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Biomedical subjects

P Friedrich

Publications and source records attributed to P Friedrich.

At least 37 records · Page 2Linked to original sources
PubMed1996

The calpain cascade. Mu-calpain activates m-calpain.

m-Calpain, which usually requires near-millimolar Ca2+ for activation, undergoes autolysis at 25 microM Ca2+ in the presence of mu-calpain. m-Calpain in itself exhibits no sign of autolysis around this Ca2+ concentration. Half-maximal rate of the reaction occurs at 30 microM Ca2+, showing that it is mu-calpain that catalyzes the limited proteolysis of m-calpain in an intermolecular reaction ("heterolysis"). This heterolytic step is accompanied by the activation of m-calpain: mu- and m-calpain preincubated together at 25 microM Ca2+ show significantly higher activity than the sum of activities of mu- and m-calpains preincubated separately. m-Calpain is sensitized to Ca2+ by mu-calpain-mediated activation: the half-maximal value of 160 microM for activation is lowered to 64 microM, which is similar to the shift found in m-calpain autoactivation. We suggest that these in vitro observations are relevant in vivo and the calpain cascade may play an important role in coordinating the functioning of calpains in living cells.

Animals↗
PubMed1996

Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2.

The C-terminal domain of microtubule-associated protein 2 (MAP2) contains a proline-rich region and the tubulin-binding domain. We have generated antibodies to follow the phosphorylation state of the proline-rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline-rich region of MAP2 and is phosphorylated in vitro by at least three different proline-directed protein kinases: p42mpk, p34cdc2, and GSK3 (glycogen-synthase kinase 3) alpha/beta. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high-molecular-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.

Animals↗
PubMed1996

Autolysis parallels activation of mu-calpain.

The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of mu-calpain large subunit at both 10 and 300 microM Ca2+, and both straight lines intersected the origin. This finding supports the view that native mu-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, K1(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 microM Ca2+), whereas the maximal value was 1.52 s-1, at 30 degrees C. The Ca(2+)-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, k1(act), was derived as the reciprocal of the lag phase ('transit time') at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 microM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of mu-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of mu-calpain. Pre-activated mu-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 microM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the mu-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.

Autolysis↗
PubMed1996

[Roxatidine acetate in therapy of reflux esophagitis].

A total of 3409 patients with gastro-oesophageal reflux disease were treated with roxatidine acetate. 60.7% of the patients received a daily dose of 2 x 75 mg roxatidine acetate, and the median duration of treatment was 5 weeks. Symptoms improved in about 90% of patients. For 1687 patients, endoscopic findings were available at the beginning and end of the treatment period. The overall endoscopic healing rate was 65.3%, and, depending on the initial finding (if), decreased from 92.9% (if: Savary-Miller stage I) to 67.5% (if: stage II), 40.7% (if: stage III), and to 22.5% (if: stage IV). Twenty-one patients experienced adverse events during the course of treatment, which, however, were either only minor or not related to the use of roxatidine acetate.

Anti-Ulcer Agents↗
PubMed1996

Mutual protection of microtubule-associated protein 2 (MAP2) and cyclic AMP-dependent protein kinase II against mu-calpain.

Phosphorylation by adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), but not by Ca(++)-calmodulin-dependent protein kinase II (CaMK II), was shown earlier to protect microtubule-associated protein 2 (MAP2) from cleavage by m-calpain (Johnson and Foley: J Neurosci Res 34: 642-647, 1993). We reinvestigated this phenomenon with the physiologically more relevant mu-calpain and found a qualitatively similar but quantitatively different picture. We further demonstrate that 1) protection is biphasically dependent on the degree of phosphorylation; 2) Ca(++)-phospholipid-dependent protein kinase (PKC) has about the same effect as PKA; 3) the effects of kinases A and C are not additive; and 4) stripping of native MAP2 from its phosphate content (17.8 +/- 2.3 mol/mol) enhances calpainolysis 3.6-fold. A reciprocal effect between kinase A and MAP2 was found: the RII, but not the RI, regulatory subunit of kinase A, which was shown to bind specifically to MAP2, is protected by MAP2 from mu-calpain attack. It is suggested that the specific anchoring of kinase A-II on MAP2 may serve not only kinase targeting in the dendrites, but also protection from calpainolytic degradation.

Animals↗
PubMed1995

An ultrasensitive, continuous fluorometric assay for calpain activity.

