Loss of nuclear flavanols during drought periods in Taxus baccata.

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current-year needles decline to zero. Nevertheless, greenish-yellow-coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue-coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl2 was studied in the presence of the H4-core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non-modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.

Flavanols in somatic cell division and male meiosis of tea (Camellia sinensis) anthers.

Young anthers excised from closed tea flower buds ( Camellia sinensis L.) were stained as fresh tissues with p-dimethylaminocinnamaldehyde reagent to localize flavanols associated with nuclei and chromosomes, apart from those flavanols stored in vacuoles. This staining reagent yields a blue colour for flavanols. In the nonsporogenic somatic cells of developing anthers, flavanols were found to be attached to chromosomes at all mitotic stages. Male meiosis started at a bud size of about 3.5 mm in diameter in pollen mother cells which displayed generally more or less pronounced blue nuclei and cytoplasm. The meiotic divisions from prophase I to telophase II were characterized by blue stained nuclei and chromosomes, but within the cytoplasm there was, if any, a random and very poor reaction for flavanols. Metaphase and telophase of meiotic divisions showed maximally condensed chromosomes staining dark blue. Early in telophase II, the cytoplasm was again stained blue; this faded at late tetrad stage. Flavanols of young mitotic and older non-mitotic anthers were determined using high pressure liquid chromatography--chemical reaction detection (HPLC-CRD). Catechin, epicatechin, B2, and epigallocatechin were minor compounds, whereas epicatechin gallate and epigallocatechin gallate were found in higher amounts. The major flavanol compound of the anthers, epicatechin gallate, exhibited a significant affinity to histone sulphate, as shown by UV-VIS spectroscopic titration.

Nuclei of tea flowers as targets for flavanols.

The tea plant (Camellia sinensis L.) is famous for its flavanol-based constituents being valuable for human health. These flavanols associate with the nuclei of tea flowers, which is demonstrated histochemically by blue colouration using the selective staining reagent p-dimethylaminocinnamaldehyde (DMACA). Sepals, petals, stamens, pollen tubes, ovaries and ovules were studied. All these organs were shown to contain flavanols in vacuolar compartments, in nuclei and, exceptionally, also in the cytoplasm of pollen tubes. In all cells, even in those lacking vacuoles, the nuclei stained blue for flavanols. The extremely divergent development, shape and function of the diverse flower organs did not basically influence the nuclear flavanol association. Nevertheless, within the limits of this study, a few tissue-dependent differences in staining intensity were obvious. Interactions between epicatechin and nuclear histone proteins (histone sulphate) were studied by UV-VIS spectroscopic titration and by means of Mauser diagrams. The results show that the observed association equilibria are strongly dependent on pH (8.0 and 7.4) and on the buffer used (Tris, phosphate).

Flavanol binding of nuclei from tree species.

Light microscopy was used to examine the nuclei of five tree species with respect to the presence of flavanols. Flavanols develop a blue colouration in the presence of a special p-dimethylaminocinnamaldehyde (DMACA) reagent that enables those nuclei loaded with flavanols to be recognized. Staining of the nuclei was most pronounced in both Tsuga canadensis and Taxus baccata, variable in Metasequoia glyptostroboides, faint in Coffea arabica and minimal in Prunus avium. HPLC analysis showed that the five species contained substantial amounts of different flavanols such as catechin, epicatechin and proanthocyanidins. Quantitatively, total flavanols were quite different among the species. The nuclei themselves, as studied in Tsuga seed wings, were found to contain mainly catechin, much lower amounts of epicatechin and traces of proanthocyanidins. Blue-coloured nuclei located centrally in small cells were often found to maximally occupy up to 90% of a cell's radius, and the surrounding small rim of cytoplasm was visibly free of flavanols. A survey of 34 gymnosperm and angiosperm species indicated that the first group has much higher nuclear binding capacities for flavanols than the second group.

Nuclei of plants as a sink for flavanols.

