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Biomedical subjects

M Jäger

Publications and source records attributed to M Jäger.

At least 55 records · Page 3Linked to original sources
PubMed2001

The scFv fragment of the antibody hu4D5-8: evidence for early premature domain interaction in refolding.

Fluorescence spectroscopy and 1H/2H-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native VL domain and an unfolded VH domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with 1H/2H-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full 1H/2H-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments.

Antibodies, Monoclonal↗
PubMed2001

A time-resolved EPR study of the electron-spin-polarization pathways of p-benzosemiquinone.

Benzoquinone (BQ), deuterobenzoquinone (d4-BQ), and hydroquinone (BQH2) are investigated in ethylene glycol by means of direct detection fast time-resolved EPR spectroscopy after laser flash photolysis. The development of the magnetization as a function of time and magnetic field is obtained and analyzed in terms of the Bloch equations and hyperfine parameters. The signals are attributed to the semiquinones BQH(*) and d4-BQH(*). The presence of 1,2-dihydroxyethyl radicals during the photolysis of BQ and d(4)-BQ is verified. No alkyl radicals are observed in solutions of BQ with excess BQH2. Detailed analysis of the chemically induced dynamic electron polarization spectra with respect to their development in time shows that polarization patterns of the semiquinones can be traced back to a superposition of triplet mechanism and radical pair mechanism, the latter arising from geminate T-pairs. Hence, two independent pathways for polarization are assumed: reaction of triplet benzoquinone with ethylene glycol leads to the semiquinone and dihydroxyethyl radicals with all signals in emission, whereas the reaction of triplet BQ and BQH2 yields two semiquinones exhibiting both net emissive and multiplet emissive/absorptive intensity distributions.

Journal Article↗
PubMed2001

Deficit states in schizophrenia and their association with the length of illness and gender.

The present study investigated the association between the frequency of deficit states (DS) and the length of illness and gender in schizophrenic patients. The following hypotheses were examined: 1) there is an association between the length of schizophrenic illness and the frequency of DS and 2) the frequency of DS in patients with comparable length of illness is higher in males than in females. Patients, included in the study, were consecutively hospitalized and diagnosed according to ICD-9 criteria (ICD-9: 295-295.9). Psychopathological assessment was performed in a standardized manner. Group differences were evaluated by using the t-test or the chi-square statistics. Variables with a possible impact on the occurrence of DS were entered into a backward multiple logistic regression model. 622 patients (total = 3914) were classified as having a DS. Logistic regression analysis revealed that the risk of having a DS was increased with a longer duration of illness (OR =1.68) and was reduced for female gender (OR = 0.56). The findings are in line with the hypothesis that apart from a neurodevelopmental origin, the schizophrenic illness may also have a progressive neurodegenerative component, which clinically emerges as DS.

Adult↗
PubMed2001

Dehydroepiandrosterone, ageing and immune activation.

The age-related decline in dehydroepiandrosterone (DHEA) production is currently attracting attention because of its possible relevance to the etiology and management of a number of age-related clinical disorders. Various abnormalities of immune system function have been described in the elderly. Among them, increased concentrations of neopterin have been reported, which is produced by human monocytes/macrophages upon stimulation by interferon-gamma. In order to examine the relation of serum DHEA to serum neopterin, we studied 281 otherwise healthy outpatients, who visited the physician's office for a medical health check-up. 10% presented with increased neopterin concentrations, 0.4% had increased DHEA sulfate (DHEAs) concentrations. DHEAs concentrations were significantly higher in patients with lower neopterin concentrations (Mann-Whitney test: U=4793, P<0.0001). There existed a rather strong inverse correlation between DHEAs concentrations and serum neopterin concentrations (Spearman's rank correlation: r(s)=-0.221, P<0.0001). The data support the concept that the decrease of DHEA with increasing age is related to immune system activation. Oxidative stress which accompanies immune response may diminish DHEA synthesis.

Adolescent↗
PubMed2001

Effects of histamine-1 receptor antagonism on leukocyte-independent plasma extravasation during endotoxemia.