A rapid, continuous assay for calcium-activated neutral protease activity is described. This assay is based on monitoring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein-labeled microtubule-associated protein 2. Tedious separation of peptide products from the protein substrate in this rapid assay is unnecessary, which thus offers two remarkable advantages over conventional caseinolytic assay procedures: (i) it raises sensitivity of detection by about three orders of magnitude, allowing the quantitative determination of calpain in the high picogram range in 10 min; and (ii) it permits a continuous detection of activity, which may prove invaluable in enzyme-mechanism studies that require pre-steady-state measurements. Other features and advantages of the assay, along with its limitations, are discussed in detail.

Animals↗
PubMed1995

CalpA, a Drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells.

Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.

Amino Acid Sequence↗
PubMed1995

Occlusive thrombus formation on indwelling catheters: in vitro investigation and computational analysis.

To determine the hydrodynamic parameters involved in thrombus formation on indwelling catheters, fibrin coagulation was investigated in an in vitro model and compared with the prevailing flow field. Computational analysis was used to simulate clot growth and identify the variables which determine its localization. The fibrin clot grew from the distal end of the catheter along the interface between the bulk stream and the annular vortex located immediately downstream of the catheter tip, finally filling the entire vortex. The calculations of flow parameters for three different growth stages showed that regions of clot formation always correlated with regions exhibiting the longest residence time. Fibrin clotting could be simulated by a mathematical model using residence time as the only variable parameter. Our results demonstrate that the residence time is the single most important flow factor for fibrin coagulation and thus probably for thrombus formation. When the residence time equals the clotting time for the fluid, thrombus formation is initiated. Conversely, it ceases when the area of long fluid residence times is replaced by a fibrin clot. Hence the fibrin clot represents a hydrodynamic optimization of the previously disturbed flow.

Animals↗
PubMed1994

Flow mediated fibrin thrombus formation in an endothelium-lined model of arterial branching.

In vivo arterial thrombosis occurs preferentially at curvatures and branchings, i.e. regions of flow separation and recirculation where blood is retained orders of magnitude longer than within straight vessel sections. To examine the effect of such disturbed flow on endothelial thromboresistance glass T-branchings lined with endothelial cells from human umbilical cord veins (HUVEC) were perfused with buffered fibrinogen solution (3mg/ml). The flow was adjusted to form a large recirculation zone and flow conditions were determined beforehand by means of flow visualization via dye injection as well as by laser ultramicroscope anemometry. Thrombus formation, which was registered on-line by video and evaluated planimetrically, was induced by injection of thrombin at concentrations ranging from 0.3 to 2.0 units/ml. Fibrin thrombus growth always began within the flow niche at the point of flow separation and extended downstream along the wall and into the vessel lumen finally occluding up to 80% of the lumen. Light and electron microscopy revealed that overall thrombus form as well as the orientation of single fibrin fibers were correlated strictly to the prevailing streamlines. Despite the integrity of the endothelial lining fibrin thrombus formation occurred. The fibrin fibers closely contacted the endothelial surface. These results indicate that recirculation zones promote fibrin thrombus formation sufficient to obstruct the vessel lumen and that intact endothelium alone is insufficient in preventing adhesion of fibrin to its surface.

Arteries↗
PubMed1994

Calpeptin, a calpain inhibitor, promotes neurite elongation in differentiating PC12 cells.

Calpain activity of nerve growth factor (NGF)-induced rat pheochromocytoma (PC12) cells shows a transient diminution in the early phase of differentiation. Calpain activity can be further decreased by a cell-permeable calpain inhibitor, calpeptin, which enhances the effect of NGF by stimulating neurite elongation. The number of neurites sprouted by one cell is not increased by calpeptin. A possible role of calpain inhibition during PC12 cells' early differentiation is discussed.

Animals↗
PubMed1994

Dephosphorylation of tau protein from Alzheimer's disease patients.

Tau protein-prepared post-mortem from brains of Alzheimer's disease patients was treated with protein phosphatase 1 catalytic subunit, 2A catalytic subunit, and 2B (calcineurin). Dephosphorylation was monitored by immunoblotting with two monoclonal antibodies, TAU-1 and SMI31, which recognize in tau the dephospho- and phospho-states, respectively, of proline-directed protein kinase phosphorylation sites. Out of the three enzymes tested, protein phosphatase 2A was only effective in dephosphorylating tau at these Alzheimer-type epitopes.

