H(2)O(2) increases de novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via GTP cyclohydrolase I and its feedback regulatory protein in vitiligo.

Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

Epidermal H(2)O(2) accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: identification of a general mechanism in regulation of all 6BH4-dependent processes?

It has been shown in vivo that patients with the depigmentation disorder vitiligo accumulate hydrogen peroxide (H(2)O(2)) accompanied by low catalase levels and high concentrations of 6- and 7-biopterin in their epidermis. Earlier it was demonstrated that epidermal 4a-OH-tetrahydrobiopterin dehydratase, an important enzyme in the recycling process of 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH(4)), has extremely low activities in these patients concomitant with a build-up of the abiogenic 7-isomer (7BH(4)), leading to competitive inhibition of epidermal phenylalanine hydroxylase. A topical substitution for the impaired epidermal catalase with a pseudocatalase effectively removes epidermal H(2)O(2), yielding a recovery of epidermal 4a-OH-tetrahydrobiopterin dehydratase activities and physiologic 7BH(4) levels in association with successful repigmentation demonstrating recovery of the 6BH(4) recycling process. Examination of recombinant enzyme activities, together with 4a-OH-tetrahydrobiopterin dehydratase expression in the epidermis of untreated patients, identifies H(2)O(2)-induced inactivation of this enzyme. These results are in agreement with analysis of genomic DNA from these patients yielding only wild-type sequences for 4a-OH-tetrahydrobiopterin dehydratase and therefore ruling out the previously suspected involvement of this gene. Furthermore, our data show for the first time direct H(2)O(2) inactivation of the important 6BH(4) recycling process. Based on this observation, we suggest that H(2)O(2) derived from various sources could be a general mechanism in the regulation of all 6BH(4)-dependent processes.

In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase.

To date there is compelling in vitro and in vivo evidence for epidermal H2O2 accumulation in vitiligo. This paper reviews the literature and presents new data on oxidative stress in the epidermal compartment of this disorder. Elevated H2O2 levels can be demonstrated in vivo in patients compared with healthy controls by utilizing Fourier-Transform Raman spectroscopy. H2O2 accumulation is associated with low epidermal catalase levels. So far, four potential sources for epidermal H2O2 generation in vitiligo have been identified: (i) perturbed (6R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) de novo synthesis/recycling/regulation; (ii) impaired catecholamine synthesis with increased monoamine oxidase A activities; (iii) low glutathione peroxidase activities; and (iv) "oxygen burst" via NADPH oxidase from a cellular infiltrate. H2O2 overproduction can cause inactivation of catalase as well as vacuolation in epidermal melanocytes and keratinocytes. Vacuolation was also observed in vitro in melanocytes established from lesional and nonlesional epidermis of patients (n = 10) but was reversible upon addition of catalase. H2O2 can directly oxidize 6BH4 to 6-biopterin, which is cytotoxic to melanocytes in vitro. Therefore, we substituted the impaired catalase with a "pseudocatalase". Pseudocatalase is a bis-manganese III-EDTA-(HCO3-)2 complex activated by UVB or natural sun. This complex has been used in a pilot study on 33 patients, showing remarkable repigmentation even in long lasting disease. Currently this approach is under worldwide clinical investigation in an open trial. In conclusion, there are several lines of evidence that the entire epidermis of patients with vitiligo is involved in the disease process and that correction of the epidermal redox status is mandatory for repigmentation.

Serum selenium levels and blood glutathione peroxidase activities in vitiligo.

It has recently been shown that patients with vitiligo can accumulate epidermal hydrogen peroxide (H2O2) in association with low catalase levels. This study examined serum selenium levels and blood glutathione peroxidase activities in 61 patients and controls. The results showed high serum selenium levels in 56% of the patients. As at least one isoform of glutathione peroxidase requires selenium for its activity, enzyme activities were also evaluated. The overall results were not significantly different compared with controls, but further age-related analysis of the data indicated significantly lower activities in patients up to 46 years. As glutathione peroxidase can also efficiently degrade H2O2, the results of this study could indicate an additional impaired H2O2 metabolism in vitiligo.

alpha-MSH can control the essential cofactor 6-tetrahydrobiopterin in melanogenesis.

In the human epidermis both keratinocytes and melanocytes express POMC m-RNA. Immunohistochemical studies of both cell types demonstrate significantly higher levels of alpha-MSH in melanocytes than in keratinocytes. Both cell types also hold the full capacity for de novo synthesis/recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4). 6BH4 is critical for the hydroxylation of the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, for nitric oxide production and in various immune modulatory processes. Recently it was shown that tyrosinase activity is regulated by 6BH4 through a specific allosteric inhibition. The tyrosinase/6BH4 inhibition can be activated by 1:1 complex formation between 6BH4 and alpha-MSH, but an excess of alpha-MSH over 6BH4 can inhibit tyrosinase due to complex formation by tyr2 in the alpha-MSH sequence. In both melanocytes and keratinocytes 6BH4 controls the L-tyrosine supply via phenylalanine hydroxylase (PAH). Recently we were able to show that the cellular uptake of L-phenylalanine and its intracellular turnover to L-tyrosine is crucial for melanogenesis. alpha-MSH can promote the production of L-tyrosine via PAH due to activation of the PAH tetramer to the more active dimer by removing 6BH4 from the regulatory binding domain on the enzyme. In conclusion, alpha-MSH can control (1) intracellular L-tyrosine formation from L-phenylalanine in both melanocytes and keratinocytes, and (2) tyrosinase activity, directly, in melanocytes.

