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J Schmitt

Publications and source records attributed to J Schmitt.

At least 37 records · Page 2Linked to original sources
PubMed2003

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coliby comparing in vivo and in vitro expression.

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

Antibody Specificity↗
PubMed2003

[A population-based survey of critical care].

OBJECTIVES: Results of a population-based survey of critical care in a German state are presented. The indication for critical care was derived using the APACHE-II score,which measures the physiologic state of patients, and the TISS-28 score as a measure for expenditure of critical care. METHODS: During 3 months all adult patients of intensive care units in Saarland, a German state with 1.08 million residents,were included. All kinds of hospitals were involved. Completeness and validity of data are proved. RESULTS: The TISS score was registered daily on 13,391 adult patients with 54,503 days, the APACHE score during admission and discharge of intensive care units only. The correlation between the scoring-systems and indication, kind of care (critical care vs. intermediate care), kind of hospital, and ICD-10 diagnoses is analysed in this paper. CONCLUSIONS: In this study the population-based critical care in a state is analysed the first time. It completes previously published studies,which were performed at the level of hospitals and departments.

APACHE↗
PubMed2003

A human flora-associated rat model of the breast-fed infant gut.

OBJECTIVES: Bacterial colonization of the infant gut may have important influences on the development of gastrointestinal, respiratory, and allergic disease. Early diet is a major determinant of the gut microflora. It is very difficult to carry out studies in human infants that can investigate the interaction of diet, flora, and mucosa. In this study we have developed an infant human flora-associated (IHFA) rat model to allow such investigation. METHODS: Germ-free infant rats were infected with fecal bacteria from exclusively breast-fed infants and were maintained on a modified infant formula for 8 weeks. The fecal and cecal contents were collected and compared with feces of breast-fed infants for bacterial populations, bacterial metabolites, and enzymes and for the ability to inhibit adhesion of pathogenic bacteria to human mucosal cells. RESULTS: The IHFA cecum and feces were dominated by lactic acid bacteria, Bifidobacterium, and lactobacilli, which were representative of the infant feces. The fecal short-chain fatty acid profile was dominated by acetic and lactic acid in a similar manner to human infant feces. Other bacterial metabolites were similar to those of the human infant. Rat intestinal samples were able to inhibit the adhesion of pathogens to mucosal cells, but to a lesser extent than the human samples. CONCLUSIONS: This IHFA infant model of the intestinal flora of the breast-fed infant is considered valid for studying the effect of diet on bacterial colonization and metabolism.

Animals↗
PubMed2003

[Systematic analysis of ground reaction forces before and after hip and knee arthroplasty].

In this prospective study we employed a newly developed gait analysis system to compare the ground reaction force patterns in 15 patients before and after total hip or knee replacement. In this system, data are measured separately for each limb. Measured data were also obtained from 30 healthy adults and compared with those obtained from the patient group. We analysed the three-dimensional force patterns, impulse, frequency, stride and double stance, and compared changes in the postoperative gait patterns. The vertical force maxima Fy identify the peak forces obtaining during walking. The results showed significantly increased (p < 0.05) postoperative force maxima Fy2 and Fy3 for both knee replacement (Fy2: 82.48 to 86.17 and Fy3: 96.09 to 99.35% body weight, pre- and postoperatively, respectively) and hip replacement (Fy2: 84.44 to 88.08 and Fy3: 98.63 to 101.96% body weight, pre- and postoperatively, respectively). The ADAL system proved suitable for the easy performance of gait analysis, and thus may be of future value in the area of clinical quality assurance.

Adult↗
PubMed2002

Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale.

Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures. Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags. Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature. For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum. Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L. In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium. It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium. Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages. Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes.

Bioreactors↗
PubMed2002

[Extracorporeal shockwave therapy (ESWT) as therapeutic option in supraspinatus tendon syndrome? One year results of a placebo controlled study].

Extracorporeal shock wave therapy (ESWT) is seen as a therapeutic option in the treatment of chronic supraspinatus tendinitis by some authors. To test whether ESWT comprising 3 x 2000 pulses with the positive energy flux density ED+ of 0.33 mJ/mm2 is clinically superior to a sham ESWT treatment, a prospective, randomized, single-blinded, placebo-controlled study with an independent observer was performed. Forty patients were treated either by verum ESWT or sham ESWT under local anesthesia. Target criteria were the age-corrected Constant score, pain at rest and during activity on a visual analogue scale, and subjective improvement. Patients who reported no subjective improvement after 12 weeks were deblinded and received verum ESWT if they had belonged to the placebo group (partial crossover). The results of the verum group lie within the range of results for ESWT published by other authors. Patients in the placebo group with local anesthetic showed equally good results. At 12 weeks, and 1 year after intervention, no difference could be found between the verum and placebo groups regarding Constant score, pain, shoulder function, or subjective improvement. The nonresponders to the placebo ESWT continued to show no improvement after receiving verum ESWT. This contradicts a specific ESWT effect. Based on the results of this placebo-controlled study, ESWT appears to have no clinically relevant effect on supraspinatus tendinitis. The study underlines the importance of a control group in evaluating new treatment methods for diseases with unknown natural history.