Onion cepa (L.) and Tsuga canadensis (L.) Carr. were investigated histochemically on the association of flavanols to nuclei. The young roots of Onion cepa are totally devoid of flavanol structures. Therefore, the excised roots tips were directly incubated into different solutions of flavanols. After 3 h of incubation a flavanol binding on the nuclei was recognizable, as seen by a yellowish-brown tanning reaction. Still to ensure the presence of flavanols on the nuclei, subsequent staining with the p-dimethylaminocinnamaldehyde reagent (DMACA) resulted in an intense blue colouration. Tsuga canadensis has significant amounts of vacuolar flavanol deposits in all parts of the tree as indicated by the DMACA reagent. It is obvious that also the nuclei were associated strongly with flavanols which can be demonstrated particularly elegant in the cells of the seed wings by histochemical methods. However, the mode of flavanol release from the original deposits is not yet clear.

Evaluation of data produced by optode arrays under flow injection analysis (FIA) conditions using a partial least squares method (PLS2).

Transient diffuse reflectance spectra in the visible wavelength region from an enzyme optode were evaluated to determine artificial mixtures consisting of penicillin and ampicillin simultaneously. In order to achieve a discrimination of the samples the optode operated at two different working pH values. Using an appropriate FIA modus a linear range in the increase and decrease of the time-dependent signal was registered which could be easily fitted by means of ordinary linear regression. In this way characteristic static and dynamic features of the enzyme catalyzed reaction could be extracted for the evaluation by partial least-squares (PLS). The pre-processed raw data were differently weighted and the optimal number of primary factors was each selected by full cross validation. The interpretation of the spectral loadings and the correlation matrix identifies redundant variables resulting in a model with an optimal number of variables. The results obtained with this model show that the straightforward linear regression can be a sufficient approach to avoid the introduction of systematic errors in on-line pre-processing of data.

Primary biomechanical influence of different sterilization methods on a freeze-dried bone-ligament transplant.

The transmission of bacteria and viruses in ligament transplants should be prevented by sterilization. In this study, the influence of two different methods on the mechanical properties of a freeze-dried medial collateral ligament was analyzed in sheep. Group I (n = 10) was treated with irradiation (26 kGy) and group II (n = 10) with ethyleneoxide. The mechanical properties changed in respect of the maximal load: group I (-29.9%; P < 0.05), group II (-7.7%), elongation: group I (-6.6%), group II (-0.3%), stress: group I (-20.1%), group II (-6.8%), strain: group I (-0.64%), group II (-0.3%), stiffness: group I (-10.2%), group II (-10.5%), energy: group I (-31.4%), group II (-6.9%) and elastic modulus: group I (-1.3%), group II (-5.0%). The irradiation dose significantly reduced the maximal load, whereas ethyleneoxide sterilization resulted only in minor changes. Because of the potential cancerogenity of ethyleneoxide, a close monitoring of aeration times and its residuals are very essential. Further studies with lower irradiation doses of between 15 and 26 kGy seem to be justified.

High incidence of pathogenic microorganisms in bone allografts explanted in the morgue.

We analyzed the influence of the environment on bacterial contamination in 431 bone allografts from 97 donors (68 multiorgan, grafts taken in the operation theater; 29 morgue donors). From each bone transplant we cultured two aerobic and two anaerobic tissue specimens. The overall contamination rate was 49% (theater 51%, morgue 40%). In grafts explanted in the morgue, we noted more pathogenic microorganisms (60%) than in multiorgan donors, explanted under aseptic surgical conditions (33%). We conclude that the harvesting environment constitutes the major source of pathogenic microorganisms.

Application of fuzzy logic in multicomponent analysis by optodes.

Fuzzy logic can be a useful tool for the determination of substrate concentrations applying optode arrays in combination with flow injection analysis, UV-VIS spectroscopy and kinetics. The transient diffuse reflectance spectra in the visible wavelength region from four optodes were evaluated to carry out the simultaneous determination of artificial mixtures of ampicillin and penicillin. The discrimination of the samples was achieved by changing the composition of the receptor gel and working pH. Different algorithms of pre-processing were applied on the data to reduce the spectral information to a few analytic-specific variables. These variables were used to develop the fuzzy model. After calibration the model was validated by an independent test data set.

Simultaneous determination of penicillin and ampicillin by spectral fibre-optical enzyme optodes and multivariate data analysis based on transient signals obtained by flow injection analysis.

