Biomedical subjects

M Kellermayer

Publications and source records attributed to M Kellermayer.

At least 19 recordsLinked to original sources

PubMed2001

[Plasma electrolytes in multiple myeloma].

The plasma cell myeloma (multiple myeloma, myelomatosis) is a progressive disease, characterized by bone marrow plasmacytomas and the presence of monoclonal antibodies (IgG, IgA, IgD, IgE), or free kappa or lambda immunoglobulin side chains. The monoclonal antibodies or Bence-Jones protein may precipitate in the tubuli and impair kidney function. In addition, the plasma protein concentration may increase at the expense of plasma water level causing unrealistically low electrolyte levels. Since the isoelectric points of immunoglobulins are higher than those of most other plasma proteins, the net charge of plasma proteins may change causing new electrolyte balance. In addition, some monoclonal antibodies are more hydrated than others, and their high concentration may cause not only increased plasma viscosity but further electrolyte imbalance. In the present work the relationship between plasma protein and electrolyte levels is studied in samples of 100 multiple myeloma patients.

Blood Proteins↗

PubMed2000

Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain.

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.

Amino Acid Sequence↗

PubMed2000

Mechanical properties of titin isoforms.

Titin is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the sarcomere. As a result of differential splicing, length variants of titin are expressed in different skeletal and cardiac muscles. Here we first briefly review some of our previous work that has revealed that titin develops force in sarcomeres either stretched beyond their slack length (passive force) or shortened to below the slack length (restoring force) and that titin's force underlies a large fraction of the diastolic force of cardiac muscle. Next we present our mechanical and immunoelectron microscopical (IEM) studies of skeletal and cardiac muscles that express titin isoforms. The previously deduced molecular properties of titin were used to model titin's extensible region in the sarcomere as serially linked WLCs: rigid segments (containing folded Ig/Fn domains) and more flexible segments (PEVK segment). The model was tested on skeletal muscle fibers that express titin isoforms with tandem Ig and PEVK length variants. The model adequately predicts titin's behavior along a wide sarcomere length range in skeletal muscle, but at long sarcome lengths (SLs), predicted forces are much higher than those determined experimentally. IEM reveals that this may result from Ig domain unfolding. Experiments were also performed on cardiac myocytes from mouse and cow that express predominantly a small cardiac titin isoform (N2B titin) or a large isoform (N2BA titin), respectively. The passive tension-SL relation of myocytes was found to increase more steeply with SL in mouse than in cow. IEM revealed an additional source of extensibility within both of these cardiac titins: the unique N2B sequence (absent in skeletal muscle). Furthermore, the PEVK segment of the N2BA isoform extended to a maximal length of approximately 200 nm, as opposed to approximately 60 nm for the N2B isoform. We propose that, along the physiological SL range, the long PEVK segment found in N2BA titins results in a low PEVK fractional extension and that this underlies the lower passive tensions of N2BA-expressing cow myocytes.

Animals↗

PubMed1999

Mechanically driven contour-length adjustment in rat cardiac titin's unique N2B sequence: titin is an adjustable spring.

The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above approximately 2.2 microm SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable.

Animals↗

PubMed1998

Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments.

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.

Chemical Phenomena↗

PubMed1998

ATP and integrity of human red blood cells.

In spite of the well known significance of ATP in the energy dependent life processes, the role of ATP in maintaining cellular integrity is poorly understood. A possible model for studying ATP dependent life processes is to monitor the kinetics of changes seen intra/extracellularly during ATP depletion. In our model system anticoagulated human whole blood was incubated at different temperatures to reduce intracellular ATP without addition of any chemicals. The red blood cells in their own plasma were incubated for several days at 4 degrees C or at 37 degrees C, and ATP, glucose, K+, Na+, hemoglobin, water content, mean corpuscular volume (MCV), pH and Ca2+ were analyzed in time-sequences. All the examined parameters remained practically unchanged at 4 degrees C, while at 37 degrees C total ATP and glucose decreased parallel and after a transient increase of MCV, the water content of red blood cells decreased. As the actual ATP fell below 10% of the initial ATP content (at 48 h), the release of potassium sharply increased. Release of hemoglobin started only after 96 hours of incubation. Maximums of changes of the examined parameters were found at different time intervals. The maximal speed of concentration changes for glucose was found at 12-24 hours of incubation and at 24-36 hours for ATP, at 48-60 hours for K+(-)Na+ and after 96 hours for hemoglobin.

Adenosine Triphosphate↗

PubMed1997

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction.

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.

Amino Acid Sequence↗

PubMed1996

Maintenance of ions, proteins and water in lens fiber cells before and after treatment with non-ionic detergents.

