Bone Regeneration Using Block-type Deproteinized Porcine Bone Mineral with Collagen Membrane Using 3,4-Dihydroxyphenylalanine as Bone Adhesive

PURPOSE: The purpose of this study was to assess the adhesiveness and cytotoxicity of 3, 4-dihydroxyphenylalanine (DOPA), and to evaluate the role of collagen membrane with DOPA in the guided bone regeneration. MATERIALS AND METHODS: Peel resistance and cell cytotoxicity test were performed. Four defect types in nine rabbit calvaria were randomly allocated: i) control, ii) membrane, iii) deproteinized porcine bone mineral (DPBM) covered by membrane with DOPA, and iv) DPBM covered by membrane with cyanoacrylate. Animals were sacrificed at 2 (n=4) and 8 weeks (n=5) for microcomputed tomography and histomorphometric analysis. DOPA showed low peel resistance but high cell viability. RESULT: Cyanoacrylate and DOPA groups showed significantly higher mineralized tissue volume (MTV) compared to control and membrane groups at 2 weeks (P < 0.05). At 8 weeks, DOPA group showed the highest MTV. Significantly higher new bone area was found in DOPA group at 8 weeks (P < 0.05). Bone formation increased from 2 to 8 weeks in DOPA group (P < 0.05). CONCLUSION: DOPA showed high cell viability and in vivo study revealed predictable performance in bone regeneration.

Designing Tyrosinase siRNAs by Multiple Prediction Algorithms and Evaluation of Their Anti-Melanogenic Effects

Melanin is a pigment produced from tyrosine in melanocytes. Although melanin has a protective role against UVB radiation-induced damage, it is also associated with the development of melanoma and darker skin tone. Tyrosinase is a key enzyme in melanin synthesis, which regulates the rate-limiting step during conversion of tyrosine into DOPA and dopaquinone. To develop effective RNA interference therapeutics, we designed a melanin siRNA pool by applying multiple prediction programs to reduce human tyrosinase levels. First, 272 siRNAs passed the target accessibility evaluation using the RNAxs program. Then we selected 34 siRNA sequences with ΔG ≥−34.6 kcal/mol, i-Score value ≥65, and siRNA scales score ≤30. siRNAs were designed as 19-bp RNA duplexes with an asymmetric 3′ overhang at the 3′ end of the antisense strand. We tested if these siRNAs effectively reduced tyrosinase gene expression using qRT-PCR and found that 17 siRNA sequences were more effective than commercially available siRNA. Three siRNAs further tested showed an effective visual color change in MNT-1 human cells without cytotoxic effects, indicating these sequences are anti-melanogenic. Our study revealed that human tyrosinase siRNAs could be efficiently designed using multiple prediction algorithms.

Effects of Gypenosides on Dopaminergic Neuronal Cell Death in 6-Hydroxydopamine-lesioned Rat Model of Parkinson's Disease with Long-term L-DOPA Treatment

The goal of this study was to determine whether gypenosides (GPS) exert protective effects against dopaminergic neuronal cell death in a 6-hydroxydopamine (OHDA)-lesioned rat model of Parkinson's disease (PD) with or without long-term 3,4-dihydroxyphenylalanine (L-DOPA) treatment. Rats were injected with 6-OHDA in the substantia nigra to induce PD-like symptoms; 14 days after injection, groups of 6-OHDA-lesioned animals were treated for 21 days with GPS (25 or 50 mg/kg) and/or L-DOPA (20 mg/kg). Dopaminergic neuronal cell death was assessed by counting tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra and measuring levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum. Dopaminergic neuronal cell death induced by 6-OHDA lesions was ameliorated by GPS treatment (50 mg/kg). L-DOPA treatment exacerbated 6-OHDA-induced dopaminergic neuronal cell death; however, these effects were partially reversed by GPS treatment (25 and 50 mg/kg). These results suggest that GPS treatment is protective against dopaminergic neuronal cell death in a 6-OHDA-lesioned rat model of PD with long-term L-DOPA treatment. Therefore, GPS may be useful as a phytotherapeutic agent for the treatment of PD.

