Purification of an inhibitor of DNA photolyase with fluorescent spectra similar to those of the enzyme.

1. The binding of DNA photolyase, the enzyme which uncouples cyclobutadipyrimidines in DNA upon illumination, to its substrate was enhanced by a substance isolated from acidified autolysates of baker's yeast. 2. This substance, referred to as activator, was partially purified by chloroform extraction, ion-exchange chromatography on Dowex 50 and DEAE-cellulose, and gel filtration on Sephadex G-10. Upon lyophilization and storage at -20 degrees C, it was converted to a potent inhibitor of enzyme binding to substrate. 3. The inhibitor appeared as a single band in two thin-layer chromatography systems and was detected by ultraviolet absorbance, fluorescence, and ninhydrin staining. 4. The similarity between the fluorescence spectra of the inhibitor and the enzyme suggested that the inhibitor was structurally analogous to a chromophore in the enzyme and that the activator from which the inhibitor was derived may be the active chromophore of the enzyme.

Purification from baker's yeast of an activator of DNA photolyase.

The activity of purified DNA photolyase from Baker's yeast is enhanced by a compound (Activator (III)) obtained from yeast by chloroform extraction ion exchange chromatography and gel filtration. Thin layer chromatography and spectral data indicate that the compound is homogeneous. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that activator (III) contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9-11, while the other chromophore has a pK of 4-5, and possibly of 9-11. The enhancement of photolytic activity by activator (III) at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator with that of heat-denatured photolyase, suggests that the activator may be the chromophore associated with the enzyme.

Opiate binding sites in the cellular immune system: expression and regulation.

The direct actions of opiates on the mammalian immune system depend on the existence of ligand binding sites either on the surface of the affected cell or in the interior of the cell. With the cloning of various opiate receptors from neuronal tissue, numerous researchers have screened leukocyte cDNA libraries for the expression of these receptors with some positive results. However, the pattern of expression of neuronal opiate receptors in the cellular immune system does not completely explain the biological action of opiates there. Several possibilities could account for this non-congruence including differential expression of the receptors as determined by such factors as cell population or prior history of the cells; the existence of sequence modified versions of the neuronal receptors such that the amplification methods miss their presence; or the opiates act by a different, non-receptor mechanism in the cellular immune system.

Binding of naloxone to human T lymphocytes.

Purified T lymphocytes have a specific binding site for naloxone, the opiate antagonist. The KD for the site was 50.6 +/- 2.4 nM, while the Hill coefficient (n) was 1.67 +/- 0.16, indicating a degree of positive cooperativity of ligand binding. The bound naloxone was partially displaceable by various opiate agonists including morphine (56%), beta-endorphin (61%), met5- and leu5-enkephalin (40% each), [D-ala2, D-leu5]-enkephalin (78%) and [D-ala2, D-leu5]-enkephalinamide (66%). Virtually all of the binding capacity was recovered in the particulate membrane fraction after sonic lysis of the cells. There was great interindividual variability in Bmax between samples, suggesting a possible mechanistic basis for the variability in drug action seen between different individuals.

Retroperitoneal renal transplantation in young children.

Children with urologic causes of renal failure constitute an increasing percentage of patients requiring transplantation. Two children weighing less than 20 Kg. underwent successful transplantation despite multiple previous intra-abdominal procedures. The retroperitoneal technique employed for these patients is described and its advantages emphasized.

The morphine-binding site on human activated T-cells is not related to the mu opioid receptor.

Mitogen activation of human T-lymphocytes induces a morphine-binding site. Morphine binding is displaceable by beta-endorphin (1--31) and (--)-naloxone but not DAMGO. This site is not stereoselective for (--)-morphine. T-lymphocytes, expressing this binding site, were assayed by reverse-transcription polymerase chain reaction (RT-PCR) for expression of hMOR-1 mRNA. Several primer sets were used and each assay compared with cells known to express human or mouse MOR-1 mRNA. Neither hMOR-1 nor any homologous receptor was detected in human T-lymphocytes. Therefore, the morphine-binding site on mitogen-activated T-lymphocytes is unlikely to be closely related to hMOR-1.

