Investigation of the peak action wavelength of light-activated gene transduction.

Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Previous studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, whereas UVA induces LAGT secondary to reactive-oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage and determine an optimal wavelength to maximize safety and efficacy. UV at 288, 311 and 320 nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4 kJ m⁻² at 311 nm. Consistent with its robust cytotoxicity, 288 nm produced significantly high levels of DNA damage at all doses tested, whereas 311, 320 and 330 nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. Although 288 nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ m⁻² at 311 nm. An in vivo pilot study assessing 311 nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (P<0.05) increase in transduction with insignificant cytotoxicity. In conclusion, 311 nm was found to be the optimal wavelength for LAGT on the basis of its superior efficacy at the peak dose and its broad safety range that is remarkably wider than the other UV wavelengths tested.

OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

Drug-Drug interactions of clinical significance in the treatment of patients with Mycobacterium avium complex disease.

Therapeutic and prophylactic regimens directed specifically against Mycobacterium avium complex (MAC) are increasingly being used in patients infected with the human immunodeficiency virus (HIV). Several of the drugs used in the management of MAC have been associated with significant drug interactions involving the cytochrome P450 (CYP) enzyme system. This enzyme system is also highly influenced by other drugs used in the management of patients with HIV, particularly the protease inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and azole antifungals. This article reviews the published concentrations or subtherapeutic concentrations of other drugs have been described. In particular, concurrent use of rifabutin with clarithromycin or fluconazole has resulted in increased concentrations of rifabutin and an accompanying increase in the incidence of rifabutin toxicities, including uveitis and leucopenia. Similar results have been seen when rifabutin is combined with protease inhibitors or delavirdine. The macrolides, clarithromycin and azithromycin, have also been associated with significant drug interactions. Clarithromycin has a higher affinity for CYP than azithromycin and, thus, is more frequently associated with clinically significant drug interactions. Clarithromycin is an inhibitor of CYP and may result in toxic concentrations of other drugs metabolised by this enzyme system. Such interactions have been described with rifabutin and the statin lipid-lowering agents. In addition, nevirapine and efavirenz have been shown to significantly reduce clarithromycin concentrations, whereas the protease inhibitors and delavirdine may increase clarithromycin concentrations. Other drugs used in the management of patients with MAC are not metabolised by CYP and thus have a lower incidence of interactions, although the absorption of ciprofloxacin may be impaired when it is given with products containing multivalent cations, such as didanosine. However, clinicians must remain vigilant for drug interactions when reviewing a patient's medication profile, keeping in mind both interactions that have been described in the literature and those that may be predicted based upon known pharmacokinetic profiles.

Novel expression of hypothalamic neuropeptide Y during postnatal development in the rat.

Neuropeptide Y (NPY) is a potent orexigenic peptide. In the normal adult rat, hypothalamic NPY mRNA expression is limited to the arcuate nucleus (ARH). The purpose of this study was to characterize the developmental expression of NPY mRNA in the hypothalamus of the rat. In contrast to the normal adult rat, NPY mRNA was observed in the ARH, the dorsomedial hypothalamic nucleus (DMH) and the perifornical region (PFR) during development. NPY mRNA expression in all three regions increased progressively from postnatal days 0-4 (P0-4) to reach maximum levels at P16 and subsequently decreased to near adult levels by P30. The unique expression of NPY mRNA in the PFR and DMH may be important in establishing the proper management of energy homeostasis and body weight in the adult animal.

Drug interactions of HIV protease inhibitors.

All the currently available protease inhibitors are metabolised by the cytochrome P450 (CYP) enzyme system. All are inhibitors of CYP3A4, ranging from weak inhibition for saquinavir to very potent inhibition for ritonavir. Thus, they are predicted to have numerous drug interactions, although few such interactions have actually been documented either in pharmacokinetic studies or in clinical reports. This article reviews the published literature with an emphasis on the magnitude of interactions and on practical recommendations for management. Many of the drugs commonly taken by patients with HIV have a strong potential to interact with the protease inhibitors. In particular, the non-nucleoside reverse transcriptase inhibitors are also metabolised by CYPand have been shown to interact with protease inhibitors. Delaviridine is an inhibitor of CYP3A4, but nevirapine and efavirenz are inducers of CYP3A4. The protease inhibitors also interact with each other, and these interactions are being explored for their potential therapeutic benefits. Other commonly used drugs are also known to affect protease inhibitor metabolism, including inhibitors such as clarithromycin and the azole antifungals and inducers such as the rifamycins. Drugs that are known to be significantly affected by the protease inhibitors include ethinylestradiol and terfenadine; many other drugs have lesser or potential interactions. Although little specific data is available on the drug interactions of protease inhibitors, this lack of data should not be interpreted as a lack of interaction. Retrospective chart reviews have demonstrated that potentially severe drug interactions are frequently overlooked. Much more clinical data is needed, but pharmacists and physicians must always be vigilant for drug interactions, both those that are already documented and those that are predictable from pharmacokinetic profiles, in patients receiving protease inhibitors.