Effect of immobilization of the recombinant human bone morphogenetic protein 2 (rhBMP-2) on anodized implants coated with heparin for improving alveolar ridge augmentation in beagle dogs: Radiographic observations / 대한치과보철학회지

PURPOSE: The aim of this study was to evaluate the effect of immobilization of the recombinant human bone morphogenetic protein 2 (rhBMP-2) on anodized titaum implants coated with heparin to enhance the vertical alveolar ridge augmentation in the supraalveolar peri-implant defect region. MATERIALS AND METHODS: 18 pure titanium implants (7.0 mm in length, 3.5 mm in diameter) were manufactured for this study. All implants were anodized and designed insertion reference line marked with laser at the apical 2.5 mm from the fixture platform. Implantation of 6 noncoated anodized implants (Control group), 6 anodized implants physically adsorbed with rhBMP-2 by dip and dry method (BMP group) and 6 anodized implants chemically immobilized 3,4-dihydroxyphenylalanine (DOPA)-heparin/rhBMP-2 (Hep-BMP group) was performed in the both mandibular of three male adult beagle dogs using split-mouth design. Radiologic examinations were performed immediately after implant placement and 4 and 8 weeks after implant placement. The amount of mesio-distal bone augmentation was evaluated by measuring the vertical distance from the platform to the marginal bone. Statistical analysis was performed using one-way analysis of variance (SPSS version 18.0) and multiple comparison analysis of The Kruskal-Wallis test and the Mann-Whitney U test. Statistical significance was established at the 5%significant level. RESULTS: At the 4 weeks vertical alveolar ridge augmentation of Control group, BMP group and Hep-BMP group is 0.09 +/- 0.22 mm, 1.02 +/- 0.72 mm, and 1.29 +/- 0.51 mm, At the 8 weeks 0.11 +/- 1.26 mm, 1.11 +/- 0.58 mm, 1.59 +/- 0.79 mm according to radiographic observations. The two experimental groups showed a significantly increasing in vertical bone height compared with the control group (P.05). CONCLUSION: The rhBMP-2 coated implants were enhanced the vertical bone growth in the supraalveolar peri-implant defect area. However, there is no significant difference between chemically and physically coating method.

Association between Antipsychotics-Induced Restless Legs Syndrome and Tyrosine Hydroxylase Gene Polymorphism

OBJECTIVE: Restless legs syndrome (RLS) has been reported to be more prevalent in schizophrenic patients who take antipsychotics. The cause of RLS is unknown but associated with dopaminergic deficiency. Tyrosine hydroxylase (TH) is the enzyme responsible for catalyzing the conversion of L-tyrosine to DOPA. The purpose of this study is to determine whether the TH gene Val81Met polymorphism is associated with antipsychotic-induced RLS. METHODS: One hundred ninety Korean schizophrenic patients were evaluated by the diagnostic criteria of the International RLS Study Group (IRLSSG). The genotyping was performed by PCR-based methods. RESULTS: Of the one hundred ninety schizophrenic patients, 44 (23.2%) were found to have RLS. Although there were no significant associations between TH genotypes or allele frequencies and RLS, when separate analyses were performed by sex (male or female), we detected significant differences in the frequencies of the genotype (chi-square=6.15, p=0.046) and allele (chi-square=4.67, p=0.031) of the TH gene Val81Met polymorphism between those with and without RLS in the female patients. CONCLUSION: These findings suggest that the TH gene Val81Met SNP might be associated with antipsychotic-induced RLS in female schizophrenic patients.