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks' balanced salt solution in either absence or presence of non-ionic detergents (0.2% Triton X-100 or 0.2% Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K+, Mg2+, Na+, Ca2+, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 degree C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K+, Mg2+, water and cytomatrix proteins.

Adenosine Triphosphate↗

PubMed1996

Nuclear magnetic resonance relaxation parameters of muscle in malignant hyperthermia-susceptible swine.

RATIONALE AND OBJECTIVES: The malignant hyperthermia (MH) syndrome involves characteristic changes in metabolism that have a significant effect on the postmortem properties of muscle tissue. We investigated the 1H nuclear magnetic resonance (MR) relaxation parameters of muscle tissue from normal (MH-) and malignant hyperthermia-susceptible (MH+) swine taken before their death to identify whether postmortem metabolic differences would be accompanied by changes in relaxation parameters. METHODS: Muscle samples from 63 MH-susceptible swine were characterized by proton MR relaxation times (T1 and T2). The water content, pH, conducting, and the "subjective meat" quality were assessed in order to differentiate the meat into one of three groups: normal, pale-soft-exudative, and dark-firm-dry. RESULTS: The T2 relaxation of MH- and MH+ muscles was significantly different, but no significant changes were detected in the water content and corresponding T1 relaxation times. CONCLUSION: These data suggest that MH-susceptible swine can be distinguished from normal swine using nuclear MR relaxometry. The results of relaxation measurements and the multiexponential analysis of the T2 curves suggest a shift between the different water compartments during MH that might predict the increased water loss of muscle tissue after death.

Animals↗

PubMed1995

Effect of non-lytic concentrations of Brij series detergents on the metabolism-independent ion permeability properties of human erythrocytes.

Subcritical micellar concentrations (sub-CMC) of Brij-series detergents alter ion movements between human erythrocytes and their environment when metabolism has been slowed down by incubation at zero degrees centigrade. The effect of nonhemolytic concentrations of detergents on the erythrocyte K+ and Na+ movements is described. Results indicate a significant difference in monovalent cation movements, depending on the number of hydrophilic polyoxyethylene units (n). There is an increasing loss of K+ and gain of Na+ as n increases from 4 to 20. Where n > or = 21, ion movements are not significantly different from those found in erythrocytes not exposed to detergents. The carbon chain length of the detergent fatty acid residue (10-18 carbons) appears to be relatively unimportant, but detergents with unsaturated (oleic acid) hydrophobic regions potentiate K+ release and Na+ uptake when compared to the corresponding saturated fatty acid (stearic acid). The erythrocyte stabilizing effect of detergents against hypo-osmotic shock correlates well with the increase of monovalent ion traffic and the mobility of membrane lipids revealed by fluorescence anisotropy measurements.

Adenosine Triphosphate↗

PubMed1994

Release of potassium, lipids, and proteins from nonionic detergent treated chicken red blood cells.

The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K+ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K+, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X-100. The lipid and protein solubilizing actions of Triton X-100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic-lipophilic balance numbers (HLB values).

Animals↗

PubMed1994

Cation distribution in mammalian red blood cells: interspecies and intraspecies relationships between cellular ATP, potassium, sodium and magnesium concentrations.

The intracellular K+/Na+ ratio of erythrocytes of different mammalian species are known to differ markedly. These differences are sustained despite plasma potassium and sodium concentrations being almost identical, i.e. high [Na+] and low [K+], in all mammalian species. Conventional cell theory couples intracellular K+ and Na+ concentrations with Na+/K(+)-dependent ATPase activity. Ling's Association-Induction (AI) hypothesis couples intracellular K+ and Na+ concentrations with the concentration of ATP, by way of an inductive adsorption of both monovalent cations on the available terminal and lateral carboxyl groups of proteins. Based on the working hypothesis, different relationships are expected between the erythrocyte K+ and Na+ concentrations and ATP levels. Our results indicate that the intracellular ATP level in erythrocytes of different species corresponds with intracellular K+ and Na+ levels, K+/Na+ ratios and total K+ + Na+ levels. Similar relationships were revealed between the erythrocyte ATP and monovalent cation contents of different individual sheep.

Adenosine Triphosphate↗

PubMed1993

Relationship between cellular ATP, potassium, sodium and magnesium concentrations in mammalian and avian erythrocytes.

Intracellular K+/Na+ ratios of erythrocytes of various mammalian species are known to differ markedly. We have measured ATP, K+, Na+, Mg2+, H2O contents of erythrocytes of twelve mammalian and three avian species. Our results indicate that the intracellular ATP concentration in erythrocytes of different species is in close positive correlation with the K+/Na+ ratios (linear correlation coefficient, r = 0.852). Furthermore, ATP levels in erythrocytes of individual sheep with different potassium concentrations correspond with their K+/Na+ ratios (r = 0.747). Intracellular magnesium concentrations also correlate with ATP concentrations in erythrocytes of different species (r = 0.629) and in different sheep (r = 0.549).

