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Biomedical subjects

A Otto

Publications and source records attributed to A Otto.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14.

Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae. The degree of in vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to the extracellular glucose concentration [Vojtek, A. B., and Fraenkel, D. G. (1990) Eur. J. Biochem. 190, 371-375]; however, a physiological role of the modification causing the dissociation of the dimeric enzyme in vitro [as effected by a serine-glutamate exchange at position 14; Behlke et al. (1998) Biochemistry 37, 11989-11995] is unclear. This paper describes a comparative stopped-flow kinetic and sedimentation equilibrium analysis performed with native unphosphorylated hexokinase 2 and a permanently pseudophosphorylated glutamate-14 mutant enzyme to determine the functional consequences of phosphorylation-induced enzyme dissociation. The use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose phosphorylation over a wide range of enzyme concentrations. The kinetic data indicated that monomeric hexokinase represents the high-affinity form of isoenzyme 2 for both glycolytic substrates. Inhibition of glucose phosphorylation by ATP [Moreno et al. (1986) Eur. J. Biochem. 161, 565-569] was only observed at a low enzyme concentration, whereas no inhibition was detected at the high concentration of hexokinase 2 presumed to occur in the cell. Pseudophosphorylation by glutamate substitution for serine-14 increased substrate affinity at high enzyme concentration and stimulated the autophosphorylation of isoenzyme 2. The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.

Amino Acid Substitution↗
PubMed2000

Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network.

Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.

Contractile Proteins↗
PubMed2000

GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis.

Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.

Amino Acid Sequence↗
PubMed2000

Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

Annexin A4↗
PubMed2000

[Chronic recurrent, locally destructive siliconomas after breast augmentation by liquid silicone oil].

At the beginning of the sixties the injection of liquid silicon oil was frequently used for breast augmentation. It was thought to be safe, simple and effective. But as complications such as local silconomas, inflammatory reactions, induration, foreign body extrusion and foreign body migration were published this procedure was not used anymore. We report about a 45-year-old female patient who suffered from late complications, chronic recurrent local destructive siliconomas, which have not been described in the literature yet. Because of the destruction forced by the primary silicone implant a bilateral subcutaneous mastectomy had to be performed and siliconomas were excised at regions different from the original injection site. Several breast reconstructions have been performed but this tissue has also been destroyed by the recurrent and aggressive siliconomas. As there can be a long period of latency before the aforementioned complications of liquid silicon injections can occur, we recommend careful follow-up for these patients.

Breast↗
PubMed2000

A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase.

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.

Adenosine Triphosphate↗
PubMed2000

TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator.

We investigated intracellular signaling events involved in fibronectin-accelerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresis and western blotting. Proteins were sequenced with electrospray ionization mass spectrometry. Apoptosis was quantitated by flow cytometry. We detected a cluster of acidic, high molecular-weight proteins that were only tyrosine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleavage, yielding a 23-kD fragment. At 8 h, intact Ly-GDI was decreased to 33% on fibronectin, compared with 69% on PolyHema (P<0.05). Inhibition of tyrosine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated apoptosis in TNF-alpha-treated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activation and that caspase-3 inhibition decreased apoptosis. We conclude that tyrosine phosphorylation of Ly-GDI, followed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alpha-mediated apoptosis on fibronectin.

Apoptosis↗
PubMed1999

T-cell-activating monoclonal antibodies, reacting with both leukocytes and erythrocytes, recognize the guinea pig Thy-1 differentiation antigen: characterization and cloning of guinea pig CD90.

A glycophosphatidylinositol (GPI)-linked differentiation antigen expressed on guinea pig T and B lymphocytes was identified by several monoclonal antibodies; it has been shown previously that this membrane protein induced strong polyclonal T cell proliferation upon antibody binding and costimulation by PMA. Purification by immunoadsorption and microsequencing revealed that this T-cell-activating protein is the homologue of Thy-1 or CD90. In contrast to the Thy-1 antigen of most other species, guinea pig Thy-1 has a much higher molecular weight, which is due to a more extensive N-linked glycosylation, bringing the molecular weight of the total antigen up to 36 kDa. Molecular cloning of guinea pig Thy-1 indicated that the deduced molecular weight of the protein backbone is 12,777 after removal of an N-terminal 19-amino-acid leader peptide and cleavage of the 31 amino acids for GPI anchoring the C-terminal end. Sequence comparison showed that guinea pig Thy-1 has an 82% homology to human and a 72% homology to mouse Thy-1 on the amino acid level. Immunohistological staining of cryostat sections revealed intensive staining with the monoclonal antibody H154 on fibroblasts, fibrocytes, Kupffer cells, alveolar macrophages, and mesangial cells. As observed in the human, mouse, and rat, Thy-1 is abundant in the guinea pig brain. Unlike Thy-1 expression in other species, guinea pig Thy-1 is strongly expressed on most resting, nonactivated B cells and, to a lesser extent, on erythrocytes. While treatment of erythrocytes and lymphocytes with GPI-specific phospholipase C largely decreased reactivity with mAb H154, T cells retained the proliferative response to antibody and phorbol esters.