The Features of Tyrosinase Expressions in Pigmentary Disorders Using TTA / 대한피부과학회지

BACKGROUND: Tyrosinase is the critical enzyme in melanin synthesis. DOPA staining has been used as a standard assay for detecting tyrosinase activity, but it exhibits several limitations. Tyramide based tyrosinase assay (TTA) is a simple and sensitive tyrosinase detecting method. OBJECTIVE: This study aimed to compare the stainability of pigmentary disorders using TTA and DOPA staining. METHODS: The subjects were composed of hyperpigmentary disorders (n=10), hypopigmentary disorders (n=7), and alopecia areata (n=7). The colocalization study of TTA and Mitf using immunofluorescence was performed on alopecia areata. DOPA staining was performed on all tissues to compare with TTA immunohistochemistry. RESULTS: TTA positive cells were correlated with Mitf positive cells in the tissue of alopecia areata. In hyperpigmentary disoders, TTA was stronger than DOPA staining. TTA and DOPA staining didn't observe the positive cells in lesion of vitiligo and piebaldism, but both showed the positive cells in normal skin. TTA staining showed positive cells in the transitional lesion of vitiligo but, DOPA staining did not. In alopecia areata, TTA positive cells were observed, but DOPA staining did not. CONCLUSION: TTA is more sensitive than DOPA staining in pigmentary disorders for detecting tyrosinase activity.

Value of Histological Staining in Differential Diagnosis of Vitiligo / 대한피부과학회지

BACKGROUND: Vitiligo is a depigmented disorder, causing serious cosmetic problems for patients. In diagnostic and therapeutic aspects, vitiligo should be differentiated from other hypopigmented disorders as the therapeutic approach and prognosis are different for each disease. OBJECTIVE: This study aimed to compare the usefulness of several markers for melanocytes or melanin in differential diagnosis of vitiligo. METHODS: Twenty-eight patients were studied, who were diagnosed clinically as suffering from one of the following diseases: vitiligo, nevus depigmentosus, pityriasis alba, postinflammatory hypopigmentation, and idiopathic guttate hypomelanosis. Skin samples (frozen or paraffin-fixed) were obtained from depigmented patches and normal neighboring skin (control). Histological staining was performed by using Fontana-Masson, S-100, MART-1, and DOPA. The staining level of lesional skin was compared with that of normal skin. RESULTS: When the staining level of vitiligo was compared with that of others, vitiligo was significantly lower in Fontana-Masson (13.3+/-17.2% vs 44.4+/-23.7%), S-100 (49.5+/-14.9% vs 74.7+/-24.2%), MART-1 (7.4+/-8.7% vs 68+/-33.9%), and DOPA (9.5+/-11.3% vs 58.2+/-29.5%) (p<0.0001). CONCLUSION: MART-1 and DOPA are valuable markers in differential diagnosis of vitiligo. However, Fontana-Masson, a marker of melanin, had some limits in detecting melanocytes, and S-100 showed non-specific staining other than melanocytes.

Mutation analysis of GCH1 gene in Chinese patients with dopa responsive dystonia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect mutations of guanosine triphosphate cyclohydrolase I (GCH1) gene in Chinese patients with dopa responsive dystonia (DRD).</p><p><b>METHODS</b>Six sporadic patients with DRD were examined. GCH1 gene mutations were detected using polymerase chain reaction (PCR), DNA sequence analysis and restriction enzyme digestion analysis. One hundred normal people were detected using PCR and restriction enzyme digestion analysis.</p><p><b>RESULTS</b>A new point mutation, 151(G-->A) in exon one was found in a patient. It lead to substitution of a methionine for isoleucine at amino acid 1(M1I). This mutation was not found in normal control people.</p><p><b>CONCLUSION</b>The authors report a new heterozygotic point mutation 151(G-->A) in GCH1 gene. There are GCH1 gene mutations in Chinese sporadic patients with DRD.</p>

A Case of Morphea Occuring in Vitiliginous Lesions / 대한피부과학회지

We report a case of morphea occuring in vitiliginous lesions. About 5 months ago, two lesions of 4X4cm-sized depigmented patches appeared on her left thigh. The depigmented patches had begun to be hardened about 3 months ago. Histopathologic examinations showed that the reticular dermis appeared thickened, closely packed, hypocellular, and stained more deeply eosinophilic than in normal skin. The epidermis had no melanin pigments. Dopa stain was negative in white lesion, whereas Dopa stain was positive in normal lesion.

