Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

Effects of isolated and combined exposures to whole-body vibration and noise on auditory-event related brain potentials and psychophysical assessment

Summary Auditory event-related brain potentials (ERP) in response to two different tone stimuli (1.1 kHz or 1 kHz, 80 dB, 50 ms; given by headphones at a regular interstimulus interval of 5 s with a probability distribution of 70:30) were recorded from 12 healthy male subjects (Ss) during four different conditions with two repetitions: A - 60 dBA white noise (wN), no wholebody vibration (WBV); B - 60 dBA wN plus sinusoidal WBV in the a_z-direction with a frequency of 2.01 Hz and acceleration of 2 m ·s^–2 root mean square; C - 80 dBA wN, no WBV; D - 80 dBA wN plus WBV. Each condition consisted of two runs of about 11 min interrupted by a break of 4 min. During the break with continuing exposure, but without auditory stimuli, Ss judged the difficulty of the tone-detection task and intensity of noise by means of cross-modality matching (CMM). Vibration-synchronous activity in the electrocardiogram was eliminated by a subtraction-technique. Noise caused an attenuation of the N1 and P2 amplitudes and prolongation of P3 latencies. The WBV did not cause systematic ERP effects. Condition B was associated with higher N1 and smaller P3 amplitudes. The factor condition had a significant effect on the peak latencies of P3 to target stimuli and the task difficulty judged by CMM. Both effects exhibited significant linear increases in the sequence of conditions A, B, C, D. For the evaluation of exposure conditions at work, it can be suggested that noise has a strong systematic effect which can be enhanced by WBV. The P3 latency is considered as an advantageous measure for the detection of objective effects of physical environmental factors, correlating with relevant subjective responses.     

Histomorphometry of bone marrow biopsies in primary osteomyelofibrosis/-sclerosis (agnogenic myeloid metaplasia) - correlations between clinical and morphological features

Summary Histomorphometry was performed on representative trephine biopsies of the bone marrow on admission of 50 patients (21 male, 29 female-age 67 years) with so-called primary osteomyelofibrosis/-sclerosis (OMF) not preceded by any other subtype of chronic myeloproliferative disorders. This study was firstly aimed at testing correlations between histological features (amount of haematopoiesis, cytological aspects of mega-karyocytes, density of reticulin and collagen fibres and degree of osteosclerosis) and laboratory data, as well as spleen size and duration of relevant prediagnostic symptoms. Secondly, we concentrated on a discrimination of OMF patients into two sub-groups according to bone marrow morphology and clinical variables. Statistical evaluation of histomorphometric variables and haematological findings disclosed that there was a progressive fibro-osteosclerotic process in the evolution of disease features. Increase in medullary fibrosis was significantly paralleled by an abnormal or pleomorphic megakaryopoiesis in the bone marrow: there was an increase in irregularity of perimeters for megakaryocytes and naked nuclei combined with smaller sizes of these elements including the nuclei. Additionally, there was a greater number of pycnotic bare nuclei. A number of morphometric features (density of fibres, degree of osteosclerosis, amount of haematopoiesis) were associated with corresponding clinical data (spleen size, length of preclinical history). By consideration of a set of basic histomorphometric variables our co-hort of 50 patients could be divided into an early hyperplastic subtype with no or minimal medullary reticulin and another group with conspicuous fibrotic and osteosclerotic alterations of the bone marrow. It was noticeable that we found no significant correlation between amount of haematopoiesis or marrow cellularity with splenomegaly. This result suggests that splenic haematopoiesis (myeloid metaplasia) may represent an autonomous or neoplastic process and not only compensation for a failing fibro-osteosclerotic bone marrow.Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-1)     

Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase)

Microtubules have been isolated from immature (3-4 weeks' old) and old (11-13 years' old) bovine brains. Quantitative studies revealed that the concentration of extractable microtubule protein per gram of wet brain decreased from 0.47 mg (immature animals) to 0.34 mg (old animals). The major components of microtubule protein (tubulin and high-molecular-weight microtubule-associated proteins) do not undergo an age-correlated change. Determination of the endogenous protein kinase activity revealed that the activity associated with "immature" calf brain microtubules was six times higher than the activity present in "old" preparations. In contrast, the stimulatory effect of cyclic AMP on protein phosphorylation in microtubules from old bovine brains exceeds nine-fold the value obtained from immature animals. After addition of casein (exogenous acceptor), the basal activities increased in both preparations without altering the age-correlated difference in the specific activity. By comparing the radioactivity pattern of sodium dodecyl sulfate polyacrylamide gels after autophosphorylation of microtubule protein with [gamma-32P]ATP, 1.5 moles of phosphate per mole of high-molecular-weight microtubule-associated protein were estimated to be incorporated in preparations from immature animals and 0.9 mole of phosphate per mole of associated protein in the experiments with "old" microtubule protein. Adenosine triphosphatase activity, associated with the high-molecular-weight microtubule-associated protein 1, was determined to be 15% reduced in preparations from old animals, compared to the activity in "young" preparations. In contrast, the guanosine triphosphatase activity increased five-fold during ageing; the higher activity of this enzyme was observed both during the initial and the steady-state phases of microtubule formation.     

