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Biomedical subjects

Werner Schmidt

Publications and source records attributed to Werner Schmidt.

At least 19 recordsLinked to original sources

Gravity-induced absorption changes in Phycomyces blakesleeanus during parabolic flights: first spectral approach in the visible.

Gravity-induced absorption changes as experienced during a series of parabolas on the Airbus 300 Zero-G have been measured previously pointwise on the basis of dual-wavelength spectroscopy. Only the two wavelengths of 460 and 665 nm as generated by light-emitting diodes have been utilised during our first two parabolic-flight campaigns. In order to gain complete spectral information throughout the wavelength range from 400 to 900 nm, a miniaturized rapid scan spectrophotometer was designed. The difference of spectra taken at 0 g and 1.8 g presents the first gravity-induced absorption change spectrum measured on wild-type Phycomyces blakesleeanus sporangiophores, exhibiting a broad positive hump in the visible range and negative values in the near infrared with an isosbestic point near 735 nm. The control experiment performed with the stiff mutant A909 of Phycomyces blakesleeanus does not show this structure. These results are in agreement with those obtained with an array spectrophotometer. In analogy to the more thoroughly understood so-called light-induced absorption changes, we assume that gravity-induced absorption changes reflect redox changes of electron transport components such as flavins and cytochromes localised within the plasma membrane.

Absorption↗

The specific FKBP38 inhibitor N-(N',N'-dimethylcarboxamidomethyl)cycloheximide has potent neuroprotective and neurotrophic properties in brain ischemia.

FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remains unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca(2+) (CaM/Ca(2+))-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38.CaM/Ca(2+) 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38.CaM/Ca(2+) inhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N',N'-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 mug/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases.

Brain↗

Prospective study on the expression of cancer testis genes and antibody responses in 100 consecutive patients with primary breast cancer.

To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohistology in selected RT-PCR-positive cases. Antibody responses against 7 CT antigens were analyzed using recombinant antigen expression on yeast surface. In 98 evaluable cases, SCP-1 and SSX-4 were expressed most frequently (both 65%), followed by HOM-TES-85/CT-8 (47%), GAGE (26%), SSX-1 (20%), NY-ESO-1 (13%), MAGE-3 (11%), SSX-2 (8%), CT-10 (7%), MAGE-4 (4%) and CT-7 (1%). One CT gene was expressed by 90% of the cases; 79% expressed > or =2, 48% > or =3, 29% > or =4, 12% > or =5, 6% > or =6, 3% > or =7, 2% > or =8 and one case coexpressed 9 antigens. Of 100 serum samples screened for CT antigen-specific antibodies, antibodies against NY-ESO-1 were detected in 4 patients, against SCP-1 in 6 patients and against SSX-2 in 1 patient, while no antibodies were detected against MAGE-3, CT-7 and CT-10. Expression of CT genes or antibody responses was not correlated with clinical parameters (menopausal status, tumor size, nodal involvement, grading, histology and estrogen receptor status) or the demonstration of CT gene expression at the protein level, by immunohistology. Our results show that breast carcinomas are among the tumors with the most frequent expression of CT antigens, rendering many patients potential candidates for vaccine trials.

Antibodies, Neoplasm↗

Serological identification of breast cancer-related antigens from a Saccharomyces cerevisiae surface display library.

A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high-through-put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild-type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies.

Amino Acid Sequence↗

The extracorporeal perfusion of the swine uterus as an experimental model: The effect of tocolytic drugs.

OBJECTIVE: Comparison of the effect of tocolytic drugs on isolated swine uterus preparations. STUDY DESIGN: Forty swine uteri were perfused with the aim to preserve a viable organ, which should be responsive to oxytocic hormones and tocolytic pharmaca. An intrauterine catheter recorded the pressure changes. After initiation of rhythmical uterine contractions we administered known tocolytic drugs (fenoterol, ritodrine, terbutaline, propofol, acetylsalicylic acid, alcohol, atosiban, verapamil, and glyceryl trinitrate) in various concentrations. RESULTS: Perfusate pH and lactate, partial oxygen and carbon dioxide tensions, and oxygen saturation in the perfusate showed good preservation of the organ for up to 8h. All substances showed a tocolytic effect on the swine uterus. The effect varied substantially with regard to the length of the contraction free intervals, which was our main effect parameter. Fenoterol, acetylsalicylic acid, and alcohol showed the most and glyceryl trinitrate the least powerful effect. CONCLUSIONS: A direct comparison of various tocolytic substances in the same experimental model showed the best effect for fenoterol. Furthermore, we could demonstrate that the swine uterus perfusion system is a suitable model to study the influence of various conditions like the administration of drugs or the induction of oxidative stress on the uterus function.