Pterins in human hair follicle cells and in the synchronized murine hair cycle.

Human dermal papilla cells (HDPC) express mRNA for the key enzymes for de novo synthesis/recycling and regulation of the pterin (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4). HDPC had significantly higher enzyme activities and 6BH4 levels in a comparative study with dermal fibroblasts, epidermal melanocytes, and keratinocytes under in vitro conditions. In addition, a significantly more rapid uptake of 14C-L-phenylalanine was demonstrated in HDPC compared with fibroblasts, whereas the differences in turnover to L-tyrosine were insignificant, suggesting a pooling of L-phenylalanine in HDPC. These results suggested that HDPC driven 6BH4 synthesis could be of major functional importance in the hair cycle. In order to follow this hypothesis in vivo, expression of enzyme activities and levels of the produced cofactor during the synchronized hair cycle were determined employing the murine model C57BL/6. These data revealed a significantly increased de novo synthesis for 6BH4 via GTP-cyclohydrolase I concomitant with high levels of 6BH4, and the induction of phenylalanine hydroxylase activities during the telogen/early anagen stage (days 0-1). Pterin levels and enzyme activities fall on day 3 and plateau during the rest of the entire cycle. In addition, thioredoxin reductase and glutathione reductase activities were measured, where the latter enzyme remained constant but thioredoxin reductase activities showed a biphasic behavior. The first peak coincided with the induction of 6BH4 de novo synthesis at the beginning of the hair cycle. The second peak was observed at mid-anagen, when melanogenesis takes place. Taken together, our results show the presence of autocrine pterin synthesis/recycling in human hair follicle cells under in vitro conditions, and a possible role for 6BH4 in the synchronized murine hair cycle.

Perturbed epidermal pterin metabolism in Hermansky-Pudlak syndrome.

In Hermansky-Pudlak Syndrome (HPS) a mutation in a 79.3 kDa transmembrane protein has been shown. The function of this protein has escaped definition so far. This study unveils a defective (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) de novo synthesis/recycling for this cofactor in HPS, where activities of the key enzyme GTP-cyclohydrolase I are in the normal range, but total biopterin levels are significantly decreased in homozygotes (n = 5) compared with unaffected controls (n = 4) (p = 0.00001). Phenylalanine hydroxylase and 4a-hydroxy-6BH4-dehydratase activities are significantly lower. mRNA of all enzymes involved in 6BH4 biosynthesis/recycling and GTP-cyclohydrolase I feedback regulatory protein were expressed in keratinocytes from homozygotes, heterozygotes, and healthy controls. Thioredoxin/thioredoxin reductase can directly control the redox status of 6BH4. These activities are allosterically controlled by calcium. Therefore calcium would directly affect this redox status. In HPS these enzyme activities are low concomitant with a defective calcium uptake, suggesting an extracellular accumulation of this second messenger. In this context phenylalanine hydroxylase is subject to phosphorylation/activation by calcium/calmodulin activated kinases. Therefore it was anticipated that calcium could directly affect the cellular L-phenylalanine turnover to L-tyrosine. A significantly more rapid L-phenylalanine uptake and its turnover to L-tyrosine was identified in normal human melanocytes (n = 5) and keratinocytes (n = 2), and was more enhanced in melanocytes in the presence of 2 x 10(-3) M calcium. The turnover to L-tyrosine was significantly slower. Based on all evidence to date, we speculate that the mutated protein in HPS could be primarily involved in maintaining calcium homeostasis in this patient group.

Pteridines in the control of pigmentation.

The influence of UVB irradiation on the metabolic pathway for the production of L-tyrosine from L-phenylalanine in the human epidermis has been examined in 12 healthy volunteers with photo skin types I-VI (Fitzpatrick classification). This metabolic pathway involves the induction of GTP-cyclohydrolase 1 (GTP-CH-1), the rate-limiting enzyme for de novo synthesis of (6R)L-erythro-5,6,7,8-tetrahydrobiopterin (6-BH4). This essential cofactor controls the production of L-tyrosine from L-phenylalanine via phenylalanine hydroxylase (PAH). The de novo synthesis of 6-BH4 depends on the induction of GTP-CH-1, e.g., by tumor necrosis factor-alpha (TNF alpha). Epidermal suction blister tissues were taken before (0 h) and after (24 and 72 h) UVB exposure with a standardized dosage [1 minimal erythema dose (MED)]. In all cases, there was a significant increase in TNF alpha release, GTP-CH-1 activity, total 6-biopterin level, and PAH activity, indicative of enhanced L-tyrosine production. The response of this metabolic cascade over baseline activities was pronounced in fair photo skin types (I-III) compared to dark skin (IV-VI). Taken together, our results suggest that UVB can control the direct supply of L-tyrosine in the epidermis, and this process may represent an important factor in de novo melanogenesis.