Adult↗
PubMed2002

Dynamics and stability of legged locomotion in the horizontal plane: a test case using insects.

Motivated by experimental studies of insects, we propose a model for legged locomotion in the horizontal plane. A three-degree-of freedom, energetically conservative, rigid-body model with a pair of compliant virtual legs in intermittent contact with the ground allows us to study how dynamics depends on parameters such as mass, moment of inertia, leg stiffness, and length. We find periodic gaits, and show that mechanics alone can confer asymptotic stability of relative heading and body angular velocity. We discuss the relevance of our idealized models to experiments and simulations on insect running, showing that their gait and force characteristics match observations reasonably well. We perform parameter studies and suggest that our model is relevant to the understanding of locomotion dynamics across species.

Animals↗
PubMed2002

Recombinant autoantigens.

Autoimmune diseases frequently manifest themselves in fulminating immune reactions involving T and B cells that lead to the appearance of circulating autoantibodies. Such antibodies are considered to be of diagnostic relevance and may be pathognomonic for their associated autoimmune diseases. The advent of recombinant protein expression technology has made it possible to economically produce high quality autoantigens for use in immunoassays to detect autoantibodies in patients' sera. This short review is intended to give the reader a brief description of the various autoantigen production methodologies with their advantages and disadvantages, and the limitations of these techniques in the diagnostics of autoimmune diseases.

Animals↗
PubMed2002

MR imaging characteristics of osteoradionecrosis of the pelvis after radiation therapy on gynecological tumors.

PURPOSE: To describe MR imaging characteristics of osteoradionecrosis (ORN) of the pelvis as a result of radiation therapy (RT) on gynecological tumors. MATERIAL AND METHODS: Radiography, computed tomography (CT) and magnetic resonance imaging (MRI) were performed on 9 women (mean age 67.5 years) with gynecological tumors to identify ORN. T1- and T2-weighted sequences and contrast-enhanced t1-weighted sequences with and without fat saturation were used. The patients began developing pain after the completion of RT indicating a possible ORN a which time MRI was performed. MR images were correlated with the results of clinical examinations. RESULTS: Depending on the time elapsed after RT, ORN presented with different signal intensities. The acquired images suggested that signal changes in T2-weighted images as well as the different enhancement behaviour of ORN could be dependent on the time elapsed after RT. Visualisation of the affected regions was best achieved with fat-saturated T1-weighted sequences. CT showed increased density in the affected regions corresponding to osteosclerosis. In all cases the sacroiliac joint was affected, some times bilaterally. CONCLUSION: MRI is helpful in detecting and characterizing ORN. Changes in signal intensity, based on histopathological tissue changes could make a chronological classification possible.

Aged↗
PubMed2002

Vessel wall MRI of the thoracic aorta: correlation to histology and transesophageal ultrasound. Preliminary results.

PURPOSE: To visualise the vessel wall of the descending thoracic aorta using magnetic resonance imaging. To evaluate the diagnostic potential of tailored T1-weighted sequences with contrast enhancement to assess systemic atherosclerotic disease. METHODS: This study was performed on a clinical 1.5 Tesla scanner using a gradient strength of 30 mT/m and the phased array spine coil. A cadaver was examined to optimise a magnetic resonance imaging (MRI) protocol to evaluate atherosclerotic aortic wall disease. The acquired MR images were compared to gross specimens and histology. Subsequently seven patients who had undergone transesophageal ultrasound (TEU) with detailed assessment of the descending thoracic aorta were examined with MRI. The optimised protocol included untriggered and fat suppressed T2-weighted turbo spin echo sequences and ECG-triggered and fat suppressed T1-weighted spin echo sequences before and after iv administration of Gd-DTPA. Findings of the MR images were compared to the results of TEU. Contrast enhancement measurements were performed in normal and thickened vessel wall segments. RESULTS: For the cadaver study a good correlation of the degree of vessel wall thickening and the extent of plaque imaged with the applied MR protocol was found. Tissue characterisation was limited due to post mortem changes. In vivo ECG-triggered T1-weighted images showed good correlation to TEU in terms of vessel wall thickness and plaque extension as verified by means of consensus reading. Differentiation of the plaque components fat, calcium and fibrous tissue was possible. In thickened aortic wall segments and fibrous caps a mean contrast enhancement of 50.4 % +/- 23.5 % was measurable while normal wall segments showed an enhancement of 6.7 % +/- 3.1 %. The difference of contrast enhancement was highly significant (p < 0.0001). CONCLUSION: Using fat suppressed T1-weighted sequences with contrast enhancement the extent of atherosclerotic vessel wall changes can be demonstrated. The suggested MR protocol contains a high potential for diagnosis and follow-up of therapy of atherosclerotic disease of the descending thoracic aorta.