Animals↗

bFGF and EGF modulate trauma-induced proliferation and neurogenesis in juvenile organotypic hippocampal slice cultures.

Since postnatal and adult mammalian brains have been shown to retain an ability to generate neurons from endogenous stem cells throughout life, these cells could play a central role in regeneration after neuronal loss. Therefore, we studied cell proliferation, glio- and neurogenesis respectively after brain injury in organotypic hippocampal slice cultures using a focal trauma by transecting Schaffer collaterals in the cornu ammonis (CA) 2 region mechanically. After determination of cell death using propidium iodide, neuroregenerative processes were quantitatively analyzed by various immunohistochemical techniques at different time points post injury. As this endogenous insult-induced neurogenesis is rather inefficient, we investigated if it can be enhanced by application of exogenous growth factors. Exogenous basic fibroblast growth factor (bFGF) enhanced neurogenesis significantly in the dentate gyrus (DG) region. A neutralizing antibody against endogenous bFGF revealed a significant decrease of basal and trauma-induced proliferation. Reverse transcription polymerase chain reaction (RT-PCR) studies exhibited a downregulation of FGF messenger ribonucleic acid (mRNA) transcription after the antibody treatment. In contrast, epidermal growth factor (EGF) increased proliferation, but not neurogenesis. A combination of bFGF and EGF displayed an EGF-like effect on proliferation and no effect on neurogenesis. These results demonstrate, that in our model bFGF but not EGF sustains neurogenesis, whereas together the two growth factors permit an increased proliferation but not neurogenesis in organic hippocampal slice cultures.

Animals↗

Relationship between nuclear chromatin decondensation (NCD) in vitro and other sperm parameters and their predictive value on fertilization rate in IVF program.

PURPOSE: The purpose of this study was to determine the in-vitro induced nuclear chromatin decondensation (NCD) of human spermatozoa and its value in combination with routine semen analysis in predicting the outcome of in-vitro fertilization (IVF). METHODS: The ejaculate of 52 couples, undergoing IVF, was incubated with lithium diidosalicylic acid (LIS) and dithiothreitol (DTT) (G.1) or with heparin and sodium dodecyl sulphate (SDS) (G.2) to induce chromatin decondensation (NCD). Smears were made at 30, 60, and 120 min after incubation. RESULTS: NCD was evaluated by morphometrical detection of the surface area of the spermatozoa using a semiautomatic image analysis system (IBAS). In both groups, the sperm heads showed a significant enlargement after 30, 60, and 120 min incubation in comparison to the initial size. However, no correlation was found between NCD at various periods of time and the fertilization rates. The mean area of the sperm heads in the native sample in the G.1 was 9.45+/- 1.33 microm(2) and 9.02+/- 1.15 microm(2) in the G.2. This area increased after incubation for 30, 60, and 120 min to 10.92 +/- 1.48, 12.26+/- 2.16, 13.54+/- 3.14, and 15.35+/- 7.78 microm(2) in the first group (G.1) and to 10.29 +/- 1.15, 11.23 +/- 1.85, 11.46 +/- 1.97, and 11.27 +/- 2.82 microm(2) in the second group, respectively. CONCLUSIONS: NCD in vitro after incubation with LIS + DTT or heparin + SDS could not be recommended as a predictive parameter for IVF outcome.

Chromatin↗

A 1-year randomized study to evaluate the effects of a dose reduction in oral contraceptives on lipids and carbohydrate metabolism: 20 microg ethinyl estradiol combined with 100 microg levonorgestrel.