Altered catecholamine synthesis and degradation in the epidermis of patients with atopic eczema.

Patients with atopic eczema have significantly higher norepinephrine levels in plasma than healthy controls. In addition, significantly higher levels of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) were found in this patient group. Cell extracts from epidermal suction blister roofs revealed only half the normal activity of phenylethanolamine-N-methyl transferase (PNMT) together with a threefold induction of the norepinephrine-degrading enzyme monoamine oxidase A (MAO-A). Taken together, these results support earlier observations of a defective catecholamine/adrenoceptor signal in patients with atopic eczema.

Antimicrobial activity of cecropins.

The lytic peptides, cecropins, were originally isolated from the haemolymph of the giant silk moth, Hyalophora cecropia and possess antibacterial and anticancer activity in vitro. This study investigated the antimicrobial activity of these peptides against human pathogens using standardised assay techniques, and the activity of cecropin B on outer and inner bacterial membranes. From a panel of 15 organisms, Gram-negative bacteria were generally more sensitive to cecropins than Gram-positive organisms, especially the lipopolysaccharide defective mutant, Escherichia coli BUE55. Cecropins B and P1 shared similar MIC values whereas Shiva-1, a cecropin B analogue, was less active. Through combination studies with hydrophobic antibiotics and electron microscopy, cecropin B was shown to disrupt the bacterial outer membrane. Protoplasts of Staphylococcus aureus and Staphylococcus epidermidis were resistant to cecropin B, suggesting that the cytoplasmic membranes of Gram-positive organisms were inherently more resistant to the peptide.

Analytical characterization of isoheroin.

The synthesis of isoheroin is presented with the analytical data (mass spectroscopy [MS], nuclear magnetic resonance [NMR], infrared spectroscopy [IR], and gas liquid chromatography [GLC]) for this compound. Comparison between analytical results for heroin and isoheroin shows differentiation is possible.

Lead intoxication 18 months after a gunshot wound.

Lead poisoning can result from a retained intra-articular bullet. A 30-year-old man who was shot with a .38-caliber pistol illustrates the symptoms, signs, and differential diagnosis of lead intoxication.

Results of nerve grafting in severe soft tissue injuries.

Nerve grafting was performed in a series of patients, 81% of whom had associated severe soft tissue injuries in the area in which nerve grafting was done. Other factors that have been shown to have an adverse effect on nerve grafting results were analyzed and were not thought to be major factors influencing results. Results were worse than those of previous reports in which the initial injury was less severe. The initial soft tissue injury is very important in predicting how well a nerve graft will function. Nerve grafting is a valuable procedure even in the face of severe soft tissue injuries, since it alone can restore protective sensation.

The trend to outpatient care: I. "In-and-out" surgery.

At Stuart Circle Hospital, one-day surgery has proved safe, cost effective, and acceptable to both surgeons and patients. Because no additional equipment is required, a similar program can be established in any hospital without great expenditure.

A biodifferential system of face-bow mechanics.

A review of the literature, both scientific and commercial, did not demonstrate the separation of the inner and outer bows into a biodifferential interchangeable system. The prime design idea, to separate the inner and outer bows, increased versatility and yielded a multiple appliance with a greater range of use and safety. The joint couple systems could be adapted to fulfill the criteria cited in the literature during preliminary treatment (deciduous dentition), partial treatment, full-banded treatment, and retention. The biodifferential design was compatible with most mechanical orthodontic appliances and systems currently used. The three joint designs presented and the three inner bow lock devices fulfilled the parameters expected from the normal use of the present face-bow in necessary strength, stability, and use. Further, the use of a joint couple between the inner and outer bows expanded the versatility of the face-bow into a biodifferential system and allowed for an easily manageable and orderly change of forces, vectors, sizes, and shapes requiring little chair time and minimum instruction to the patient. The inner bow could be used as an intraoral appliance for bumpers, shields, and oral screens. These easily made, preformed, interlocking parts could be marked or color coded as to type and size and keyed, grooved, and designed to prevent inverted interlocking of parts. The inner bow could be tied in, as an aid in patient cooperation, or the inner and outer bows could be pinned or ligated together to prevent separation. One of the most important factors of the biodifferential system is protection of the patient from severe trauma to the eyes, face, and mouth due to misuse or accident, as has occurred in the one-piece face-bow. The biodifferential system fulfills the five criteria of the investigation: (1) a system of interchangeable, interlocking joint parts, (2) safety and dependability, (3) easy changes in force, vectors, and moments, (4) enhanced patient cooperation, and (5) oral hygiene.