Alzheimer Disease↗
PubMed1993

Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B.

Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAP1B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline-directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.

Animals↗
PubMed1993

Protein phosphatase 1-deficient mutant Drosophila is affected in habituation and associative learning.

The learning and memory of Drosophila melanogaster strains carrying the Su-var(3)6(01) mutation, which is known to affect the structural gene of a protein phosphatase 1 isoenzyme, PP1(87B), were studied in various behavioral paradigms. Three lines of Drosophila comprising the Su-var(3)6(01) mutation in different genetic backgrounds were shown to have diminished protein phosphatase 1 activity and behavioral anomalies. Associative olfactory learning and visual conditioning were impaired. Olfactory acuity for the odorants used and response to electric shock were largely unchanged in the mutant lines. The motility and flight activity of the mutants were reduced. Habituation of the landing response, a nonassociative learning process, was more pronounced in heterozygotes of the mutants than in the wild-type control strains. Taken together with earlier data, the results indicate that protein phosphatase PP1(87B), while affecting several cellular processes, is also part of the biochemical machinery of various forms of neuromodulation in Drosophila.

Animals↗
PubMed1992

Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster.

A Ca(2+)-activated thiol protease was purified from Drosophila melanogaster. The procedure involves Phenyl-Sepharose, Reactive Red-Agarose and Q-Sepharose fast flow (or MonoQ) chromatographic steps. The enzyme eluting from Q-Sepharose fast flow seems to be homogeneous as judged by silver staining on SDS-PAGE: it consists of a single polypeptide chain of M(r),app = 94K and pI = 5.46. The proteolytic activity of the purified enzyme is absolutely Ca(2+)-dependent, characterized by 0.6 mM free Ca2+ at half-maximal activity. Ca2+ ions cannot be replaced effectively by the divalent cations Mg2+, Mn2+, Zn2+, Ba2+, and Cd2+. The enzyme shows the inhibitor pattern of thiol proteases. Human recombinant calpastatin (domain I) completely inhibits the enzyme at a nearly 1:1 molar ratio. Several of these properties resemble those of vertebrate calpain II. However, various attempts to detect a small subunit of M(r) approximately 30K, common with vertebrate calpains, remained unsuccessful. We suggest that the Drosophila enzyme is a novel calpain II-like protease.

Animals↗
PubMed1992

Mouse microtubule-associated protein 2 expression in transgenic Drosophila.

Full-length mouse microtubule-associated protein 2 complementary DNA inserted between Drosophila heat-shock protein 70 promoter and trailer was introduced in the germ-line of Drosophila melanogaster by P element-mediated transformation. Three transformant lines contained microtubule-associated protein 2 complementary DNA in sense orientation and two in antisense orientation. All sense lines produced microtubule-associated protein 2 after heat-shock in several tissues at low level. In the adult brain, high-level microtubule associated protein 2 expression independent of heat induction occurred in specific neuron types in each sense transformant, suggesting the action of neuron-specific enhancer genes. High microtubule-associated protein 2 levels are expected to interfere with the cells' activity by forming unphysiological cross-links, which may help elucidate the function of the given neurons.

Animals↗
PubMed1991

MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture.

Microtubule-associated protein 2 (MAP2), a long, filamentous molecule thought to cross-link dendritic cytoskeleton, is rich in PEST sequences, putative signals for rapid proteolytic degradation. It is suggested that MAP2 is indeed highly susceptible to protease, e.g. calpain, attack, which is needed for a plastic change, but actual breakdown depends on the regulation of protease(s). Phosphorylation is expected to make the molecule longer and rigid, similarly to what was observed with the related tau protein. Such a structural transition may provide a mechanism for the putative role of MAP2 in dendritic branching.

Amino Acid Sequence↗
PubMed1991

Signal convergence on protein kinase A as a molecular correlate of learning.

The response of a reaction network composed of protein kinase A, calpain, and protein phosphatase to transient cAMP and Ca2+ signals was studied. An essential feature of signal convergence is that the regulatory subunit of cAMP-dissociated protein kinase A undergoes limited proteolysis by the Ca(2+)-activated proteinase calpain. A dynamic model of this system based on kinetic differential equations was built and simulated by computer. The system shows analogies to typical features of associative learning such as acquisition, contiguity detection, extinction, and memory decay, suggesting that these biochemical reactions may be part of the molecular mechanism of learning in Drosophila.

Calpain↗