PURPOSE: The purpose of this study was to investigate the role of histamine in mediating leukocyte-independent microvascular permeability and mast cell activation during endotoxemia. Microvascular permeability and mast cell activity were determined after inhibition of the L-selectin mediated leukocyte-adherence by fucoidin and after inhibition of histamine effects by the histamine H1-receptor antagonist diphenhydramine. MATERIALS AND METHODS: In male Wistar rats, leukocyte rolling, leukocyte adherence, microvascular permeability, and mast cell activity were determined in mesenteric postcapillary venules using intravital microscopy. After pretreatment with the histamine H1-receptor antagonist diphenhydramine, animals in the ETX/H1-ANT group received a continuous infusion of endotoxin. Animals in the ETX group underwent the same procedure, but received saline 0.9% instead of diphenhydramine. In both groups, leukocyte adherence was prevented by administration of fucoidin. Animals in the control group received volume-equivalent saline 0.9%. RESULTS: In the endotoxin-challenged groups, fucoidin prevented leukocyte rolling and reduced leukocyte adherence to values comparable to control group. In the ETX group and the ETX/H1-ANT group both microvascular permeability and mast cell activity increased significantly, starting at 60 minutes. Differences in mast cell activity between the ETX group and the ETX/H1-ANT group were significant at 60 minutes and at 120 minutes. Differences in microvascular permeability between the ETX/H1-ANT group and the ETX group were not significant. CONCLUSIONS: The leukocyte-independent microvascular damage during early endotoxemia cannot be inhibited efficiently by the H1-receptor antagonist diphenhydramine, indicating that histamine seems to play only a minor role in that pathophysiology. Furthermore, mast cells do not seem to be involved in the development of leukocyte-independent plasma extravasation during endotoxemia.

Animals↗
PubMed2000

[Typical anticipation in type 7 spinocerebellar ataxia].

Spinocerebellar ataxia type 7 (SCA7) belongs to the category of autosomal dominant cerebellar ataxias (ADCA). The clinical picture is characterised by progressive ataxia and macular degeneration. Other common signs are slow saccades, external ophthalmoplegia, and pyramidal tract signs. The disease is caused by the expansion of an unstable CAG trinucleotide repeat in the gene for ataxin 7 on chromosome 3. SCA7 is a rare disorder. The first case in Germany was described only recently. We report two additional patients, father and son, with the molecular genetic diagnosis of SCA7. The father carries a trinucleotide expansion of 42 CAG repeats, the son 51. Normal alleles range from 7 to 35 CAG repeats. Both patients show the typical picture with progressive ataxia and macular degeneration. We found a pronounced anticipation (earlier disease onset in subsequent generations), which is highly characteristic of CAG repeat disorders.

Adult↗
PubMed2000

Suicidality in bipolar compared to unipolar depressed inpatients.

The aim of the present analyses was to evaluate differences in suicidality (past suicide attempts, suicidal thoughts at time of admission and completed suicides during the hospital stay) between bipolar and unipolar depressed inpatients. Apart from a higher frequency of past suicide attempts in bipolar depressed patients (26.6% in bipolar vs. 17.8% in unipolar patients), findings do not indicate any further differences in suicidality (suicidal thoughts (about 40% in both groups) and completed suicides during the hospital stay (0.8% in both groups)) between bipolar and unipolar patients. Factors with a predictive value for suicidal thoughts at the time of admission were a positive family history for affective disorders, past suicide attempts, and the depressive and paranoid hallucinatory syndrome (all associated with an increased risk). Female gender, an older age at hospitalisation and a longer duration of the illness were found to be associated with a lower probability for having suicidal tendencies at the time of admission. The risk for committing suicide during the hospital stay was increased if the patients had a history of past suicide attempts and suicidal thoughts at the time of admission. A more pronounced depressive syndrome at time of admission was slightly associated with a lower risk of committing suicide.

Bipolar Disorder↗
PubMed2000

Methodologies for evaluating electromyographic field data in ergonomics.