Adenosine Triphosphate↗

PubMed1993

[Experimental study of postoperative scarring around the tendon sheath].

Authors have compared two kind of exposures of the flexor tendons. The results were assessed by measuring the flexion oft the given finger on pulling, by light and electron microscopic examination. It was found that in fingers where the tendon sheat was detached from the subcutis the scarring was greater and its effect, limiting the motion has its maximum on the fourth postoperative week. It is raised that the role of the tendon sheat in respect of the success of the flexor tendon reconstruction is mainly mechanic and most effective if its highly integrated connection with its surrounding is not disturbed.

Animals↗

PubMed1993

Transient cytoplasmic vacuolization in cultured normal and neoplastic cells treated with low molecular weight human serum ultrafiltrate: is out inside?

Human embryonal fibroblasts (HEF) and epithelial carcinoma cells (HEp-2) were exposed to low molecular weight (LMW) fractions (M(R) < 50-kDa) of human serum, generated by ultrafiltration. A prominent change in cytoplasmic morphology was detected by microscopic analysis of both cell types. Within 1-3 h of incubation an extensive vacuolization was induced, which phenotype was maintained for 6-12 h in the presence of the LMW filtrate in a cell type dependent manner, followed by a spontaneous reconstitution of the original cytoplasmic morphology by 24 h. Reversibility occurred by incubation in medium without filtered serum fractions. Transmission electron microscopy revealed vesicles without continuous bordering membrane or electron dense interna and demonstrated vacuoles in close contact with filamentous structures. Taken together the light- and electron microscopic characteristics of the observed vacuolization, potential interpretations of our results are discussed in the context of general cytoplasmic organization. Rearrangement of the cytoplasmic protein-water system as a consequence of an inductive effect of serum filtrates is proposed.

Blood↗

PubMed1992

Motional characteristics of K+ and Na+ in intact and sucrose-permeabilized rat lymphocytes.

Most, if not all, cells maintain an unequal distribution of Na+ and K+ against their environment. These two monovalent ions are in constant exchange between the cell and the extracellular space since both ions have proved to be permeable through the cell membrane. The distribution of Na+ and K+ in intact and "sucrose-permeabilized" rat lymphocytes were studied ("sucrose-permeabilization" means homogenization in isotonic sucrose solution). Both the intact and the permeabilized lymphocytes were incubated in Hanks' solution and then transferred into K+, Na(+)-free isotonic sucrose solution. Alternatively, the cells were incubated only in the sucrose solution or in Hanks' solution. The Na+ and K+ content of the cells were determined at the conclusion of each period of incubation in the same or different medium. We found that K+ did not equilibrate under any conditions in intact lymphocytes but Na+ responded to changes of the incubation media. In the permeabilized cells Na+ freely equilibrated with the extracellular medium while K+ did not, although its concentration decreased compared to that of intact cells.

Animals↗

PubMed1992

Human and dog erythrocytes: relationship between cellular ATP levels, ATP consumption and potassium concentrations.

The intracellular K+/Na+ ratio of various mammalian cell types are known to differ remarkably. Particularly noteworthy is the fact that erythrocytes of different mammalian species contain entirely different potassium and sodium concentrations. The human erythrocyte is an example of the supposedly "normal" high potassium cell, while the dog erythrocyte contains ten times more sodium than potassium ions (Table I). Furthermore, this difference is sustained despite the plasma sodium and potassium concentrations being almost identical in both species (high Na+ and low K+). In spite of these inorganic ion differences, both human and dog erythrocytes contain 33% dry material (mostly Hb) and 67% water. Conventional cell theory would couple cellular volume regulation with Na+ and K+ dependent ATPase activity which is believed to control intracellular Na+/K+ concentrations. Since the high Na+ and low K+ contents of dog erythrocytes are believed to be due to the lack of the postulated Na/K-ATPase enzyme, they must presumably have an alternative mechanism of volume regulation, otherwise current ideas of membrane ATPase activity coupled volume regulation need serious reconsideration. The object of our investigation was to explore the relationship between ATPase activity, ATP levels and the Na+/K+ concentrations in human and dog erythrocytes. Our results indicate that the intracellular ATP level in erythrocytes correspond with their K+, Na+ content. They are discussed in relation to conventional membrane transport theory and also to Ling's "association-induction hypothesis", the latter proving to be a more useful basis on which to interpret results.

Adenosine Triphosphate↗

PubMed1991

Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non-ionic detergent Triton X-100.

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.

Animals↗