inhibit reproductive behaviour or nociception, CCK facilitates. Because oestrogen is a powerful regulator of the expression of CCK and EOP, we examined whether oestrogen‐state also modulated the interactions of these neuropeptides. In this paper we present new data and review previous work that demonstrates oestrogen modulation of functional CCK – opioid interactions that regulate reproductive behaviour, cortical function and nociception.
UVA1 irradiation induces deoxyribonuclease dependent apoptosis in cutaneousT‐cell lymphoma in vivo
Authors: Kobyletzki Gregor , Heine Oliver , Stephan Harald , Pieck Carsten , Stücker Markus , Hoffmann Klaus , Altmeyer Peter , Mannherz Hans Georg
Published: March 2003

Cutaneous T‐cell lymphoma (CTCL) is a malignancy of mature T‐cells, predominantly of the helper phenotype, that primarily invade the skin. Different photo‐ and chemotherapeutic treatments are known to be beneficial in early‐stage CTCL. This observation has initiated prospective investigations into the efficacy of phototherapeutic regimens. The purpose of our study was to investigate the ability of medium‐dose UVA1 phototherapy (60 J/cm2) to induce apoptosis (programmed cell death) in skin infiltrating T‐cells of CTCL in vivo. We describe the results of three different staining methods for formalin‐fixed, paraffin‐embedded tissue sections. The in situ end‐labeling (ISEL) procedure, nuclear staining using the DNA‐binding fluorochrome Hoechst 33342, and immunohistochemistry using polyclonal antibodies against recombinant mouse deoxyribonuclease I (DNase I) demonstrated that UVA1 irradiation was able to induce marked apoptosis in CTCL. Thereby, ISEL and Hoechst staining clearly revealed DNA‐condensation and nuclear fragmentation, accompanied by the formation of typical “apoptotic bodies”. The accumulation of DNase I immunoreactivity in the cytoplasm of lymphocytes in UVA1 irradiated skin indicated that DNase I or DNase I‐related endonucleases may have acted as apoptotic endonuclease(s) which were synthesized after UVA1 irradiation prior to their apoptotic elimination.
Photochemotherapy of vascular cells with 8‐methoxypsoralen and visible light: differential effects on endothelial and smooth muscle cells
Authors: Lee David M. , Gasparro Francis P. , Wang Xiu Jie , Kopec Chris , DeLeo Kier , Sumpio Bauer E.
Published: March 2003

Background: The long‐term efficacy of percutaneous transluminal coronary angioplasty is limited by the restenosis which occurs in approximately 40% of patients, usually within 6 months of the procedure.

Purpose: The present study was designed to evaluate the effects of 8‐methoxypsoralen (8‐MOP) activated with visible light on the properties of bovine aortic smooth muscle cells (SMC) and endothelial cells (EC) in vitro.

Methods: Cells were seeded in polystyrene wells, allowed to attach over a 24‐h period, incubated with 1, 20, or 50 µg/ml 8‐MOP and then exposed to 12 J/cm2 visible light (447 nm). Cell counts were performed for up 14 days (n = 4–6 wells per time point), and each experiment was performed in triplicate. Cellular migration, morphology, and size were also analyzed.

Results: The lowest 8‐MOP dose (1 µg/ml) had no significant effect on SMC proliferation, while the highest dose (50 µg/ml) induced cytostasis. An intermediate dose of 8‐MOP (20 µg/ml) produced a transient and reversible inhibition of proliferation. There was no significant effect on proliferation of EC at lowest dose of 8‐MOP (1 µg/ml). However, in contrast to the SMC experiments, a transient and reversible inhibition of EC proliferation was seen at both 20 and 50 µg/ml 8‐MOP.