OBJECTIVES: To evaluate the impact on lipid and carbohydrate variables of a combined one-third ethinyl estradiol (EE)/levonorgestrel (LNG) dose reduction in oral contraceptives. METHODS: In an open-label, randomized study, a dose-reduced oral contraceptive containing 20 microg EE and 100 microg LNG (20 EE/100 LNG) was compared with a reference preparation containing 30 microg EE and 150 microg LNG (30 EE/150 LNG). One-year data from 48 volunteers were obtained. RESULTS: We found a decrease of HDL2 cholesterol and increases of low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol and total triglycerides in both treatment groups from baseline to the 13th treatment cycle. Although for four of six variables, the changes in the 20 EE group were lower compared with the 30 EE group, none of the differences between the two treatments were statistically significant. The median values for the fasting levels of insulin, C-peptide and free fatty acids slightly increased or remained unchanged while the fasting glucose levels slightly decreased after 13 treatment cycles. While the glucose area under the curve (AUC) (0-3 h) was similar in both groups during the OGTT, the insulin AUC(0-3 h) was less increased in the 20 EE/100 LNG group compared with the 30 EE/150 LNG group. None of the differences between the treatment groups for any of the carbohydrate metabolism variables were statistically significant at any time point. Both study treatments were safe and well tolerated by the volunteers. CONCLUSION: Similar effects on the lipid and carbohydrate profiles were found for both preparations. The balanced one-third EE dose reduction in this new oral contraceptive caused slightly lower, but insignificant, changes in the lipid and carbohydrate variables compared with the reference treatment.

Adult↗

A 1-year study to compare the hemostatic effects of oral contraceptive containing 20 microg of ethinylestradiol and 100 microg of levonorgestrel with 30 microg of ethinylestradiol and 100 microg of levonorgestrel.

OBJECTIVES: To comparatively evaluate the impact of a balanced one-third dose-reduced oral contraceptive on hemostatic variables. METHODS: In an open-label, randomized study, a dose-reduced oral contraceptive containing 20 microg of ethinylestradiol (EE) and 100 microg of levonorgestrel (LNG) was compared with a reference preparation containing 30 microg of EE and 150 microg of LNG. One-year data were obtained from 48 volunteers. RESULTS: The direction and magnitude of the changes (increase or decrease) in most of the hemostatic variables were similar in both treatment groups. The majority of changes of all investigated variables remained within the reference ranges of variation. The procoagulatory variables increased to some extent from baseline to treatment cycle 13, while the anticoagulatory variables slightly decreased. In particular, thrombin turnover measured by prothrombin fragments 1+2 increased during treatment by 35% in the 20 microg of EE group and by 38% in the 30 microg of EE group. Statistically significant differences between the two treatment groups were found only for TAT. For the profibrinolytic variables, plasminogen was increased by 42% (20 microg of EE) and 49% (30 microg of EE). While the plasma levels of tPA antigen were reduced during treatment, the levels of its activity were increased by 54% (20 microg of EE) and 20% (30 microg of EE). For PAI, both antigen and activity were decreased, somewhat more pronounced with 20 microg of EE. D-Dimer remained virtually unchanged. Finally, the median FbDP levels were elevated by 30% (20 microg of EE) and 38% (30 microg of EE).

Adult↗

Biochemical indexes of the B vitamins in cord serum are predicted by maternal B vitamin status.

BACKGROUND: The concentration of total homocysteine (tHcy) is higher in newborns than in older children. Vitamin B-12 is the major determinant of tHcy in newborns. Maternal status of folate, vitamin B-12, and vitamin B-6 during pregnancy may affect the biochemical markers of these micronutrients in newborns. OBJECTIVE: Our objective was to study the relation between concentrations of the metabolites and B vitamins in maternal sera and concentrations in the umbilical venous blood of the corresponding newborns. DESIGN: We studied healthy pregnant women at the time of labor who were expecting healthy, full-term, appropriate-birth-weight babies. Samples were available from 82 mother-infant pairs. RESULTS: Concentrations of B vitamins were higher in cord samples than in maternal blood (folate, 2-fold; vitamin B-12, 1.5-fold; and vitamin B-6, 6-fold). Concentrations of cystathionine and methylmalonic acid (MMA) were also higher in the infants than in the mothers (x +/- SD: cystathionine, 462 +/- 189 and 343 +/- 143 nmol/L; MMA, 353 +/- 144 and 233 +/- 110 nmol/L). No significant differences in tHcy concentrations were observed between fetal and maternal samples. Concentrations of vitamin B-12 did not differ significantly between mothers of infants from different quartiles of cord MMA. Higher fetal MMA concentrations were related to higher maternal MMA and vitamin B-12 concentrations and lower fetal concentrations of vitamin B-12. Fetal concentrations of cystathionine were predicted by maternal cystathionine, gestational age, fetal vitamin B-6, and fetal tHcy. CONCLUSIONS: Maternal concentrations of the metabolic markers of B vitamins predict values in fetal blood at delivery. Fetal tHcy concentrations were low but were predicted by the vitamin status of the mother. The effect of increasing maternal intake of vitamins B-12 and B-6 during pregnancy on the fetal concentrations of the metabolites should be investigated.