Two Cases of Oculocutaneous Albinism with Congenital Nystagmus / 대한이비인후과학회지

Albinism is a hereditary disease caused by the defect of tyrosinase that converts tyrosine to dihydroxyphenylalanine (DOPA). `Oculocutaneous albinism' is classified as hypopigmentation of skin, hair and eyes, but incidences of `ocular albinism' where hypopigmentation is limited to eyes are found rarely. Biochemically, albinism is caused by the tyrosinase activity. Typical findings in oculocutaneous albinism include not only ophthalmologic problems such as hypopigmentation of skin, foveal hypoplasia, photophobia and decreased visual acuity but also congenital nystagmus. We cannot determine distinctive characteristics of nystagmus of albinism because domestically, there are only a few reports that have been recorded correctly about nystagmus of albinism. Merely, we present our experience of two cases of albinism with congenital nystagmus because we think that these two cases, showing different types of nystagmus and electronystagmography, stand for the two representative types of nystagmus found in the literature up to date.

6-pyruvoyl tetrahydropterin synthase deficiency: a case report.

A 5 day old girl screened positive for hyperphenylalaninemia on routine newborn screening. Initial diagnostic work-up showed elevated blood phenylalanine of 1100 mmol/L and low tyrosine. Limited protein diet and phenylalanine-free formula were prescribed. Further investigation revealed a defect in biopterin metabolism. Urine had no detectable biopterin (BH4) and an elevated level of neopterin at 24.31 mmol/mole Cr. Enzymatic assay showed zero level of 6-pyruvoyl tetrahydropterin synthase. The activity in the mother was 3.5 or 19.9% of controls consistent with heterozygosity. The concentrations of 5-hydroxyindoleacetic acid and homovanillic acid in the cerebrospinal fluid were below the reference ranges. A treatment regimen of BH4 tablets, 5 hydroxytryptophan and DOPA was initiated. The diagnostic evaluation, management and follow-up of patients with this disorder will be outlined. This is the first reported case of a Filipino with a defect in biopterin metabolism.

Experimental study on inhibition of neuronal toxical effect of levodopa by ginkgo biloba extract on Parkinson disease in rats / 华中科技大学学报（医学）（英德文版）

In order to observe neuronal toxical effect of Levodopa and investigate if using Levodopa together with Ginkgo Bilobar Extract (EGb) would be an workable method to treat Parkinson disease, rat models of Parkinson disease (PD) were made by injecting 6-OHDA stereotaxically to right side of the mesencephic ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Rotational behavioral observation, TUNEL, immunocytochemistry, Nissl's body staining were performed to measure the difference between group treated by Levodopa (50 mg/kg every day for 3 days, 5 days, 7 days, L-dopa group) and group treated by Levodopa combined with EGb (100 mg/kg every day, E-D group). The results showed that in the L-dopa group, the numbers of apoptosis of substantial nigra, rings of rotational behavior were more than those in the E-D group (P < 0.05). The numbers of Nissl's cells in L-dopa group were fewer than in E-D group (P < 0.05). The results suggested that Levodopa had neur toxic effect and EGb may decrease the toxicity of levodopa. The combined use of EGb with Levodopa may be a workable method to treat PD and may be better than using Levodopa alone.

Microscopic analysis of epidermal melanocytes in human abdominal skin

Microscopic analysis of epidermal melanocytes in human abdominal skin with respect to age and sex. Design: Cross sectional study. Place and Duration of Study: Department of Anatomy. BMSI, JPMC, Karachi. About one year in 1998. Subjects and Demonstration of epidermal melanocytes in 5mm thick vertical paraffin embedded sections of thirty-eight skin samples from different age and sex groups, using dihydroxyphenyl alanine [Dopa] reagent. The melanocytes count per unit area of skin was significantly higher in the younger than older age groups. No significant difference was noticed between males and females epidermal melanocytes counts. Distribution of epidermal melanocytes was inversely proportional to the advancing age. However, there was no significant gender differences in the distribution of epidermal melanocytes

The measurement of tyrosine hydroxylase activity in the brain of conscious rats / 中国中药杂志