A multicomponent detection system using optical biosensors and flow injection analysis is described. The analysis of mixtures containing penicillin and ampicillin was realised by evaluating dynamic measurements of Phenol Red spectra in penicillinase optodes in combination with a diode array spectrometer. A variety of optodes has been produced by changing the composition of the receptor gel and the working pH. A set of characteristic quantities (describing dynamic and static features) could be obtained for each optode. These were used to compare the predictivity of classical multivariate calibration methods as well as of an artificial neural network. In addition, different algorithms were applied for the evaluation of the spectral data in order to select the most appropriate method for feature extraction. In consequence, the information obtained from the multivariate calibration models was used to set up an optimal sensor array consisting of four optodes with different types of penicillinase at different working pH.

[Allograft transplantation in pelvic instability following prosthesis exchange]. / Allografttransplantation bei Beckeninstabilität nach Prothesenwechsel.

The collective consists of 19 patients with 21 operations after multiple revision operations with complete pelvic instability. The reconstruction was done with tibia (11x) and femur allografts (6x). The bridging was reached by pressfit contact. The junction side was supplemented with autologous bone chips. The allografts were fixed with screws or plates. In all patients a good primary stability was achieved. Partial weight bearing was gained within 10.1 week and full weight bearing in 15.1 weeks. We notice one allograft fracture after 4 months and one infection after 3 months. The reoperation rate was 4. The clinical results are encouraging. No lytic lines were seen between the acetabulum cup implant and the allograft within 20.1 months time.

Long term results of uncemented Judet hip endoprostheses.

We reviewed 94 patients in whom 113 Judet uncemented total hip endoprostheses had been inserted; 83 had a mean follow up of 10.8 years (range 6 to 13 years). The Merle dAubigne and Harris hip scores were used for clinical evaluation. In the long term follow up, severe or moderate pain was present in 38.6% of hips. Radiographs were studied and radiolucent lines, and other evidence of loosening, were recorded. Twenty hips (24.1%) showed radiological signs of loosening of the acetabular cup and a displacement of more than 5 mm in 37.3%. The loosening rate of femoral stems was 32.5%, but only 8.4% showed subsidence. The cumulative survival rate at 6 years was 84.1%, decreasing to 55.1% after the eleventh year. Complications and other aspects of the outcome are discussed. Our results were on the whole not satisfactory and we cannot recommend this type of uncemented endoprosthesis for routine total hip replacement.

[Organization of a bone and tissue bank]. / Organisation einer Knochen- und Gewebebank.

The regional bone bank offers a good foundation for covering the expanding clinical demands in tumor and revision surgery. Since 1980 the tissue bank of Münster consists of 32 cadaveric and 41 multi organ donors. In the guidelines of the tissue bank the responsibility, organisation form, donor selection, quality control, sterilisation, documentation and all questions of finances are fixed, in accordance to the American Association of Tissue Banks. A fixed staff will be helpful to offer a secure and not expensive donor tissue for the clinical demands.

Software FIACRE: bioprocess monitoring on the basis of flow injection analysis using simultaneously a urea optode and a glucose luminescence sensor.

Various computer programs for large-scale bioprocess control and optimization have been developed as well as software for simple laboratory routine analysis. In comparison, software can hardly be found that works on laboratory scale and provides the control of complex flow injection analysis (FIA) systems, multisubstrate determination, data evaluation as well as minimal process control abilities. The sensors applied can be of different type (luminometric or other optical as well as electrochemical biosensors). The development of such a software may be very helpful for the transfer of FIA/biosensor systems from the state of development to industrial processes. Hence, each analysing system--even a well established biosensor--has to be individually adapted to the process, a task which is best done under laboratory conditions. Such a flexible, computer-controlled FIA system for research level based on the software FIACRE is presented. Five FIA/(bio)sensor system can be controlled simultaneously. Additionally, common temperature and pH recordings are possible. Determinations of substrate concentrations are performed by means of calibration curves which can be recorded at different times. This allows supervising the activities of the sensors during a cell cultivation and controlling the bioprocess, e.g. by adding substrate to a cell culture. The automated monitoring of the degradation of glucose and urea by two different optical sensing principles during a cell cultivation under the control of one microcomputer is presented for the first time. For this purpose, already well examined biosensors (a urease optode and a luminometric glucose sensor) were employed and their properties discussed under the aspect of working in real cultivation media. It will also be shown that substrates being of interest for bioprocess control can be detected by slight modifications of known reactions. For example, substrates of NADH-dependent enzymatic reactions can be detected by the luminol chemiluminescence system, and optodes can be employed for pH, penicillin and glucose determination.

