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S Klumpp

Publications and source records attributed to S Klumpp.

At least 19 recordsLinked to original sources
PubMed2001

Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

Animals↗
PubMed2001

Random walks of cytoskeletal motors in open and closed compartments.

Random walks of molecular motors, which bind to and unbind from cytoskeletal filaments, are studied theoretically. The bound and unbound motors undergo directed and nondirected motion, respectively. Motors in open compartments exhibit anomalous drift velocities. Motors in closed compartments generate stationary nonequilibrium states with spatially varying densities of the motor concentrations and currents. "Traffic jams" on the filaments lead to a maximum of the motor current at an optimal motor concentration. Quantitative estimates based on experimental data for bound motors indicate that these transport phenomena are accessible to experiments.

Algorithms↗
PubMed2001

Noise-induced transport of two coupled particles.

We study the motion of two harmonically coupled particles in a sawtooth potential. The particles are subject to temporally correlated multiplicative noise. The stationary current is calculated in an expansion about the limit of rigid coupling. For two coupled particles a driving mechanism occurs which is different from the one occurring in the case of a single particle. In particular this mechanism does not need diffusion. Depending on the equilibrium distance of the particles and the coupling constant, a current reversal occurs. Possible relevance as a model for motor proteins is discussed.

Biological Transport↗
PubMed2001

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-kappaB.

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor-kappaB (NF-kappaB) and how this activation contributes to preconditioning-induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine-induced (200 nM, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF-kappaB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IkappaBalpha as well as by electrophoretic mobility shift assay. Preconditioning-mediated neuroprotection was abolished by antioxidants, inhibitors of NF-kappaB activation and cycloheximide suggesting the involvement of ROS, an activation of NF-kappaB and de novo protein synthesis in preconditioning-mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn-superoxide dismutase which could be blocked by antioxidants, cycloheximide and kappaB decoy DNA. Our data suggest that inhibition of staurosporine-induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF-kappaB and an increase in the protein level of Mn-superoxide dismutase.

Acetylcysteine↗
PubMed1998

Protein phosphatase type 2C active at physiological Mg2+: stimulation by unsaturated fatty acids.

Type 2C serine/threonine protein phosphatases (PP2C) so far require unphysiologically large amounts of Mg2+ ions for activity. Activators and inhibitors are not available, targeting subunits unknown. Studying the regulation of PP2C isozymes in bovine retinae, we found that the activity of PP2C increased specifically by the addition of mono- and polyunsaturated fatty acids. Activation was most pronounced at low Mg2+ levels (10-fold stimulation of PP2Calpha by 0.5 mM arachidonic acid at 0.7 mM Mg2+). Sensitivity of PP2Cbeta was 30-50% less, revealing for the first time enzymatic differences among the PP2C isozymes. Combining unsaturated fatty acids with physiological Mg2+ concentrations resulted in PP2C activity that by far exceeded the dephosphorylation rates obtained otherwise. This suggests that PP2C activity has been severely underestimated in the past. In the presence of fatty acids, Ca2+ ions became inhibitory in the micromolar range. We conclude that unsaturated fatty acids may play a role in the regulation of PP2C activity.

Animals↗
PubMed1998

Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization, and localization in photoreceptors.

Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular. This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade. Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11-cisretinal and by dephosphorylation of the opsin moiety. Phosphatases type-1 and type-2A have been identified in vertebrate retinae. Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type-2C (PP2C, PPM) do exist in retinal tissue. In this report, we have purified PP2Calpha and PP2Cbeta from bovine retinae. Thirty to 40% of PP2C was recovered in the cytosolic fraction. Biochemical properties of native and heterologously expressed recombinant enzymes were similar. Enzymatic activity is strictly dependent on the presence of Mg2+. Addition of Ca2+ ions inhibits Mg2+-sustained activity. Antiserum raised against a C-terminal peptide of PP2Cbeta specifically labeled the outer segments of rod photoreceptor cells. PP2C protein levels were significantly reduced in RCS rats, which develop age-dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa. Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery.

Amino Acid Sequence↗
PubMed1998

Serine/threonine protein phosphatases type 1, 2A and 2C in vertebrate retinae.

A number of retinal proteins are phosphorylated by a variety of kinases, resulting in well-known regulatory effects. The identity and role of corresponding phosphatases is less clear. We simultaneously measured the activity of serine/ threonine protein phosphatases type 1, 2A and 2C in bovine retinae. The enzymes were classified according to substrate specificity, divalent cation requirement and the effect of phosphatase subtype-specific inhibitors. The total- and specific activity of phosphatase type 2A was prevalent. Type 2C was 10-fold less abundant. 80% of type 1 and 50% of type 2A and type 2C, respectively, were soluble. An economic purification scheme was developed. We demonstrated the presence of phosphatase isozymes 2Calpha and 2Cbeta in bovine rod outer segments by enzymatic analysis as well as immunological techniques. The results suggest a yet unknown role of phosphatase type 2C in phototransduction. On the other hand, the immense amount of protein phosphatases found to be soluble - therefore not associated with rod outer segment membranes - points towards participation of these enzymes in the process of visual transduction not considered thus far.