<p><b>OBJECTIVE</b>To set the measuring method of tyrosine hydroxylase activity in the brain of conscious rats.</p><p><b>METHOD</b>By using microdialysis and High Performance Liquid Chromatography-Electrochemical Detector system, the 3, 4-dihydrioxphenylalanine (DOPA) formation in the striatum of 6-hydroxdopamine-pretreated rats during infusion of an L-aromatic amino-acid decarboxylase inhibitor (NSD1015) was monitored.</p><p><b>RESULT</b>The absence of DOPA in dialysates of 6-hydroxdopamine-pretreated rats, the measurable DOPA and the steady decreasing of 3,4-dihydroxyphenylacetic acid(DOPAC) during infusion of NSD1015 and the disappearance of DOPA after administration of alpha-methyl-rho-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. After intraperitoneal administration of dopamine receptor agonist apomorphine the DOPA output was deseased. After intraperitoneal administration of dopamine recepter antagonist haloperidol, the DOPA output was increased. The study showed that twenty-four hours ofter implantation of the probe with infusion of 0.01 mmol.L-1 NSD1015, the DOPA level in the striatum of 6-hydroxdopamine-pretreated rats was 0.39 +/- 0.12 pmol/min (X +/- S, n = 5).</p><p><b>CONCLUSION</b>The DOPA concentration in striatal dialysates could be considered as an index of tyrosion hydroxlase activity during infusion of 0.01 mM NSD1015. The method in vivo to monitor tyrosine hydroxlase activity in the brain is reliable.</p>

Effects of Fluoxetine on Tyrosine Hydroxylase Expression in Schedule-Induced Polydipsia Rats / 신경정신의학

OBJECTIVE: It has been suggested that fluoxetine inhibits the dopaminergic neurotransmission by serotonergic mediation. And also, it has been shown to inhibit synthesis of DOPA in dopamine-rich areas of the rat forebrain. These dopamine-antagonistic capacity of fluoxetine is only supported by anecdotal report that the increased amount of motor disability in patients with idiopathic Parkinson's disease after exposure to fluoxetine. However, there is still no evidence of the direct effect of fluoxetine on dopaminergic neuronal cell body in the substantia nigra, VTA, caudate & putamen. This study was designed to evaluate the effects of fluoxetine in rat brain which showed decreased numbers of dopaminergic neuronal cell body induced by schedule-induced polydipsia(SIP). METHODS: We incidentally found that 4 weeks of schedule-induced polydipsic rats revealed the suppression of tyrosine hydroxylase expression in the substantia nigra, VTA, caudate & putamen with the immunohistochemistric measures. After 3 weeks of intraperitoneal injection of 10mg/kg of fluoxetine to the schedule induced polydipsic rats, the tyrosine hydroxylase expression was also measured with immunohistochemistry. We compared the tyrosine hydroxylase expression among the normal control, the polydipsic rats, and the rats with fluoxetine treatment. RESULTS: 1) By contrast with the control, the polydipsic rats revealed the evidence of decreased tyrosine hydroxylase expression in the substantia nigra, VTA, caudate & putamen. 2) After daily injection of fluoxetine for 3 weeks, the polydipsic rats showed increment of tyrosine hydroxylase expression in those areas. CONCLULSION: In previous studies, a great deal of results suggest that fluoxetine negatively influence the dopaminergic systems indirectly via serotonergic activation such as inhibition of dopamine synthesis or transport system. Although our results are obtained from rodents, we suggest that fluoxetine directly and positively enhance the dopamine system in the substantia nigra, VTA, caudate & putamen. The chronic adminstration of fluoxetine may be helpful to dopamine-depleted condition in clinical situations. We anticipate the replication studies of our findings and well-controlled clinical trial.

