Band 382, Heft 11

The evolutionary processes underlying the differentness of prokaryotic and eukaryotic cells and the origin of the latters organelles are still poorly understood. For about 100 years, the principle of endosymbiosis has figured into thoughts as to how these processes might have occurred. A number of models that have been discussed in the literature and that are designed to explain this difference are summarized. The evolutionary histories of the enzymes of anaerobic energy metabolism (oxygenindependent ATP synthesis) in the three basic types of heterotrophic eukaryotes those that lack organelles of ATP synthesis, those that possess mitochondria and those that possess hydrogenosomes play an important role in this issue. Traditional endosymbiotic models generally do not address the origin of the heterotrophic lifestyle and anaerobic energy metabolism in eukaryotes. Rather they take it as a given, a direct inheritance from the host that acquired mitochondria. Traditional models are contrasted to an alternative endosymbiotic model (the hydrogen hypothesis), which addresses the origin of heterotrophy and the origin of compartmentalized energy metabolism in eukaryotes.

A dualaffinity method was established to purify, for the first time, a microsomal ecdysonebinding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on ωoctylaminoagarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazolebased general P450 inhibitor (typeII ligand) followed by elution with the substrate ecdysone (typeI ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDSPAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergentextracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysoneinduced typeI difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrixbound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17β hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dualaffinity chromatography described here may be generally applicable to the isolation of cytochromes P450.

Alanine scanning mutagenesis and the introduction of deletions and insertions were used to address the role of the large cytoplasmic loop in 2-deoxyDglucose (2-DOG) uptake by GLUT1 expressed in Xenopus oocytes. Alanine scanning mutagenesis of 29 amino acid residues that are identical or homologous in GLUT1 to GLUT4 demonstrated that the transport activities of only a few variants were affected. Progressive truncation of the loop by six deletions leaving intact 59 (Δ236241), 49 (Δ231246), 39 (Δ226251), 28 (Δ221257), 18 (Δ216262), or 10 (Δ213267) amino acid residues resulted in a progressive decrease in 2-DOG uptake. Compared with wildtype GLUT1 the uptake rates varied between 33% for the Δ236241 mutant and 4% for the Δ213267 mutant. Insertional mutagenesis using hexaalanine or hexaglycine to fill in the deletion 236D-241L restored 2-DOG uptake to 73% of wildtype GLUT1 in the case of hexaalanine, whereas hexaglycine insertion was without effect. Confocal laser microscopy demonstrated that a deletion of six amino acid residues did not influence the expression level in the plasma membrane (Δ236241 mutant), whereas the plasma membrane fluorescence of the Δ213267 mutant was comparable with that of waterinjected Xenopus oocytes. Computeraided secondary structure prediction of the loop suggested that it consists of a long αhelix bundle interrupted or kinked by the highly conserved glycine-233.

Atomic force microscopy (AFM) allows the observation of biological material without fixation procedures. Here we present AFM images of ribonucleoproteins (nucleocapsids) derived from a plant infecting RNA virus (tomato spotted wilt virus, TSWV), which have been recorded in contact mode. The nucleocapsids, prepared from systemically infected leaves of tobacco, were spreaded on a glass surface and dried in air, and appeared as regularly formed rings, resembling the proposed pseudocircular and panhandle structure of encapsidated genomic RNA. Average values between 1300 and 2200 nm of nucleocapsid lengths could be related to dimensions estimated by electron microscopy, thereby validating a filamentous configuration of the TSWV ribonucleoproteins. However, to our knowledge regular, ringlike forms of ribonucleoproteins have not been obtained by electron microscopy, which rather showed an amorphous structure of the virus particles. Hence, the AFM approach provides a starting point for further detailed studies on TSWV ribonucleoprotein complexes.

Human cervical epithelial cells transfected and immortalized with human papillomavirus type 16 DNA (HCE16/3) can be, like many other epithelial cells, normally grown in medium supplemented with epidermal growth factor, cholera toxin, hydrocortisone, insulin, transferrin, thyroid hormone and serum. We found that hydrocortisone diminished tissue plasminogen activator (tPA) production to an undetectable level. The removal of hydrocortisone increased urokinase plasminogen activator (uPA) activity within 2448 h and tPA activity within 4872 h, and converted the cells to a more elongated and fibroblastic phenotype. Upregulation of uPA mRNA was seen as early as at 3 h and of tPA mRNA within 4872 h. Higher molecular weight forms (97110 kDa) of plasminogen activators were seen in zymograms, apparently complexed with PAI-1, starting at 6 h both in the presence and absence of hydrocortisone. Immunoprecipitation with a PAI-1 monoclonal antibody confirmed that both uPA and tPA were complexed. We also studied normal diploid human bronchial epithelial cells (NHBE) and NHBE cells transformed with an adeno-12/SV40 hybrid virus (BEAS-2B). In both types of nonmalignant epithelial cells, the removal of hydrocortisone increased uPA activity. The omission of hydrocortisone increased tPA levels significantly in BEAS-2B cell cultures, and in NHBE cell cultures tPA became detectable at 72 h. No PA complexes were seen in these two cell types. We conclude that normal and immortalized nonmalignant epithelial cells produce tPA, but only if hydrocortisone is omitted in the growth medium.