[Pathogenesis of and causal surgery for senile entropion by en bloc resection (author's transl)]. / Zur Pathogenese und Kausalchirurgie des Entropiums senile durch en-bloc-Resektion.

The pathogenetic factors responsible for senile entropion are: age-induced relaxation of the lower lid structures with elongation of the lid and improper loose adaptation between lid margin and globe, a weak tarsal plate and a lower stimulus threshold of the facial nerve. A surgical intervention which consists of radical lid-shortening by an bloc tarsal resection seems to be the most logical procedure. It was performed on 285 patients. The methods results in a lower rate of recurrence, normalizes lacrimal flow and has an excellent cosmetic side-effect improving the configuration of the lid-fissure. A special indication exists for cases in which there is a relapse following surgery.

Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization.

The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the enzymatic activities of molybdoenzymes. In all organisms studied so far Moco is synthesized by a unique and evolutionary old multistep pathway that requires the activities of at least six gene products. In eukaryotes, the last step of Moco synthesis, i.e., transfer and insertion of Mo into molybdopterin (MPT), is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals. Both domains (E and G) of these proteins are able to bind MPT in vitro. Here, we show the identification and mutational dissection of functionally important regions within the Cnx1 G domain that are essential for MPT binding, the conversion of MPT to Moco, and Moco stabilization. By functional screening for mutants in the Cnx1 G domain that are no longer able to complement Escherichia coli mogA mutants, we found two classes of mutations in highly conserved amino acid residues. (i) The first class affects in vitro binding of MPT to the protein and the stabilization of Moco, the product of the G domain. (ii) The second class is represented by two independent mutations in the aspartate 515 position that is not affected in MPT binding and Moco stabilization; rather the conversion of MPT to Moco by using bound MPT and a yet unknown form of Mo is completely abolished. The results presented here provide biochemical evidence for a purified Cnx1 G domain catalyzing the insertion of Mo into MPT.

Young hospital doctors after night duty: their task-specific cognitive status and emotional condition.

Sleep deprivation is an unpleasant burden of young hospital doctors during their medical training. It may disrupt the balance between coping strategies available to them and the professional demands encountered. Impaired medical care offered by sleep-deprived juniors may be a consequence. Valid research work on this subject is rare and surprisingly contradictory. Therefore, we evaluated the task-specific cognitive status and emotional condition of 40 young hospital doctors (27 men and 13 women, 29.9 +/- 2.9 years of age) at the University of Tuebingen, all of whom were in the beginning of their academic career. Subjects were tested twice acting as their own control, once at 8.00 am after a night off duty (OD) (at least 6 hours of uninterrupted sleep), and once at a similar time after a night on call (OC) being in the hospital for 24 hours. Standardized and reliable psychometric tests thought to represent daily routine medical function were performed. On-call activities were recorded by means of a sleep diary, whereas a questionnaire interrogated aspects of private and professional life. Neuropsychological function deteriorated significantly: number connection test (per cent of norms +/- SD, 103.2 +/- 9.8 OC vs 107.8 +/- 10.5 OD, F = 27.7, P < 0.001), things-to-do list (correct items +/- SD, 6.7 +/- 1.2 OC vs 7.4 +/- 1.5 OD, F = 12.7, P < 0.01), Vienna reaction timer (per cent of norms +/- SD, 95.6 +/- 9.0 OC vs 97.7 +/- 10.4 OD, F = 4.8, P < 0.05), Stroop test (T-values +/- SD, 59.7 +/- 6.3 OC vs 64.6 +/- 7.1 OD, F = 37.1, P < 0.001), ECG test (correct responses +/- SD, 38.3 +/- 7.3 OC vs 43.4 +/- 6.5 OD, F = 45.2, P < 0.001) and status of mood (T-value +/- SD, 60.3 +/- 9.0 OC vs 54.0 +/- 6.6 OD, F = 19.6, P < 0.001). Cognitive function and mood status of young hospital doctors after a night on call decrease considerably. In view of the special vulnerability of medical trainees to occupational stress all efforts are warranted to reduce sleep deprivation in the medical profession.

High average power active-mirror amplifier.

We report operation of the first high average power Nd:glass active-mirror amplifier, a scalable laser device that may be used to configure solid-state laser systems with high average power output into the kilowatt regime. An extractable average power of over 120 W was achieved at the device laser material fracture limit and at a repetition rate of 5 Hz.

Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis.

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.

The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton.

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.