Surface electromyography (SEMG) is an important tool for work load assessment in ergonomics. Several different approaches using amplitude as well as frequency parameters give fruitful information depending on question at issue in the laboratory as well as in field studies. One basic factor determining the choice of analysis method is whether the SEMG is interpreted as an indicator of forces/torques or pure muscular activation. Two methods for occupational SEMG data reduction representing two different approaches to SEMG applications in ergonomics, Exposure Variation Analysis (EVA), and Joint Analysis of EMG Spectrum and Amplitude (JASA), applied on the same SEMG recording from three muscles during urology surgeon work, have been compared. The EVA method categorised the three muscle recordings as too static with no EMG gaps while the JASA method identifies fatigue in two of the three recordings. The practical relevance of these findings is discussed.

Electromyography↗
PubMed2000

[3D-spectrogram of the doppler signal in real time].

AIM: Doppler signals are normally displayed as an x/y-graph. The frequency is plotted in the y-direction, and the intensity is represented by the brightness. This represents less information than is contained in the acoustic signal: often one can hear more one can see. We are investigating the question as to whether it is feasible to render the complete information visible without the use of expensive additional equipment. METHOD: The sound card (SB16 P&P value edition) in a Pentium-PC (200 MHz 32 MB RAM) was used as a 16 bit dual channel a/d converter and connected to the ultrasound system Sonoline Elegra (Siemens, Erlangen, Germany). We used a standard 32 bit dual channel real-time FFT-software (SpectraPro, Sound Technology, California) to display a 3D-spectrogram on the PC-screen in real-time mode. This system was first tested on a flow model both with laminar steady flow and with pulsatile flow. Following this, measurements were made of the arteries and veins in healthy volunteers. RESULTS: In the 3D-Spectrogram the character of the flow can easily be seen. The quantitative parameters of velocity (Doppler frequency), direction and intensity of flow can be readily determined. Additional information about quality of flow, erythrocyte clusters and changes occurring in areas of slight stenoses can be used for diagnostic evaluation. The orthograde and retrograde flow are displayed separately using the dual channel technique. The arterial and venous flow in small and closely neighbouring vessels, such as the central retinal artery and vein, can easily be differentiated. The venous flow in particular is more turbulent in the 3D-spectrogram than would be expected from the normal display mode. CONCLUSION: The 3D-spectrogram provides much additional information in comparison with the normal Doppler spectrogram of the ultrasound system. This 3D-spectrogram can be easily obtained using inexpensive standard hard- and software.

Arteries↗
PubMed2000

Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment.

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of V(L), which contains two cis-prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V(L) and V(H). Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.

Deuterium↗
PubMed1999

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria.

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.

Dose-Response Relationship, Drug↗
PubMed1999

Localization of 5-oxo-L-prolinase mRNA in the murine choroid plexus by in situ hybridization.

The gamma-glutamyl cycle is thought to play a significant role in the transport of amino acids between the blood and the cerebrospinal fluid. In the present study we investigated the expression of 5-oxo-L-prolinase, an enzyme of this pathway, in mouse brain and kidney by in situ hybridization using digoxygenin-labeled RNA probes. Expression of 5-OPase mRNA was found in epithelial cells of the proximal tubules in kidney as well as in the epithelium and vascular endothelial cells of the choroid plexus but not in periventricular capillaries.

Amidohydrolases↗
PubMed1999

Immunohistochemical localization of 5-oxo-L-prolinase, an enzyme of the gamma-glutamyl cycle, in porcine brain microvessels.

The immunohistochemical analysis of the distribution of 5-oxo-L-prolinase in porcine brain at the light microscopic level was performed with an antibody raised against the enzyme purified from pig kidney. The present study reveals the specific expression of 5-oxo-L-prolinase in brain capillaries with an average diameter of 4.1+/-0.9 microm, while larger blood vessels remain unstained. Porcine kidney and skeletal muscle show no endothelial-specific staining with the antibody. In some cases, the asymmetrical staining pattern in cross and longitudinal sections of brain microvessels indicate endothelial- but also pericyte-specific expression.

Animals↗
PubMed1999

Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen.