The effect of topical melatonin on epidermal melanocytes in uv-irradiated black mice

Melatonin was derived from pineal gland, act as hormone and is known to be extremely active in certain biological systems. To observe the depigmentation effet of melatonin, author induced activation of melanocytes and melanin by UVB irradiation for seven days and then melatonin ointment or ointment base was applied topically on the ear skin of C57 B1 mouse. Tissue samples were obtained from 4 mice in each group before and after UVB irradiation for given days(1,3,5 and 7 week). The morphological, numerical, and ultrastructural changes of epidermal melanocytes were observed with light microscope and electron miroscope after DOPA stain. The results were as follows: 1) In all groups, the number of DOPA positive melanocytes began to decrease from 1 week. 2) The number of DOPA-positive melanocytes in melatonin ointment applied group was significantly lower than in control group. 3) In the electron microscopic findings, the number of melanosomes markedly decreased from 1 week to 7 weeks and a small amount of destructed melanosomes(vacuoles) and destroyed or swollen mitochondria in cytoplasm of th melanocyte appeared more significant in 3% melatonin ointment applied group compared to ointment base applied group from 5 weeks.

Growth of Human Melanocytes in Human Epidermis Reconstructed by Culture / 대한피부과학회지

BACKGROUND: Melanocytes grown in pure monolayer culure lack many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. OBJECTIVE: The objective of the present study was to grow human melanocytes in human epidermis reconstructed on dermal substrates in vitro and to examine their response to UV radiation. METHODS: The skin equivalents were prepared by seeding cultured human keratinocytes together with cultured human melanocytes(in a ratio of 5%) onto de-epidermized dermis. After 7 days of culture, they were exposed to UVB irradiation(total 150m J/cm over 5days). On day 12 of air exposure the sections of the skin equivalents were prepared for histology. The structure of the skin equivalents was studied following staining with hematoxylin and eosin. Melanocytes were characterized by DOPA staining and by immunohistochemistry. RESULTS: Melanocytes were localized singly within the basal layer of the reconstructs. Melanin was also visible both in the melanocytes and in neighboring keratinocytes. There was an increase in melanocyte size and dendricity following UV irradiation. Melanocytes became positive to staining with HMB-45 antibody following UV irradiation. CONCLUSION: Our results indicate that melanocytes grown in reconstructed human epidermis are functional and capable of responding to UV irradiation.

Lag kinetics of tyrosinase: its physiological implications.

The various theories put forward to explain the characteristic lag kinetics of oxidation of L-tyrosine by tyrosinase a rate regulatory step in the biosynthesis of melanin are reviewed Examination of the evidence in the literature and from experiments in the author's laboratory indicate that one of the hypotheses, that is, competition of tyrosine and dopa for met-tyrosinase and the formation of a dead-end complex of met-enzyme with tyrosine as explanation for lag kinetics is not consistent with available information. The alternative hypothesis that tyrosinase is an allosteric enzyme with tyrosine having negative effector site on the enzyme and dopa competing for it as an explanation for lag kinetics of tyrosinase is not yet disproved. Irrespective of the actual explanation for the lag kinetics of tyrosinase, it is suggested that the highly conserved lag kinetics may serve a physiological function. It is suggested that this function is to keep the enzyme essentially inactive during its transport to the specific organelle, namely the melanosome, in which an acidic environment exists. Only at acidic pH is the enzyme able to catalyze the biosynthesis of melanin.

DOPA-stained melanocytes in the macular lesions of early leprosy

The mechanism of association of hypopigmentation and sensorial loss in a leprosy macular lesions has not been clarified yet. The biopsy of a macular lesion on the medial face of the right forearm of a fourteen-year old male leprosy patient was submitted to DOPA-staining for melanocytes, which is specific for the melanocytic tyrosinase enzyme and it is a proper method for identifying and couting these cells in the skin. A contralateral specimen of the same patient went through the same procedure as a control experiment. The specimen from the macular lesion showed a higher number of DOPA-stained melanocytes than the control fragment. Dermal melanocytes were present in high amounts in the abnormal specimen. Increased expression of tyrosinase by melanocytes in the macular lesions may reflect a positive feed-back stimulus represented by the lack of substance tyrosine, which may in turn be utilized by the myocobacterial agent. Ultrastructural study of the normal and pathological specimens showed no significant differences in the morphological appearance of melanocytes and their melanosomes. These results suggests that the utilization of phenolic compound by the Mycobacterium leprae may be involved in the mechanism of hypopigmentation. A higher number of cases will be necessary to confirm this hypothesis.