Heparin treatment mitigates radiation-induced oral mucositis in mice by interplaying with repopulation processes.

PURPOSE: To investigate the mechanistic background of the muco-protective effect of systemic heparin treatment on the development of radiation-induced oral mucositis in mice. MATERIALS AND METHODS: Fractionated irradiation was given to the snouts of male C3H/Neu mice over 2 weeks (10â€¯× 3 Gy), either alone or in combination with daily subcutaneous application of unfractionated or low molecular weight heparin (40 or 200 I.U./mouse, respectively). Over this course of 14 days, groups of mice (n = 3) were sacrificed every second day, their tongues excised and processed for histological analysis. The epithelial radiation response with and without heparin treatment was evaluated in terms of tissue morphology, proliferation and expression of cell contact molecules. RESULTS: Systemic treatment with heparins significantly reduced the cellular effects of irradiation to the oral epithelium. Heparin treated animals showed significantly higher total epithelial cell numbers and thickness throughout the study course. Bromodeoxyuridine (BrdU) incorporation analyses revealed that markedly more epithelial cells retained their proliferative capacity in the beginning of the first treatment week, but the proliferation of the mucosa was not stimulated during the rest of the study course. The expression of the adherens junction protein ß­catenin was slightly elevated in heparin treated animals, on day 2 the increase was statistically significant. The expression of e­cadherin and occludin was mostly unaffected by the concomitant heparin treatment. CONCLUSION: The findings of this study indicate an interplay of additional heparin treatment with the repopulation processes, leading to an earlier onset of this adaptive radiation response in oral mucosa. Importantly, we could demonstrate that the protective potential of heparin did not rely on stimulation of normal tissue proliferation. Since both heparin preparations are already approved for clinical use, they are considered as promising candidates for future clinical studies.

Analyzing your complexes: structure of the quinol-fumarate reductase respiratory complex.

The integral membrane protein complex quinol-fumarate reductase catalyzes the terminal step of a major anaerobic respiratory pathway. The homologous enzyme succinate-quinone oxidoreductase participates in aerobic respiration both as complex II and as a member of the Krebs cycle. Last year, two structures of quinol-fumarate reductases were reported. These structures revealed the cofactor organization linking the fumarate and quinol sites, and showed a cofactor arrangement across the membrane that is suggestive of a possible energy coupling function.

Recommendations for the forensic diagnosis of sex and age from skeletons.

In both diagnostic fields a two-stage strategy is recommended: to first use "field" methods that are quick and easy but more imprecise and then "laboratory" methods that are time consuming but more precise. In preparing skeletal work, individuality of a skeleton should be checked, traces of diseases sought and time since death assessed. For sexing non-adults, the field methods are tooth mineralisation, long bone length and a few morphological skull and pelvis characteristics, for adults it is the morphology of pelvis and skull, and for both age groups the advanced laboratory method is molecular biology. For ageing non-adults the methods are mineralisation of teeth, long bone length and epiphysis development. For ageing adults the advanced laboratory method is aspartic acid racemisation. Less accurate laboratory methods are cement ring counts and histology of bones and teeth. Quick morphological methods using the pubic symphysis and other traits in combinations follow. Finally, cranial sutures and tooth number give a quick and rough impression. For the selection of a method and the assessment of its value the stochastic error produced for the reference sample is the decisive criterion; it should also be used to assess the reliability of a single diagnosis. Prerequisites for all work with skeletons are not only a complete knowledge of the relevant biology as well as specific techniques but also initial detailed instructions and with forensic applications, personal experience.

Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+.

BACKGROUND: Studies performed within the last decade have indicated that microbial reduction of Fe(III) to Fe(II) is a biologically significant process. The ferric reductase (FeR) from Archaeoglobus fulgidus is the first reported archaeal ferric reductase and it catalyzes the flavin-mediated reduction of ferric iron complexes using NAD(P)H as the electron donor. Based on its catalytic activity, the A. fulgidus FeR resembles the bacterial and eukaryotic assimilatory type of ferric reductases. However, the high cellular abundance of the A. fulgidus FeR (approximately 0.75% of the total soluble protein) suggests a catabolic role for this enzyme as the terminal electron acceptor in a ferric iron-based respiratory pathway [1]. RESULTS: The crystal structure of recombinant A. fulgidus FeR containing a bound FMN has been solved at 1.5 A resolution by multiple isomorphous replacement/ anomalous diffraction (MIRAS) phasing methods, and the NADP+- bound complex of FeR was subsequently determined at 1.65 A resolution. FeR consists of a dimer of two identical subunits, although only one subunit has been observed to bind the redox cofactors. Each subunit is organized around a six-stranded antiparallel beta barrel that is homologous to the FMN binding protein from Desulfovibrio vulgaris. This fold has been shown to be related to a circularly permuted version of the flavin binding domain of the ferredoxin reductase superfamily. The A. fulgidus ferric reductase is further distinguished from the ferredoxin reductase superfamily by the absence of a Rossmann fold domain that is used to bind the NAD(P)H. Instead, FeR uses its single domain to provide both the flavin and the NAD(P)H binding sites. Potential binding sites for ferric iron complexes are identified near the cofactor binding sites. CONCLUSIONS: The work described here details the structures of the enzyme-FMN, enzyme-FMN-NADP+, and possibly the enzyme-FMN-iron intermediates that are present during the reaction mechanism. This structural information helps identify roles for specific residues during the reduction of ferric iron complexes by the A. fulgidus FeR.

Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin.

The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.

Evidence for non-cysteinyl coordination of the [2Fe-2S] cluster in Escherichia coli succinate dehydrogenase.

The consequences of replacing Cys65 in the FrdB subunit of Escherichia coli fumarate reductase by Asp or Ala have been investigated in terms of bacterial growth, enzymatic activity, and the ERP/redox properties of the [2Fe-2S] cluster. An aspartic acid residue occupies the equivalent position in E. coli succinate dehydrogenase, and the FrdBCys65Asp mutation has little effect on cell growth, enzyme activity or the physical properties of the Frd [2Fe-2S] cluster. In contrast, the [2Fe-2S] cluster was not observed in the FrdBCys65Ala mutant showing that a coordinating residue is required at this position for assembly of this cluster and significant levels of enzymatic activity. These results support the presence of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster in E. coli succinate dehydrogenase.

Effect of tungstate on nitrate reduction by the hyperthermophilic archaeon pyrobaculum aerophilum

Pyrobaculum aerophilum, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that P. aerophilum requires the metal oxyanion WO42- for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 &mgr;M MoO42- did not replace WO42- for the growth of P. aerophilum. However, cell growth was completely inhibited by the addition of 100 &mgr;M MoO42- to the culture medium. At lower tungstate concentrations (0.3 &mgr;M and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO42- concentrations (<0.7 &mgr;M). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO42- levels of 0.7 &mgr;M or higher. The high specific activity of the nitrate reductase enzyme observed at low WO42- levels (0.3 &mgr;M or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in P. aerophilum occurs in the presence of high concentrations of tungstate.

Inhibition of human and duck hepatitis B virus by 2',3'-dideoxy-3'-fluoroguanosine in vitro.

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) has been shown to have an effect on duck hepatitis B virus (DHBV) in vivo and in vitro. In this study the inhibitory effect of FLG on DHBV and human hepatitis B virus (HBV) was evaluated in vitro. Cell lines transfected either with DHBV or HBV DNA and primary duck hepatocyte cell cultures were used. Virus production was analysed by PCR and a quantitative PCR was established for DHBV for determination of the inhibitory concentrations of the drug. 50% inhibition was achieved with an FLG concentration of 0.2 microg/ml (0.7 microM) and 90% inhibition was observed with an FLG concentration of 1.0 microg/ml (3.7 microM) using the DHBV transfected cell line. FLG showed an effect on DHBV production in primary duck hepatocyte cell cultures at concentrations down to 0.1 microg/ml (0.4 microM). However, the DHBV production returned to pre-treatment levels within a few days after cessation of treatment. HBV production in transfected cell lines was also inhibited by FLG. Both DHBV and HBV DNA-polymerases were inhibited by FLG triphosphate and 50% inhibition was observed at a concentration of 0.05 microg/ml (0.1 microM) for DHBV and 0.03 microg/ml (0.05 microM) for HBV. FLG is an efficient inhibitor of DHBV replication both in vivo and in vitro and of HBV in vitro which makes it a good candidate for treatment of HBV infections. However, it does not completely eliminate the virus since a relapse in virus production was observed when treatment was withdrawn. Therefore it would be interesting to evaluate FLG in combination with other types of anti-HBV drugs.

The NIST Cold Neutron Research Facility.

The Cold Neutron Research Facility (CNRF) at the National Institute of Standards and Technology (NIST) Research Reactor (NBSR) is now coming on line, with the first seven experimental stations operational, and more stations scheduled to be installed during 1992. The present article provides an introduction to the facility, and to other articles in the current issue that give more details on some of the research opportunities that the facility will bring to NIST.

["I protect others' and my life". Adolescents and their adaptation to the dangers of AIDS. A survey of trade school students]. / "Ich schütze anderes Leben und meins". Adoleszente und ihre Anpassung an die Gefährdung durch AIDS. Eine Befragung von Berufsschülern.

170 trade school students (main inquiry 142) between 18 and 21 in eight cantons were questioned via group discussions and questionnaire on their attitude to the STOP AIDS campaign, to the messages put across by the campaign and to the use of condoms. About half the trade school students recognise the personal appeal. 77% consider the recommended protection by using condoms personally acceptable and correct. However, the inquiry disclosed that this is only a relative acceptance. In practice, a considerable minority is developing individual behaviour concepts with a view to circumventing the recommended condom protection whenever possible. There is a lack of insight into the advantages of using condoms as standard practice in risk situations as a manifestation of the solidarity of all concerned to prevent AIDS from spreading further. The analysis of the results considers, inter alia, to what extent partner loyalty, confidence and abstinence are integrated in these behavioural concepts and attempts to assess their value with a view to reliable AIDS prevention. Prejudice against the condom and a gap in the STOP AIDS campaign reasoning are considered as possible grounds for the resistance to the recommended condom protection.