We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human α-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both α-1-antitrypsin and HMWkininogen, but neither α-1-antichymotrypsin nor antithrombin III.

Human cathepsin L (hCATL) mRNA occurs in vivo in at least three splice variants. They differ in the length of exon 1, which comprises 278 nucleotides (hCATLA), 188 nucleotides (hCATLA2) and 132 nucleotides (hCATLA3), respectively. We describe here the shortest variant for the first time. This form is predominant in all tissues and cells examined so far, including malignant tumors. We studied the expression rate of the three mRNA variants in order to explain why malignant kidney tumors show low cathepsin L activity despite of high mRNA levels. The variant hCATLA3 showed the highest expression rate in vitro and in vivo. Based on these results, we suggest a cisacting element on human cathepsin L mRNA which can be bound by a negative transacting regulator, thus leading to reduced expression rates.

Protein misfolding and aggregation play an integral role in many diseases. The misfolding of the serpin (SERine Proteinase INhibitor) α1-antitrypsin results in the accumulation of insoluble polymers within hepatocytes and α1-antitrypsin deficiency in plasma, predisposing patients to liver cirrhosis and emphysema. We have examined the effect of three naturally occurring osmolytes, sarcosine, glycine betaine and trimethylamine Noxide, on conformational changes in α1-antitrypsin. All three solutes protected native α1-antitrypsin against thermally induced polymerisation and inactivation in a concentrationdependent manner. Further spectroscopic analysis showed that sarcosine stabilises the native conformation of α1-antitrypsin, thus hindering its conversion to an intermediate state and subsequent polymerisation. On refolding in the presence of sarcosine, α1-antitrypsin formed a heterogeneous population, with increasing proportions of molecules adopting an inactive conformation in higher concentrations of the osmolyte. These data show that sarcosine can be used to prevent abnormal structural changes in native α1-antitrypsin, but is ineffective in facilitating the correct folding of the protein. The implications of these results in the context of conformational changes and states adopted by α1-antitrypsin are discussed.

Oxidative stress is a widespread phenomenon in the pathology of neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis. Neuronal cell death due to oxidative stress may causally contribute to the pathogeneses of these diseases. Therefore, neuroprotective antioxidants are considered to be a promising approach to slow down disease progression. We have investigated different aromatic amine and imine compounds for neuroprotective antioxidant functions in cell culture, and found that these compounds possess excellent cytoprotective potential in diverse paradigms of oxidative neuronal cell death, including clonal cell lines, primary cerebellar neurons, and organotypic hippocampal slice cultures. Aromatic amines and imines are effective against oxidative glutamate toxicity, glutathione depletion, and hydrogen peroxide toxicity. Their mode of action as direct antioxidants was experimentally confirmed by electron spin resonance spectroscopy, cellfree brain lipid peroxidation assays, and intracellular peroxide measurements. With halfmaximal effective concentrations of 2075 nM in different neuroprotection experiments, the aromatic imines phenothiazine, phenoxazine, and iminostilbene proved to be about two orders of magnitude more effective than common phenolic antioxidants. This remarkable efficacy could be directly correlated to calculated properties of the compounds by means of a novel, quantitative structureactivity relationship model. We conclude that bridged bisarylimines with a single free NHbond, such as iminostilbene, are superior neuroprotective antioxidants, and may be promising lead structures for rational drug development.

By using the ESR spin trapping technique with the NmethylDglucamine dithiocarbamate (MGD)2-Fe(II) complex, the generation of nitric oxide (NO), a gaseous free radical, was observed in NO spin trapping solution bubbled with the filtered mainstream of cigarette smoke. The ESR signal with a threeline spectrum characteristic of an NO radical, which was not observed immediately after bubbling of smoke, started rapidly increasing with time up to around 25 min after the last addition of ferrous ions Fe(II), and then slowly approached a peak value dependent on the burned cigarette mass and on the smoking speed. The production of NO was, however, much affected by air oxidation and enhanced by the addition of ascorbic acid. A certain concentration of sodium nitrite (NaNO2) solution, in which nitrite NO2 is assumed as the main origin of the NO, mimicked closely the time course of NO generation resulting from the smoke of one cigarette. The cigarette smoke that was passed through alkaline pyrogallol solution as a deoxidizer; however, it exhibited an unchanged intensity of NO signal throughout the measurement. These results strongly suggest that NO would be gradually reproduced from NO2 in the reductive aqueous solution containing excess Fe(II) through NO2, which is initially formed and is concomitantly oxidized from NO in cigarette smoke.

We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxyterminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by singlestep affinity chromatography on streptavidinagarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidinbinding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3x 1010M). Similarly, the streptavidinbinding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.