[HIV-screening in bone transplantation]. / HIV-Screening bei Knochentransplantation.

Tissue transplantation obviously always involves the risk of HIV transmission. Since 1980, the Orthopaedic University of Muenster has conducted careful anamnestic and laboratory screening in 62 donors. Epidemiological investigations show that 90.1% of HIV-positive people in Germany are between 20 and 50 years old. For 25 donors (40%) the results of the HIV antigen test performed in the recipients of the other organs guaranteed HIV negativity. A further 17 donors (28%) were outside the epidemiologic risk group aged between 20 and 50 years. Tissue from all 20 other donors (32%) was subjected to a secondary sterilization method, e.g. fumigation with ethylene oxide or irradiation with 25 kGy.

[Long-term results of Judet's cementless total endoprosthesis of the hip joint]. / Langzeitergebnisse der zementlosen Totalendoprothese des Hüftgelenkes nach Judet.

AIM: Control of the long term results of the Judet-TEP. METHODS: From 1980-1985 124 Pat. have been operated. The follow up was done in the same collective of 83 patients after 2-6 years and > 6 years. RESULTS: The intraoperative complications have been fractures of the femur (19.3%) and the trochanter major (3.6%) and luxation (1.9%). In the follow up time the clinical results declined. The patients complained increasing pain in the thigh (26.5%) and the groin (13.2%). Radiological loosening of the acetabulum occurred especially in the medio-caudal area (61.5%) and in the proximal femur (32.5%). Cranial movement of the cup (31.3%) and varus deviation in the femur (40.9%) predominated. 28 prostheses were revised. After 11 years the cumulative success rate was 55.1%. CONCLUSIONS: The long term results are disappointing.

Kinetic analysis of second order reactions using UV-VIS spectroscopy.

A new method was recently developed for the spectroscopic kinetic analysis of reactions with two linearly independent concentration variables. Firstly, the results of this theory applied to experimental data are represented. For this investigation, reactions of the mechanism type A + Bk (1)--> C + D and A + E uc-->|k(2)|F + G were chosen since both reaction steps can be studied separately. The evaluation of the reaction system consisting of both reaction steps gives satisfactory results when the methods of "formal integration" and "singular value decomposition" (SVD) are used. Absorption coefficients of absorbing species are not needed for the evaluation. Only time dependent spectroscopic measuring values and initial concentrations are used in the presented practical example.

Inflammatory reactions in primary osteoarthritis of the hip and total hip prosthesis loosening.

In 14 patients [9 with loosened total hip arthroplasty (THA), 5 with primary osteoarthritis], the inflammatory cell infiltrate was characterized in cryostat tissue sections using antibodies against subsets of macrophages and lymphocytes. In all patients the abundance of a mature type macrophage (25F9+) was observed, whereas 27E10+ macrophages, characteristic of acute inflammatory reactions, were not seen in primary osteoarthritis but were found in 4 out of 9 patients with THA loosening. The RM3/1 + macrophage phenotype was seen in some of both sets of patients (osteoarthritis 4/5; THA loosening 4/9). The markers MRP8 and MRP14 typical of immature macrophages, were found in all patients with osteoarthritis and in some (4/9 and 8/9 respectively) of those with THA loosening. IOT4+ T-helper cells were only present in 1/5 5 patients with osteoarthritis and in 2/9 patients with THA loosening. IOT8+ T-suppressor lymphocytes were more frequently present (in 4/5 and 5/9 respectively). The data reveal remarkable differences between the two groups of patients. While the inflammatory infiltrate in patients with osteoarthritis lacks the characteristics of acute inflammation, in patients THA loosening the inflammatory process seems to be determined by periods of acute reactions. The occasional presence particularly of T-helper cells suggests that the inflammatory process is less T-cell but rather macrophage-driven.

[Biomechanics of the hip joint]. / Biomechanik des Hüftgelenkes.

The load of the hip joint is related to the density of bone, thickness of cartilage and pattern of the cancellous bone. The X-rays of the hip joint must provide all information for the calculation of the individual strain and stress.