Variations of sitting posture and physical activity in different types of school furniture.

Inadequate school furniture is frequently taken to be the reason of severe posture problems in adulthood. Therefore, chairs and desks used by children for considerable periods of time need to be evaluated carefully. In this study we investigated the postures and movement patterns of schoolchildren sitting on two distinctly different types of school furniture. The analysis of our observations and records showed numerous differences of movement patterns with respect to sex, age, and type of furniture. These results include overall movement frequencies, the possibility of choosing extreme postures in order to interrupt monotonous permanent positions, and freedom of movements for the legs. In general, the ordinary standardized school furniture under investigation turned out to meet the ergonomic demands better than a second type of furniture, which had been advertised to be especially "ergonomic". It can be concluded that the analysis of movement patterns offers additional criteria for the evaluation of the quality and suitability of school furniture.

[Adjuvant properties of transfer factor preparations from human tonsillar lymphocytes]. / Ad"iuvantnye svoistva preparatov transfer-faktora iz limfotsitov mindalin cheloveka.

Low molecular components (Mol. m below 10 000 dalton) of the extract prepared from the human tonsils lymphocytes produced an adjuvant effect on th formation of tuberculin sensitivity in guinea pigs immunized with BCG. This interspecies adjuvant effect was proportional to the dose of the "transfer-factor" preparations administered, depended on the method of their preparation, was expressed in administration of the preparations simultaneously with the immunization, before it or after the tuberculin tests, and under definite conditions was replaced by the immunodepressive effect.

A subsequent fit of time series and amplitude histogram of patch-clamp records reveals rate constants up to 1 per microsecond.

Fast gating in time series of patch-clamp current demands powerful tools to reveal the rate constants of the adequate Hidden Markov model. Here, two approaches are presented to improve the temporal resolution of the direct fit of the time series. First, the prediction algorithm is extended to include intermediate currents between the nominal levels as caused by the anti-aliasing filter. This approach can reveal rate constants that are about 4 times higher than the corner frequency of the anti-aliasing filter. However, this approach is restricted to time series with very low noise. Second, the direct fit of the time series is combined with a beta fit, i.e., a fit of the deviations of the amplitude histogram from the Gaussian distribution. Since the "theoretical" amplitude histograms for higher-order Bessel filters cannot be calculated by analytical tools, they are generated from simulated time series. In a first approach, a simultaneous fit of the time series and of the Beta fit is tested. This simultaneous fit, however, inherits the drawbacks of both approaches, not the benefits. More successful is a subsequent fit: The fit of the time series yields a set of rate constants. The subsequent Beta fit uses the slow rate constants of the fit of the time series as fixed parameters and the optimization algorithm is restricted to the fast ones. The efficiency of this approach is illustrated by means of time series obtained from simulation and from the dominant K+ channel in Chara. This shows that temporal resolution can reach the microsecond range.

[Therapy with transfer factor in diseases with disorders of the cellular immune reaction]. / Zur Therapie mit Transfer-Faktor bei Erkrankungen mit Störung der zellulären Immunreaktion.

On the basis of publications on experimental therapy with transfer factor is tried to show limits and perspective of a new therapeutic principle. The now 10-year-old therapeutic experiences with dialysates of leucocytes and chromatographically cleansed fractions were confirmed by the new investigations of the last years which were evaluated. They show the transfer factor as an effective immune therapeutic for the stimulation of the cellular immunity in a large number of diseases. In these cases a combination with other therapeutic measures proved particularly favourable. Concerning the breadth of use and the chances of success chronic infections are in the foreground. A practical importance of the transfer factor for the treatment of malignant diseases is at present not to be seen. Comprehensive double blind studies with large groups of patients are further necessary, in order to exclude accidental findings for the corresponding picture of the disease and to estimate possible chances of success.

Subjective responses to light sources of different colour temperatures and illuminances.

User attitudes towards certain lighting conditions have as yet been predominantly a field of lighting engineering research, aiming at an optimisation of lighting conditions on the basis of averages. Therefore, data on the interindividual variability of subjective responses hardly exist. In contrast to this, the methodological approach of industrial anthropology is based on the consideration of individual variation to provide basic data which are required to design our dwelling environment. Against this background we simulated an office work situation in the light laboratory and-using semantic differentials-studied subjective responses to four different lighting conditions in which different illuminance levels were combined with different colour temperatures of the light source. Colour temperature is an aspect of artificial illumination that has rarely been considered in lighting studies. The results of our study indicate that (1) subjective judgements of the four lighting situations differ significantly, (2) relatively long, repeated exposures of test participants to lighting situations lead to a change of attitudes, and (3) subjective judgements and preferences show a considerable interindividual variation. These results suggest that lighting designs of office rooms that are merely orientated towards averages are inadequate and support lighting concepts that allow for individually adjustable light sources.