Single-stage reconstruction of mandibular and soft tissue defects using a free osteocutaneous groin flap.

Microvascular reconstruction of the mandible and soft tissues using the composite groin flap is ideal in selected patients. No other available bone so closely approximates the mandible in both thickness and curvature as does the iliac crest. The soft tissues are available for reconstruction and may allow the surgeon to avoid a second flap, except in cases where both lining and cover are needed. The deep circumflex artery is of generous size, usually 2 to 2.5 mm in diameter, allowing greater reliability in the microvascular anastomoses. The flap has a fairly long vascular predicle, 6 to 8 cm. The ability of this flap to withstand irradiation and infection because of its blood supply permits early institution of postoperative radiotherapy and prevents bone loss due to small intraoral wound dehisicence or total flap loss. Although the donor site requires extensive dissection, it can be closed primarily, eliminating the need for skin grafts or other flaps. As further experience is gained with this flap, both the functional and cosmetic results should be improved. In patients undergoing resection of the remaining portion of the mandible, the symphysis or the anterior portion of the mandible, a procedure of this type should be done primarily to prevent deformity and to minimize disability for the patient.

Pectoralis major musculocutaneous flap: a new flap in head and neck reconstruction.

The pectoralis major musculocutaneous flap described by Ariyan has great potential in single stage reconstructions of the head and neck. The advantages of the flap are greater length, improved vascularity, bulk, and one-stage reconstruction of oropharyngeal defects. The flap was used successfully in eight patients to reconstruct large defects in the head and neck area. Experience to date indicates that this flap has greater versatility than the deltopectoral flap in one-stage head and neck reconstructions.

Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts.

To refine previous studies of chromosome damage (CD) and sister-chromatid exchanges (SCE) in heroin addicts, we applied new methods developed in our laboratory to enhance detection of the cytogenetic effects of low-level radiation exposure in hospital workers. For CD analysis, we applied our thymidine-fluorodeoxyuridine-caffeine (TFC) enhancement procedure in which cells at setup receive 1 x 10(-7) M fluorodeoxyuridine to inhibit thymidylate synthetase and 4 X 10(-5) M thymidine to satisfy the induced requirement, and then in G2 receive 2.2 mM caffeine to modulate DNA repair. For SCE enhancement, caffeine treatment was initiated in G1 at 19 h before harvest. Using both standard and enhanced procedures for CD and SCE analysis, blood samples were evaluated from 20 street heroin addicts and 22 controls. Standard 2-day CD and 3-day SCE assays showed small, insignificant genotoxic increases in addicts while the enhanced CD and SCE assays showed highly significant increases. Most CD events were in the form of chromatid and chromosome breaks. There were no rings and only a few dicentrics were observed in the TFC-enhanced cultures. Although quadriradials are rare, 10 were found in addict TFC-cultures and 3 in control TFC-cultures. With the standard CD assay, the mean number of chromosome breaks per 100 cells was 0.727 for controls and 1.056 for addicts (not significant). With the TFC-enhanced assay, the same measure showed 1.483 chromosome breaks for controls and 5.143 for addicts (highly significant, ANOVA: p less than 0.0001). A highly significant difference was also observed for chromatid-type damage with the TFC-enhanced assay (chromatid breaks per 100 cells: 16.793 for controls; 48.191 for addicts). The SCE data also showed significant differences with the enhanced assay. Scoring 25 cells/condition, standard SCE cultures showed 10.892 SCE/cell for controls and 11.732 SCE/cell for addicts (not significant). With CAF enhancement there were 13.08 SCE/cell for controls and 17.05 SCE/cell for addicts (ANOVA: p less than 0.008). These findings indicate that detection of CD and SCE effects can be significantly enhanced by the use of these new procedures. The finding of greatly increased chromatid damage in the addicts with the TFC procedure suggests that at least part of the CD detected occurred in vitro and is not a product of prior in vivo damage. Therefore exposure to this drug and perhaps other environmental agents may not only leave a residue of DNA or chromosome damage but may also induce a sensitivity to further genotoxic damage that is revealed by using the enhanced procedures.