Animals↗
PubMed1996

Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide.

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 microM.

Amino Acid Sequence↗
PubMed1996

Cloning and expression of a bovine adenylyl cyclase type VII specific to the retinal pigment epithelium.

A cDNA of a type 7 adenylyl cyclase isoform was cloned from a bovine retinal pigment epithelium cDNA library using oligonucleotides developed to conserved regions common to mammalian adenylyl cyclases. A 6.7 kb mRNA of very high abundance was uniquely present on Northern blots containing mRNA or total RNA from the pigment epithelium. This transcript was undetectable in all other tissues examined. The cDNA encoded a protein of 1,097 amino acids and exhibited the known doublet of 6 transmembrane-spanning regions in a hydrophobicity plot. The novel member of the type 7 adenylyl cyclase isoform was expressed in COS-1 cells. It was stimulated 10- and 20-fold by 10 microM GTP gamma S and 100 microM forskolin, respectively. The high expression rate exclusively in the retinal pigment epithelium suggests that this adenylyl cyclase isoform is involved in processes specific to this functionally exceedingly important subretinal cell layer.

Adenylyl Cyclases↗
PubMed1996

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.

Amino Acid Sequence↗
PubMed1994

A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. Purification, characterization, and cloning.

We isolated the first membrane-bound type 2C serine/threonine protein phosphatase from the ciliated protozoan Paramecium tetraurelia (PtPP2C). Three isozymes of 33, 32, and 31 kDa with a specific activity of 1 mumol.min-1.mg1 were purified from the ciliary membrane. All enzymatic properties including (a) insensitivity toward inhibitors of other protein phosphatase families such as okadaic acid and microcystin, (b) absolute requirement for divalent cations, and (c) substrate specificity tested with synthetic phosphopeptides were identical to mammalian PP2C enzymes and identified the PtPP2C as a canonical PP2C in spite of it being about 25% smaller. The NH2-terminal was blocked. Microsequencing of six tryptic peptides established a relationship to other PP2C enzymes. The PtPP2C gene was obtained using degenerate oligonucleotide primers and the polymerase chain reaction. The gene coded for a 33-kDa protein with 300 amino acids and had an (A+T) content of 62%, typical for this protozoan. Nine of 15 Gln residues are encoded by TAA, a universal stop codon which codes for Gln in Paramecium. A large truncation at the COOH-terminal is responsible for the smaller size of the PtPP2C. Only a single transcript of 1 kilobase was detected with a Northern blot indicating that the 32- and 31-kDa proteins were proteolytic products of the 33-kDa enzyme. Sequence comparisons with PP2C enzymes from rat, rabbit, yeast, Arabidopsis, and Leishmania defined a highly diverged enzyme family which shares three conserved domains, I, II, and III, accounting for about 25% of the primary structure. We demonstrated further that the distances between domains I/II and II/III are very similar in all PP2C enzymes (9-13 and 74-80 amino acids, respectively). However, the amino acid sequences of the spacer regions are unrelated. In addition, the COOH-terminal ends of 100-200 amino acids which comprise 30-50% of the enzyme, display no identity. A dendrogramm shows that PtPP2C surprisingly is most closely related to the mammalian PP2C, and enzymes from Leishmania, Arabidopsis, and yeast are more distant relatives.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain.

Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach. One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids). The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT. Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids). When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid. As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs. This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis. mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver. It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase.

3T3 Cells↗
PubMed1993

Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant.

Simian immunodeficiency virus (SIV) induces an immunodeficiency syndrome similar to human AIDS. Although the disease course of SIV-induced immunodeficiency is generally measured in months to years, a disease syndrome that results in death in 5 to 14 days has been described in pig-tailed macaques infected with the SIVsmmPBj (PBj) strain. The purpose of this study was to derive an acutely lethal PBj molecular clone in order to study viral genes involved in pathogenesis. Six infectious molecular clones were generated; acutely fatal disease was induced by experimental inoculation of pig-tailed macaques with virus stocks derived from either of two clones, PBj6.6 or PBj14.6. Molecular chimeras were constructed by exchange of regions of the genome of PBj6.6 and a nonlethal, related clone, SIVsmH4. Only a chimera expressing the PBj genome under the control of a SIVsmH4 long terminal repeat induced death soon after inoculation. These studies suggest that multiple viral genes of PBj are critical for development of acute disease. More specifically, the env gene but not the long terminal repeat PBj was required for acute disease induction; however env must act in concert with another gene(s) of the PBj genome.

Animals↗