Failure of BCL-2 up-regulation in proximal tubular epithelial cells of donor kidney biopsy specimens is associated with apoptosis and delayed graft function

SUMMARY: In renal transplantation, postischemic acute renal failure (ARF) develops in more than 20% of patients. We investigated whether tubular epithelial cells obtained from donor kidneys without subsequent ARF express a different pattern of survival genes, compared with cells from kidneys exhibiting ARF. Donor kidney biopsy specimens were obtained before transplantation from eight recipients of cadaveric kidneys with primary graft function (CAD-PF), eight patients with biopsy-proven ARF without rejection (CAD-ARF), and eight recipients of living donor kidneys with primary graft function (LIV). One thousand proximal tubular epithelial cells per biopsy specimen were isolated by laser capture microdissection. Quantitative analysis of apoptosis and the apoptosis regulatory genes Bcl-2, Bcl-xL, and Bax were performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling staining and real-time PCR, respectively. Primary cultures of human proximal tubular epithelial cells served as calibrator. The number of apoptotic cells was significantly higher in CAD-ARF compared with LIV and CAD-PF (1.5 +/- 1.1% [p < 0.05] vs. 0.3 +/- 0.2% vs. 0.4 +/- 0.2%; mean +/- SD). The apoptosis inhibitors Bcl-2 and Bcl-xL were significantly up-regulated in renal tubular cells of recipients without ARF compared with CAD-ARF. The ratios of Bcl-2/GAPDH normalized to calibrator were as follows: LIV 48 +/- 30, CAD-PF 38 +/- 55, and CAD-ARF 5 +/- 7 (p < 0.05). The corresponding ratios for Bcl-xL were as follows: LIV 6 +/- 6, CAD-PF 5 +/- 3, and CAD-ARF 1 +/- 1 (p < 0.05). No difference in the expression of the proapoptotic Bax could be observed. These data suggest that failure of proximal tubular cells to respond to injury by up-regulation of survival factors from the Bcl-2 family contributes to postischemic ARF in patients after cadaveric renal transplantation.     

Zur Katalyse der stereospezifischen Butadienpolymerisation mit den Katalysatorsystemen aus Nd(η3-C3H5)3 · dioxan und methylaluminoxan (MAO) sowie Hexaisobutylaluminoxan (HIBAO)

The activation of the tris(allyl)neodymium complex Nd(η³-C₃H₅)₃ · dioxane with alkylaluminoxanes (MAO or HIBAO) results in highly selective catalysts for the 1,4-cis-polymerization of butadiene (cis-selectivity up to 80%). Under standard conditions (50°C, toluene), the turnover frequency (TOF) of the catalyst/MAO system amounts to 10–15000 mol butadiene/(mol Nd · h). Molecular weight determinations indicate the formation of only one polymer chain per neodymium center as in a living polymerization reaction, and for the catalyst/HIBAO system the rate law r_p = k_p [Nd][C₄H₆] with k_p = 8,7 · 10^?2 mol/(L · s) (at 25°C) has been derived. As the catalytically active species, a cationic monobutenyl neodymium(III) complex is discussed, which is stabilized through coordinative interaction with the counter anion as well as the growing polybutadiene chain. This cationic complex reacts under insertion with butadiene in a bimolecular fashion.     

Irreversible shock of rats after acute renal vein thrombosis

Summary After occlusion of the renal veins rats die quickly in progressive shock (within 4.5 h), but after ligating the renal hilum of both Kidneys they survive 27 h. To learn why renal vein occlusion is so rapidly lethal, and what substances are given off and by what method from the hemorrhagically infarcted kidneys, we studied eight groups of rats, each containing at least seven animals. The groups differed in the combination of hilar structures (renal veins, ureters, lymphatics) ligated. We compared: survival times, changes in blood pressure, blood volume, levels of plasma kinins, adenosine, and lactate, changes of blood pH, responses to Indomethacin, Trasylol^®, and plasma expanders, tubular and capillary flow rates, histopathological changes in organs and cerebral blood flow and changes in the blood coagulation system. Our results suggest that the venous stasis, anoxia, and hemorrhagic necrosis caused by bilateral venous occlusion release into renal lymphatics toxic substances which reach the systemic circulation and induce irreversible shock. We have excluded prostaglandins and adenosine as the toxic substances inducing shock but could not rule out an action of the kallikrein-kinin-system. We postulate that the striking degenerative changes occurring in the arterioles of the brain after bilateral venous occlusion may mean these vessels are especially susceptible to high levels of lactic acid and that this may explain why these animals die so quickly. Our conclusions should help not only in understanding why high levels of lactate in shock portend a poor prognosis but also help in formulating appropriate therapy for circulatory failure of renal origin and for protracted hypotension after extensive tissue injury.The studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres SystemPresented in part: Jäckh and Steinhausen, 1976; Dallenbach et al., 1978; Zimmerhackl et al., 1979We dedicate this paper to Wilhelm Doerr, Dr. med., Professor of Pathology, University of Heidelberg on the occasion of his 65th birthday (August 25th, 1979)