Parallel increases in sister-chromatid exchanges at base level and with UV treatment in human opiate users.

The SCE base level frequency and SCE levels induced by far-UV (254 nm) treatment of cells in early G1 and early S phases of the cell cycle were significantly higher in leukocytes from heroin addicts as compared to controls. The increased SCE levels in addicts was greatest at base level and smallest after UV irradiation of cells in S phase. These results corroborate and extend our previous findings of increased chromosome damage and reduced DNA-repair synthesis in heroin users. Since opiates do not directly damage DNA, the elevated cytogenetic effects associated with opiate use probably arise from secondary promotional effects related to opiate-mediated alterations in leukocyte metabolism.

Vascular basis for a compound flap of the finger incorporating a bone graft.

A defect of soft tissue and bone is closed by a primary arterialized flap. The flap was constructed to include a vascularized bone graft. The vascular anatomy of the hand permitting creation of this flap is reviewed.

Use of the free dorsalis pedis flap in head and neck repairs.

Many defects of the head and neck can be readily repaired with a free dorsalis pedis flap, and we report success with these flaps in 9 of 12 cases. A precise knowledge of the anatomy of the arterial supply of the flap is necessary. Preoperative arteriography is recommended if the dorsalis pedis artery is not easily palpable, or if an anomalous distribution of the artery along the dorsum of the foot is sus pected. However, the transfer of the flap should be delayed for two weeks after preoperative arteriography is performed. The one-stage soft tissue reconstruction with a free dorsalis pedis flap has been associated with minimal morbidity and good acceptance by patients. A delay procedure for the flap seems to enhance the chances of complete survival which is so necessary in the repair of intraoral and pharyngeal defects. Careful attention to details and close monitoring of the flap will minimize morbidity. In case of an early failure of a flap, a secondary reconstruction by a different flap can be done in the first 48 to 72 hours. Early postoperative radiotherapy has been well tolerated over these free flaps.

Immediate reconstruction of the pharynx and cervical esophagus with the pectoralis major myocutaneous flap following laryngopharyngectomy.

The "island" pectoralis major myocutaneous flap has been used in nine patients for immediate hypopharyngeal and cervicoesophageal reconstruction following laryngopharyngectomy. Two patients underwent total hypopharyngeal and cervicoesophageal reconstruction. Postoperative evaluation revealed adequate lumens with no evidence of stricture. There was retained innervation of the flaps through the lateral pectoral nerve, but no additional innervation from the cricopharyngeal musculature could be demonstrated. Normal esophageal motility was maintained, but cervicoesophageal pressures were diminished. Donor site morbidity was minimal, and the complication rate was low. We present the pectoralis major myocutaneous flap as an alternate method for hypopharyngeal and cervicoesophageal reconstruction.

The use of nonneuronal cells as an in vitro model system for studying the genetic component of cellular response to opiates and other drugs of abuse.

Nonneuronal cells, such as the human T lymphocyte, react directly with opiates in vitro causing significant alterations in the metabolism of these cells. Morphine and cocaine, for example, can modulate the repair of DNA damage caused by ultraviolet light (UVC)--morphine in the negative direction and cocaine in the positive. The mechanism by which these drugs cause these metabolic changes is not yet known, but a simple receptor mechanism such as is found in the central nervous system (CNS) can not be demonstrated. Binding studies using lymphocyte membrane preparations or whole cells do not support the premise that T lymphocytes have opiate binding sites with specificities comparable to those identified in the CNS--the mu, delta and kappa receptors. Even without knowing the mechanism for the opiate-induced metabolic changes, the alterations can be used as the basis for a test of the genetics of opiate metabolism. If the interindividual variation in the opiate-induced repair response is greater than the intraindividual variation as assessed by repeated measures on the same subject, it may be possible to utilize this assay in the classic sorts of family or twin studies to determine the genetic component of the response to opiates.