The folding and assembly of the Fv fragment of the phosphorylcholine binding antibody McPC603, a non-covalent heterodimer of the variable domains VH and VL, was investigated. Since both domains, each engineered for stability and folding efficiency, could now be obtained in native and soluble form by themselves, fluorescence spectra of VH and VL in unfolded, folded and associated states can be reported. VH and VL only associate when they are native, and the stability of the heterodimer is strongly increased in the presence of antigen. VH rapidly folds into an hyperfluorescent intermediate, and the native state is reached in two parallel, proline-independent reactions. VL displays two fast refolding reactions, which are followed by two slower phases, limited by proline cis/trans-isomerization. The rate-limiting step for both the Fv and the scFv (single-chain Fv) fragment is the formation of the native VH-VL interface, which depends on ProL95 being in cis. The folding of the Fv fragment is fast after short-term denaturation or in the presence of proline cis/trans-isomerase catalysis, but the scFv fragment falls into a kinetic trap, observed by the persistence of the slow phases under all conditions. Furthermore, the scFv fragment, but not the Fv fragment, gives rise to premature interface formation, indicated by the fluorescence spectra and a much higher transient binding of 8-anilino-1-naphthalene sulfonate. The analysis of the folding pathway of the domains VH and VL in isolation and in non-covalent and covalent assemblies should provide helpful insights into the folding of multimeric proteins in general, and for the further engineering of stable and well-folding antibody fragments in particular.

Anilino Naphthalenesulfonates↗
PubMed1999

Kinetic parameters and tissue distribution of 5-oxo-L-prolinase determined by a fluorimetric assay.

5-Oxo-L-prolinase (5-OPase) catalyses the hydrolysis of 5-oxo-L-proline to glutamate with concomitant stoichiometric cleavage of ATP to ADP, a reaction which is known to be part of the gamma-glutamyl cycle-an interrelated series of reactions involved in the synthesis and metabolism of glutathione. As recent studies indicate, this cyclic pathway plays a crucial role in the regulation of amino acid transport. Apparently, the intermediate product 5-oxo-L-proline functions as a second messenger molecule that upregulates the activity of certain amino acid transport systems. Thus, the degradation of 5-oxo-L-proline by 5-OPase leads to the downregulation of this stimulus. In this study, a new sensitive fluorimetric assay for 5-OPase activity was established which is based on the derivatization of glutamate with o-phthaldialdehyde in the presence of thiols and subsequent separation of the products by HPLC. The method is suitable for the screening of chromatography fractions as well as for the determination of the kinetic parameters Km and Vmax of purified 5-OPase. Additionally, it can be used for the measurement of enzyme activity in crude cell extracts and evaluation of tissue distribution.

Amidohydrolases↗
PubMed1999

Serum levels of neuron-specific enolase and s-100 protein after single tonic-clonic seizures.

Serum neuron-specific enolase (s-NSE) and s-100 protein (s-100) are sensitive markers of various brain diseases. We investigated both of these markers in nine patients within 5 min, 6 h, 12 h, and 48 h after a single tonic-clonic seizure. The mean peak s-NSE level was significantly higher after 5 min (11.97 +/- 8.56 microg/l) and 48 h (10.31 +/- 8.92 microg/l, P < 0.05) than the levels of seizure-free, age-matched controls. Five patients had increased s-NSE levels regarding the upper limit of normal as mean + 3 SD. s-100 was not detected either in controls or epileptic patients. These data indicate that s-NSE in contrast to s-100 may be an in vivo marker after generalized seizures in some patients.

Adult↗
PubMed1999

A case of severe chronic active infection with Epstein-Barr virus: immunologic deficiencies associated with a lytic virus strain.

Infectious mononucleosis (IM) is a self-limiting, lymphoproliferative disease induced by primary infection with the Epstein-Barr virus (EBV). Infection with EBV leads in general to lifelong asymptomatic persistence of the virus. We report the case of a woman who acquired IM at the age of 15 years and then suffered from recurrent high fever, fatigue, and signs of immunologic disorder for more than 12 years until she died of liver failure. In an attempt to describe and to define the course of chronic active infection with EBV, we performed immunologic and molecular assays that demonstrated lytic replication of EBV in the B and T cells of the peripheral blood. In addition to signs of humoral and cellular immune deficiency, we detected an EBV strain with an impaired capability to immortalize B cells and a tendency to lytic replication, thus contributing to the pathogenesis of this chronic active infection.

Adult↗