Adult↗

Cataracts in a laboratory colony of African green monkeys (Chlorocebus aethiops).

Three cases of spontaneous cataracts were investigated in a colony of African green monkeys (Chlorocebus aethiops). Clinical, pathological and microbiological investigations were conducted in two cases of juvenile cataracts and in one case of a mature cataract. These investigations revealed no indication of an infection as the cause of cataract development. A genetic correlation existed between the affected individuals. Clinical chemistry gave a hint that calcium might be a "key factor" in the development of these cataracts: in both cases of the juvenile cataracts, the calcium content in the serum and in the aqueous humor was clearly decreased in the affected babies. The calcium uptake in the affected baby itself was not altered but the calcium content in the mother's milk was low.

Animals↗

Relationship between cytokine concentration in serum and preovulatory follicular fluid and in vitro fertilization/intracytoplasmic sperm injection outcome.

Differences in follicle cell function and follicular fluid biochemistry have been suggested to influence the developmental potential of human oocytes at both cellular and chromosomal levels. Follicular fluids provide the environment in which oocyte maturation occurs. Therefore, follicular fluids may affect fertilization and early embryonic development. In humans, it has been shown that follicular fluid exerts chemotactic activity towards neutrophilic granulocytes and that the concentrations of this activity are related to the outcome of in vitro fertilization (IVF) treatment. The present study focuses on growth and cytokine presence in serum and follicular fluid of patients of reproductive age and their role in ovarian follicles, fertilization, embryo development, and implantation and to find out which growth factor could be a useful predictive parameter of IVF/intracytoplasmic sperm injection outcome.

Animals↗

Application of silicon sensor technologies to tumor tissue in vitro: detection of metabolic correlates of chemosensitivity.

Silicon sensor technologies, developed during the 1990s, allow measurement of extracellular chemical changes related to cell metabolism. Exposition of tumor cells in vitro to anticancer drugs modifies cell metabolism, making it possible to detect on-line with sensor chips patterns of metabolic activity, which depend on drug sensitivity, or drug resistance of the cells. Sensor devices are composed of an incubation chamber with a sensor chip and a fluidic system for medium supply. Basically, two sensor types are available: (1) monosensor systems to detect extracellular acidification; and (2) multisensor arrays for many parameters such as pH, oxygen consumption, and impedance. Two companies have developed such systems: Molecular Devices (USA) and Bionas (Germany). In this chapter, in addition to operation of the sensor devices, we describe techniques for tissue (tumor and non-tumor) preparation. Basically, three procedures are described: (1) tissue dissociation and further cultivation on the sensor chip or on Transwell inserts; (2) preparation of tissue slices (300 microm thick) and attachment to the sensor chip or to inserts, and (3) cultivation of cells in dialysis tubes, a procedure necessary for nonadherent cells and cell suspensions to avoid their washing away. Evaluation of results and selection of controls are also discussed.

Antineoplastic Agents↗

Identification of an HLA-DR-restricted peptide epitope with a promiscuous binding pattern derived from the cancer testis antigen HOM-MEL-40/SSX2.

The SSX2 gene encodes the tumor-specific antigen HOM-MEL-40/SSX2 expressed in a broad spectrum of tumors of different origin, against which humoral and CD8+ T-cell-mediated MHC-I-restricted responses have been demonstrated. Searching for promiscuous MHC-II-restricted peptides that might be suitable as a CD4+ stimulating vaccine for many patients, we used the SYFPEITHI algorithm and identified a HOM-MEL-40/SSX2-derived pentadecamer epitope (p45-59) that induced specific CD4+ T-cell responses restricted by the HLA-DRB1 subtypes *0701, *1101 and *1302 that have a cumulative prevalence of approximately 25% in the Caucasian population. The CD4+-mediated response against p45-59 and its DR restriction was demonstrated by inhibition with anti-CD4 and HLA-DR antibodies, respectively, and by blocking experiments using HLA-specific antibodies. The natural processing and presentation of p45-59 was demonstrated by recognition of the SSX2+ melanoma cell line Me 275 as well as autologous and allogeneic dendritic cells pulsed with whole-protein SSX2 by T cells with specificity for p45-59. p45-59 was able to induce responses in 3/6 breast cancer patients and 1/5 healthy controls. No correlation was found between CD4+ T-cell responses against p45-59 reactivity and anti-SSX2 antibody titers in the serum of patients, suggesting that CD4+ and B-cell responses are regulated independently. p45-59 holds promise as a broadly applicable peptide vaccine for patients with SSX2-positive neoplasms.