Conclusions: These experiments demonstrate that while 8‐MOP photoactivated with 447 nm visible light can reversibly inhibit the proliferation of both SMC and EC in a dose‐dependent fashion, SMC are more sensitive to the treatment than EC.
Influence of wetness on the ultraviolet protection factor (UPF) of textiles: in vitro and in vivo measurements
Authors: Gambichler Thilo , Hatch Kathryn L. , Avermaete Annelies , Altmeyer Peter , Hoffmann Klaus
Published: March 2003

Background/Purpose: Clothing is an important product for sunburn protection and skin cancer prevention. The moisture content of a fabric, which can increase during its wearing, may decrease the fabric's capability of protecting the skin from solar UV radiation, that is, lower its UPF (ultraviolet protection factor). Due to limited data about the effect of fabric wetness on UPF, this study was undertaken to investigate the following: (a) the effect of saturating a variety of fabrics with tap water and with salt water on fabric UPF and (b) whether wetted‐fabric UPF values reflect only the fact that the fabric is wet during testing or the fact that the skin is hydrated and the fabric is wet.

Methods: For objective a, 69 summer fabrics were spectrophotometrically (in vitro) assessed when “dry” and when saturated with tap and salt water. In vitro UPFs, percent UVA transmission and percent UVB transmission values were calculated from the transmission data. For objective b, 100% cotton and 100% polyester fabrics were tested in vivo to determine in vivo UPF values. The minimal erythema dose (MED) was determined for each of the 12 subjects on unprotected “dry” skin and on “hydrated” unprotected skin. MEDprotected was determined when the subject's skin was covered with “dry” and with saturated fabric. In vivo UPFs were calculated using this data. Student's paired t‐tests were used to determine the effect of wetting.

Results: With one exception, in vitro UPF values were the same when the fabrics were saturated with tap water and when they were saturated with salt water. However, saturating the fabrics with water had different effects on the UPF, UVA transmission, and UVB transmission values. For linen, viscose and polyester fabrics, UPF significantly increased. For the cotton fabrics and the polyester + TiO2 fabrics, UPF significantly decreased. For the modal + TiO2 fabrics and the polyester crepe + TiO2 fabrics, UPF significantly increased. From the in vivo testing, the MED of the “hydrated unprotected” skin was not different than the MED of “dry unprotected skin.” Values obtained from subtracting dry‐fabric in vivo UPF values from dry‐fabric in vitro values and subtracting wet‐fabric in vivo UPF values from wet‐fabric in vitro values are not different.

Conclusion: Fabrics do not need to be tested when saturated with tap and with salt water. Testing fabrics wet and dry should be done, as the effect of saturating fabric on UPF value varies. Fortunately, UPF values for wetted fabrics reveal only the effect of increased moisture content in the fabric and have nothing to do with wetting of the skin by the fabric.
Effect of propranolol plus exercise on melatonin and growth hormone levels in children with growth delay
Authors: Muñoz‐Hoyos A. , Hubber E. , Escames G. , Molina‐Carballo A. , Macías M. , Valenzuela‐Ruiz A. , Fernández‐García J.M. , Acuña‐Castroviejo D.
Published: March 2003

The pineal gland in humans is under both α‐ and β‐adrenergic control, although it seems that β1‐adrenoceptors are mainly implicated in melatonin secretion. In the present study, we evaluated the role of β‐adrenergic innervation on melatonin production and its relation with the production of growth hormone (GH). Thirty‐four children (15 males and 19 females, mean age 10.5±0.8 years) from the University of Granada Hospital were studied. The children were included in a protocol for the evaluation of growth delay using the propranolol+exercise test. This standardized test allowed us to study simultaneously the role of an unspecific β‐adrenergic blocker such as propranolol and of an adrenergic stimulus such as exercise on the pineal production of melatonin. Changes in plasma levels of melatonin and GH were determined at basal, 120 and 140 min after the test was applied. Hormonal determinations were carried out by commercial radioimmunoassay kits previously standardized in our laboratory. The results show a significant decrease in plasma melatonin levels at 120 and 140 min after the test (P<0.05), whereas GH levels increased significantly at 140 min (P<0.001). The decrease of melatonin levels was a consequence of the test, since in a control group, the circadian decay of melatonin was significantly less pronounced (P<0.05). These data suggest an inverse relationship between melatonin and GH after the propranolol+exercise test, and the reduction in melatonin may be related to its depletion by exercise‐induced oxidative stress.
Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathway
Authors: Blumenau Cirstin , Berger Eva , Fauteck Jan‐Dirk , Madeja Michael , Wittkowski Werner , Speckmann Erwin‐Josef , Mußhoff Ulrich
Published: March 2003

