Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.     

Determination of the viremia threshold for dental cross-infection in a mouse model

An animal model of dental virus transmission was developed using the lactate dehydrogenase-elevating virus (LDV) of mice to study cross infection. Mouse-to-mouse cross-infection was carried out by scaling the teeth of LDV-infected donor mice with dental instruments, immediately prior to using the contaminated instruments on the teeth of recipient indicator mice. The level of donor viremia was found to correlate with the rate of virus cross-infection, with a viremia threshold level of 10(7.5) ID50/ml observed for dental cross-infection. The blood volume transferred during dental cross-infection was approximately 10(-4) to 10(-5) ml, demonstrating the inefficiency of virus cross-infection, since deposition of about 1000 virions on dental instruments was associated with the threshold limit. Virus transferred during dental cross-infection rapidly entered the blood circulation, showing that dental cross-infection was not dependent on an oral infection. The results from these model studies predict the general inefficiency of dental instrument virus cross-infection, and a further reduced likelihood of dental cross-infection with appropriately cleaned instruments.     

Dual immunodetection and hybridisation in situ of opsin mRNA and rhodopsin protein in retinal sections

We describe a simple method for combining in situ hybridisation and immunohistochemistry on the same retinal section. The technique was developed using a radiolabelled cDNA probe for opsin and an antibody (ROS1F4) against rhodopsin. This method retains the antigenic sites if immunocytochemistry is performed prior to in situ hybridisation. Opsin mRNA was found in the photoreceptor inner segment with rhodopsin immunolocalised to the photoreceptor outer segments. The technique should be applicable to numerous situations including analysis of the sequence of events in the expression and synthesis of the various opsins during retinal development and degeneration.     

Esterified estrogens with and without methyltestosterone decrease arterial LDL metabolism in cynomolgus monkeys

OBJECTIVES: To assess retinal complications and to identify risk factors for retinal complications following aqueous shunt procedures. MATERIALS AND METHODS: Records of 38 consecutive aqueous shunt procedures that were performed on 36 patients at the Eye Institute of the Medical College of Wisconsin, Milwaukee, from June 1993 to March 1995 (minimum follow-up, 6 months) were reviewed. The mean +/- SD follow-up was 11.4 +/- 5.2 months (median, 10.5 months). RESULTS: Twelve patients (32%) had the following retinal complications: 4 serous choroidal effusions (10%) that required drainage, 3 suprachoroidal hemorrhages (8%), 2 vitreous hemorrhages (5%), 1 rhegmatogenous retinal detachment (3%), 1 endophthalmitis (3%), and 1 scleral buckling extrusion (3%). Surgical procedures for retinal complications were required in 8 (67%) of these 12 patients. Visual acuity decreased 2 lines or more in 9 (75%) of these 12 patients. The median onset of a postoperative retinal complication was 12.5 days, with 10 patients (83%) experiencing complications within 35 days. Serous choroidal effusions developed in 10 other patients (26%), and these effusions resolved spontaneously. Visual acuity decreased 2 lines or more in 2 (20%) of these additional 10 patients. Patients who experienced serious retinal complications were significantly older, had a higher rate of hypertension, and postoperative ocular hypotony. Serious retinal complications were distributed evenly among patients with Krupin valves with discs and Molteno and Baerveldt devices. Experience with the Ahmed glaucoma valve implant was limited. CONCLUSION: Aqueous shunt procedures may be associated with significant retinal complications and subsequent visual loss.     

In vivo cartilage formation from growth factor modulated articular chondrocytes

Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.     

Assessing declarative memory in schizophrenia using Wisconsin Card Sorting Test stimuli: the Paired Associate Recognition Test

The Paired Associate Recognition Test (PART) was developed to measure declarative memory using Wisconsin Card Sorting Test (WCST) stimuli, so that both tasks could be administered during functional neuroimaging to differentiate memory and executive function, and associated frontal and temporal lobe activation in schizophrenia. The current study was designed to compare PART and WCST performance in schizophrenic patients and to examine effects of medication and symptomatology. The PART, WCST, and standard declarative memory tasks were administered to 30 chronic schizophrenic patients and 30 matched healthy control subjects. Supporting task validity was the finding that patients were equally impaired on the PART and the WCST. Neuroleptics did not appear to affect performance. The effect of anticholinergic medication correlated negatively with WCST performance in a small subsample. Severity of schizophrenia-specific symptoms measured at intake on the Brief Psychiatric Rating Scale correlated negatively with performance on the WCST. These results support the application of the PART and WCST in future functional neuroimaging studies.     

Paraneoplastic encephalomyelitis and seminoma: importance of testicular ultrasonography

We report two patients with paraneoplastic limbic and brainstem encephalitis associated with occult nonmetastatic testicular seminoma. In each patient, the neoplasm was detectable only by testicular ultrasonography. Male patients with this syndrome in whom lung cancer is not found should undergo testicular ultrasonography as part of the search for an extrapulmonary neoplasm. A normal clinical testicular examination is insufficient to exclude an occult seminoma.     

Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe

Sinus pericranii is an anomalous extracranial vascular malformation that is in continuity with the intracranial dural venous sinuses. Five case reports, three congenital and two traumatic, are described. Clinical management, including evaluation, diagnosis, and treatment, is discussed. Awareness of this entity by plastic surgeons will allow for earlier diagnosis and appropriate surgical management, resulting in decreased risk of complications.