Antibody Formation↗

Optospectroscopic detection of primary reactions associated with the graviperception of Phycomyces. Effects of micro- and hypergravity.

The graviperception of sporangiophores of the fungus Phycomyces blakesleeanus involves gravity-induced absorbance changes (GIACs) that represent primary responses of gravitropism (Schmidt and Galland, 2000). GIACs (DeltaA(460-665)) of sporangiophores were measured in vivo with a micro-dual wavelength spectrometer at 460 and 665 nm. Sporangiophores that were placed horizontally displayed an instant increase of the GIACs while the return to the vertical position elicited an instant decrease. The GIACs are specific for graviperception, because they were absent in a gravitropism mutant with a defective madJ gene. During parabola flights hypergravity (1.8 g) elicited a decrease of the GIACs, while microgravity (0 +/- 3 x 10 (-2) g) elicited an instant increase. Hypergravity that was generated in a centrifuge (1.5-6.5 g) elicited also a decrease of the GIACs that saturated at about 5 g. The GIACs have a latency of about 20 ms or shorter and are thus the fastest graviresponses ever measured for fungi, protists, and plants. The threshold for eliciting the GIACs is near 3 x 10 (-2) g, which coincides numerically with the threshold for gravitropic bending. In contrast to gravitropic bending, which requires long-term stimulation, GIACs can be elicited by stimuli as short as 20 to 100 ms, leading to an extremely low threshold dose (acceleration x time) of about 3 x 10 (-3) g s, a value, which is four orders of magnitude below the ones described for other organisms and which makes the GIACs of Phycomyces blakesleeanus the most sensitive gravi-response in literature.

Gravitation↗

A mini-rapid-scan-spectrophotometer.

The mini-rapid-scan-spectrophotometer (Mini-RSS) is a scanning single-beam spectrophotometer that has been patented. It is based on a minimum of reflections and involves exclusively mirrors as beam-deflecting components. This way stray light is minimized, which results in an excellent light-throughput, high dynamics, low cost, compactness and rigidity. The Mini-RSS has been designed as a multi-purpose instrument that allows absorption, transmission, reflection, fluorescence and luminescence measurements in a single-beam mode. Its spectral range extends from the UV and visible spectrum to the IR. This provides for the possibility to measure even optically unfavorable, highly turbid or scattering samples that would be otherwise inaccessible to investigations with commercial spectrophotometers. A miniaturized and very sensitive photomultiplier-module (PM) of high dynamics allows in the visible spectral range absorbance measurements that cover up to four OD units. The Mini-RSS is capable of scanning up to 100 spectra per second with a resolution of 12 bit and 500 points. The linear dispersion is currently 5 nm and the stray light level <0.01%.

Calibration↗

A high performance micro-dual-wavelength-spectrophotometer (MDWS).

The dual wavelength spectrophotometer (DWS) has proven to be the most sensitive device to monitor minute optical absorbance changes, which are inaccessible to conventional single or double beam spectrophotometers. The typical set ups, e.g. extensively used for Ca2+ or phytochrome measurements, are huge, expensive and cumbrous. Therefore, a novel high performance micro-dual-wavelength spectrophotometer (MDWS) was developed. It is miniaturized and no moving parts such as vibrational mirrors or rotating filter wheels involved. Its specifications are superior compared to the conventional set up being capable of detecting minute optical changes (reflection, absorbance, transmittance) at particular wavelengths.

Calibration↗

Quickly changing acceleration forces (QCAFs) vibration analysis on the A300 ZERO-G.

Experiments that are done under microgravity, e.g. during space or parabola flights, are invariably accompanied and affected by ubiquitous vibrations of the surroundings. Vibrations induce Quickly Changing Acceleration Forces (QCAFs) that interfere with the perception of the earth gravitational field. To investigate their impact on experiments under microgravity we monitored the vibrations of the airplane A300 ZERO-G during parabola flights and analyzed them in their spectral and frequency domains. Power spectra obtained with Fast-Fourier Transforms (FFT) display a complex pattern of various vibrations whose origin, relative phases and intensities remain unidentified. During the zero-g phases (parabolas), when the engines of the airplane are throttled, the vibrations still elicit residual QCAFs of at least +/- 1 g. By means of adequate damping procedures the QCAFs could, however, be reduced by approximately 95%.

Acceleration↗