Amino Acid Sequence↗
PubMed1999

Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry.

We paper describe a mass spectrometric approach generally applicable for the rapid identification and characterization of proteins isolated by two-dimensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electrospray mass spectrometry employing a quadrupole-time of flight mass spectrometer was used for the direct identification of proteins from the peptide mixture generated from only one high resolution 2-DE gel without high performance liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this technique proved to be a powerful tool for the identification of proteins from femtomole amounts of materials. We applied the technique for the investigation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 36 protein spots were examined, from only one micropreparative Coomassie Brilliant Blue R-250 stained gel, after proteolytic digestion by matrix assisted laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully identified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databases. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The given technique and the protein data are the basis for construction of a database to compare normal and apoptosis-induced cells and, further, to enable fast screening of drug impact in apoptosis-associated processes.

Amino Acid Sequence↗
PubMed1999

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Animals↗
PubMed1999

Megalin knockout mice as an animal model of low molecular weight proteinuria.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

Amino Acid Sequence↗
PubMed1999

Preparative high-resolution two-dimensional electrophoresis enables the identification of RNA polymerase B transcription factor 3 as an apoptosis-associated protein in the human BL60-2 Burkitt lymphoma cell line.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.

Amino Acid Sequence↗
PubMed1999

[Successful ear replantation without venous anastomosis by using leeches].

Replantation of the ear is a challenging problem for the reconstructive surgeon. Avulsion injury and intima tears of these small vessels often make back cuts and venous interposition necessary. Furthermore, identification and differentiation of the vessels are difficult. The volume of the ear is large in relation to the wound surface and the absence of axial venous outflow makes replantation prone to venous congestion. On the basis of a case report we present the problems, the specific management and final outcome of a successful ear replantation and we discuss the alternative possibilities of reconstruction.

Adult↗
PubMed1999

Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a gene (mspA) encoding the major sheath protein of Treponema maltophilum ATCC 51939(T).

The major sheath protein-encoding gene (mspA) of the oral spirochete Treponema maltophilum ATCC 51939(T) was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The mspA gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an mspA-specific probe showed no cross-reactivity with the msp gene from Treponema denticola.

Antigens, Surface↗
PubMed1998

Molecular cloning and tissue distribution of rat sarcosine dehydrogenase.

Sarcosine dehydrogenase (SarDH) is a mitochondrial flavoenzyme involved in the oxidative degradation of choline to glycine. The absence of SarDH activity in humans is genetically transmitted and is the cause of an amino acid metabolism disorder called sarcosinemia. Tryptic fragments of the purified enzyme from rat liver were subjected to Edman degradation and the sequences obtained were used to clone the cDNA encoding the full length protein. The deduced amino acid sequence of SarDH shares an overall similarity of 47% with dimethylglycine dehydrogenase (Me2GlyDH), another flavoenzyme involved in the mitochondrial choline catabolism with a similar FAD-binding domain. Covalent binding of FAD to SarDH was demonstrated by the observation of strong fluorescence at 530 nm under excitation at 450 nm of the enzyme immunoprecipitated under denaturing conditions from liver extracts. The localization of SarDH immunoreactivity in the mitochondrial matrix was confirmed by Western-blot analysis of purified mitochondrial fractions. Finally, the tissue distribution of SarDH was investigated by Northern-blot analysis of total RNA and Western-blot analysis of total protein from several rat tissues. A strong expression in the liver, but also in the lung, pancreas, kidney, thymus, and oviduct was observed. We therefore suggest that the enzymes of the choline catabolism pathway are important also for metabolism in nonhepatic tissues.

Amino Acid Sequence↗