Aorta, Thoracic↗
PubMed2002

[Comparison of expertises between long-term care insurance (SGB XI) and the law on ambulatory care for disabled individuals in Berlin (HPG)].

A long-term care insurance (SGB XI) was established in Germany in 1995. Previously, care for predominantly disabled and disabled elderly people in Berlin was regulated by a law enforced by the Berlin government (HPG). The objective of this study was to investigate the differences in age, sex, diagnosis and care of the disabled individuals at home. In a cross-sectional study, the social medicine certificates of 3.916 disabled individuals were evaluated. The certificate assesses the grade of disability and the care needed for a person entitled to benefit from either the HPG or later from SGB XI. Disabled females benefiting from the HPG and SGB XI regulations are in the majority. The male: female ratio for HPG was 1:2.215 and for SGB XI 1:2.759. The differences between male and female as well as between HPG and SGB XI are statistically significant. The even higher proportion of females for those benefiting from SGB XI is due to the demographic change over time. The results of the study also show that the certificates differ in diagnosis, in the care needed at home, and in the grades of disability. By logistic regression analysis, the univariate method shows the amount of care needed at home to be greater for the HPG than for the long-term care insurance. The collective of disabled persons benefiting from HPG were derived from the ambulatory and the stationary sector. In this study, those individuals who were investigated and supported by SGB XI originated from the ambulatory sector, although the law also supports those under hospital care. By calculating analysis of multivariate logistic regression with grades of disability taken as dependent and disability of a given person as independent variables related to the grades of disability when applying SGB XI were compared to HPG. The independent variables have different hierarchies for both laws and need to be divided into more specific subgroups. Interestingly, age and sex did not influence the grading of disability. Diseases leading to disability differ between male and females. Males suffer mostly from neurologic and psychiatric and females from orthopaedic diseases. The criteria for the particular grades are too broad and a better specification is suggested, in that the grades should be increased from three to five. For exceptional cases, a more flexible procedure should be allowed, which is possible under the present SGB XI regulations.

Adolescent↗
PubMed2001

Cavernous angioma associated with ipsilateral hippocampal sclerosis.

We report two cases with extratemporal cavernous angioma (CA) and coexisting ipsilateral hippocampal sclerosis. Classically dual pathology is defined as the association of hippocampal sclerosis with an extrahippocampal lesion. Subtle changes in hippocampus might be overlooked in the presence of an unequivocal extrahippocampal abnormality. Seizure outcome after epilepsy surgery in cases with dual pathology is less favourable if only one of the lesions is removed. Dual pathology must always be considered in diagnostic imaging of patients with intractable epilepsy and CA.

Adult↗
PubMed2001

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris.

In this report, we describe the high-yield secretory expression ( approximately 40 mg x l(-1)) of pure, atrazine-specific Fab fragments (K411B) from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells. Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5-l scale. Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media. Approximately 30% of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions. Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 microg x l(-1), which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in Escherichia coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and to the parental mAb K4E7.

Atrazine↗
PubMed2001

Cloning, functional expression, and characterization of recombinant pig liver esterase.

The N-terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline-beta-naphthylamidase from pig liver. Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N-terminal sequence were used to clone the 1698 base pairs of PLE cDNA. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Umg(-1) and a Vmax/Km value of 139 micromolmin(-1)mM(-1) with p-nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline-beta-naphthylamide. This is a substrate specificity for the so-called gamma subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degrees C, respectively. The determined molecular weight of the secreted enzyme was approximately 61-62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-Gly).

Amino Acid Sequence↗
PubMed2001

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Bacillus megaterium↗
PubMed2001

[Incidence of occupation-related skin diseases in skin-exposure occupational groups].

BACKGROUND AND OBJECTIVE: Occupational skin diseases (OSD) rank at the top of occupational diseases in Germany. We studied the cases with initial medical report of an OSD reported to our Register of OSD in North Bavaria (BKH-N). PATIENTS/METHODS: From 1990-99 5.285 cases were closed and recorded. Since the number of employees in the different occupations was known, a population-based study was performed to investigate incidences and demographic characteristics in occupations classified into 24 groups. RESULTS: In the occupational groups in 3.097 cases a work-related skin disease was stated. The estimated overall incidence was 6.7 cases per 10,000 workers per year. The highest incidence was in hairdressers (97.4), bakers (33.2), and florists (23.9), while the largest number of cases was in hairdressers (856), health services (481), and metal-surface workers (260). The median age of all cases was 25 years. In 1.611 cases (52%) a delayed-type sensitisation with occupational relevance was perceived. CONCLUSIONS: The descriptive data of the BKH-N highlight occupational groups being at risk for OSD. The results assist in working out prevention measures aiming on lowering the incidence of OSD.

Adult↗
PubMed2001

Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones.

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.

Burkholderia cepacia↗