Melatonin‐sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1–100 μmol/L melatonin to voltage‐clamped oocytes activates calcium‐dependent chloride currents via a pertussis toxin‐sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2‐iodomelatonin and 2‐iodo‐N‐butanoyl‐5‐methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5‐methoxycarbonylamino‐N‐acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2‐phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4‐phenyl‐2‐propionamidotetralin (4‐P‐PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA‐injected oocytes (83%). Co‐injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P<0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway.
The effects of day‐time exogenous melatonin administration on cardiac autonomic activity
Authors: Harris Angela S. , Burgess Helen J. , Dawson Drew
Published: March 2003

Melatonin has a functional role in the nocturnal regulation of sleep and thermoregulation. In addition to its action on peripheral receptors, melatonin may act by altering autonomic activity. To determine the effect of melatonin on cardiac autonomic activity, 5 mg of melatonin or placebo was orally administered to 12 young subjects at 14:00 hr, in a repeated measures design. Melatonin decreased sleep onset latency to Stage 2 sleep by 4.92±1.81 min (measured by Multiple Sleep Latency Tests), rectal temperature by 0.19±0.05°C, and increased foot temperature by 0.74±0.45°C (all P<0.05). Melatonin decreased heart rate by 3.66±1.68 beats/min (P<0.05) and pre‐ejection period (measure of cardiac sympathetic activity) by 16.48±4.28 ms (P<0.05), but had no effect on respiratory sinus arrhythmia (measure of cardiac parasympathetic activity) (P>0.05). As the decrease in pre‐ejection period is likely to have resulted from a decrease in blood pressure, these results do not confirm an effect of melatonin on cardiac sympathetic activity. However, the results do clearly indicate that melatonin is unlikely to drive the previously observed presleep increase in cardiac parasympathetic activity.
Altered melatonin production in TGR(mREN2)27 rats: on the regulation by adrenergic agonists, antagonists and angiotensin II in cultured pinealocytes
Authors: Enzminger Helene , Witte Klaus , Lemmer Björn
Published: March 2003

Transgenic TGR(mREN2)27 rats (TGR), carrying an additional mouse renin gene, are characterized by severe hypertension, an inverse circadian blood pressure profile, a blunted response to photic entrainment signals, and an increased nocturnal production of the pineal hormone melatonin. In order to evaluate the contribution of the over‐expressed renin‐angiotensin system to the function of the pineal gland in TGR, we studied the adrenergic and angiotensin II (Ang II)‐mediated regulation of melatonin synthesis using dispersed pinealocytes from TGR and from Sprague‐Dawley control rats (SDR). Isoproterenol was more effective in stimulating melatonin release in pinealocytes from TGR than from SDR, whereas the maximum effect of norepinephrine (NE) stimulation did not differ between the strains. Prazosin reduced the NE‐mediated melatonin release only in SDR but not in TGR pinealocytes. Competition experiments with (±)‐, (+)‐, (−)‐propranolol and (±)‐atenolol revealed one homogenous population of β1‐adrenoceptors. Ang II had no significant effect on basal or isoproterenol‐induced melatonin release in either strain. In conclusion, TGR pinealocytes were more sensitive to β‐adrenergic stimulation than SDR pinealocytes, but lacked the α1‐adrenergic potentiation of β‐adrenergic induced melatonin release. The renin‐angiotensin system was not directly involved in the regulation of melatonin synthesis by rat pinealocytes in vitro.