Structural and mechanistic studies on chloroplast translational initiation factor 3 from Euglena gracilis

Chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis contains a central region (homology domain) that is homologous to prokaryotic IF3. The homology domain is preceded by a long NH2-terminal extension (head), and followed by a 64 amino acid COOH-terminal extension (tail). Sequences in these extensions reduce the activity of the homology domain. To gain insight into these effects, a possible three-dimensional structure for the homology region of IF3chl has been modeled using the X-ray coordinates from the N- and C-domains of Bacillus stearothermophilus IF3. In B. stearothermophilus IF3, these two compact domains are thought to fold independently and are separated by a helical lysine-rich linker. The modeled structure suggests that IF3chl has a similar overall fold although some subtle differences are predicted to occur. Both the head and tail regions of IF3chl are oriented toward the linker region in the homology domain where they may potentially interfere with its function. Circular dichroism spectropolarimetry (CD) indicates that the lysine-rich linker region in IF3chl is not in a helical conformation and is probably a random coil. CD analysis indicates that a portion of the tail region of IF3chl is helical and that the tail has a direct interaction with the linker region in the homology domain. Site-directed mutagenesis of the linker indicates that two conserved lysine residues are important for the function of IF3chl and play a role in the binding of IF3chl to the 30S ribosomal subunit. Mutation of these residues affects the interaction of the homology domain with the tail.     

Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis

Aquacobalamin reductase (NADPH), which catalyzes the reduction of aquacobalamin to cob(II)alamin in the synthesis of cobalamin coenzymes, has already been purified from mitochondria of Euglena gracilis and partly characterized. Here, the enzyme was further characterized to clarify its enzymatic properties. The enzyme reduced 2 mol of aquacobalamin per mole of NADPH and had NADPH diaphorase-like activity. The 16 amino acid residues at the NH2-terminal of the enzyme were identical with those of the NADPH diaphorase domain of pyruvate: NADP+ oxidoreductase, which is involved in Euglena wax ester fermentation. Peptide mapping of the aquacobalamin reductase showed that elution during C-18 reversed-phase high-performance liquid chromatography was identical to that of the NADPH diaphorase domain. Immunoblotting indicated that the Euglena aquacobalamin reductase had a higher molecular weight (166,000) in the intact mitochondria than the purified enzyme (65,000), and that the molecular weights of the native and purified enzyme were identical with those of the subunit and the NADPH diaphorase domain, respectively. These results showed that the aquacobalamin reductase isolated earlier was the NADPH diaphorase domain, cleaved by trypsin during preparation of the mitochondrial homogenate from the native enzyme. Purified pyruvate:NADP+ oxidoreductase also had the activity of aquacobalamin reductase, which suggests that the enzyme in Euglena mitochondria has more than one function in the synthesis of cobalamin co-enzymes.     

U1 small nuclear RNA and spliceosomal introns in Euglena gracilis

In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, we have identified three spliceosomal-type introns that have GT-AG consensus borders. Furthermore, we have isolated a small RNA from E. gracilis and propose, on the basis of primary and secondary structure comparisons, that it is a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5' splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. Our observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote.     

Influence of tetracyclines or cetylpyridinium bromide on activity of fluoroquinolones in Euglena gracilis

To verify the hypothesis that the fluoroquinolone inhibitors of DNA gyrase generate some oxidant species and subsequently free radicals, the effects of simultaneous action of fluoroquinolones (ofloxacin, fleroxacin) and tetracyclines (tetracycline, doxycycline, thiatetracycline) or cetylpyridinium bromide as possible antioxidants on the flagellate Euglena gracilis were examined. Chloroplasts due to their bacterial character were injured and subsequently eliminated by fluoroquinolone. The loss of chloroplasts was accompanied by bleaching of originally green euglena cells. Tetracyclines, as well as cetylpyridinium bromide, decreased the euglena bleaching caused by ofloxacin or fleroxacin. The bleaching induced by ofloxacin or fleroxacin was most effectively inhibited by thiatetracycline and cetylpyridinium bromide. Control treating of cells with tetracyclines or cetylpyridinium bromide alone did not exert any bleaching effect. In vitro weak but significant interactions between examined substances were established as well.     

Multifunctional tryptophan-synthesizing enzyme. The molecular weight of the Euglena gracilis protein is unexpectedly low

After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995) Biotechnol. Appl. Biochem. 22, 179-190), we here describe structural and catalytic properties of the multifunctional tryptophan-synthesizing enzyme. The kinetic parameters kcat of all five activities and Km for the main substrates were determined. The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer. Neither intersubunit disulfide linkages nor glycosylations were detected. On the other hand, the polypeptide chains are blocked N-terminally. Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed. Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, N-terminally blocked fragment with a molecular weight of 119,000. In addition, an increase in beta-elimination accompanied by a decrease in beta-replacement activity of the beta-reaction during proteolysis was observed.     

Cadmium resistance of achlorophyllous Euglena gracilis cells: constitutive overexpression of two heat-shock proteins

The heat-shock response of Euglena gracilis was studied by cell labeling at both the normal growth temperature (23 degrees C) and an elevated temperature (35 degrees C). Analysis of the labeled proteins by two-dimensional polyacrylamide gel electrophoresis indicated that the rate of synthesis of two polypeptides p55 (55 kDa) and p40 (40 kDa) increased in cells labeled at the highest temperature studied. These polypeptides are also overexpressed in Cadmium-resistant Euglena gracilis cells labeled at the normal growth temperature (23 degrees C). On the basis of these results, p55 and p40 appear to be heat-shock proteins involved in some steps of the acquired Cd-resistance process in Euglena gracilis cells.     

Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60

To elucidate the role of ras gene mutations during the early stage of colorectal tumour progression, K-ras gene mutations were analysed in 32 benign adenomas and 36 adenomas with focal carcinoma in the colorectum by microscraping of histologically pure regions from tissue sections, polymerase chain reaction-restriction fragment length polymorphism and in part by direct sequencing. Several regions were scraped out and analysed when an adenoma contained areas with different grades of dysplasia. The frequencies of K-ras gene mutation in mild dysplasia, moderate dysplasia and focal carcinoma were 19% (7/36), 51% (25/49) and 39% (14/36) respectively. The K-ras gene status was heterogeneous in 4 of the 11 benign adenomas from which multiple samples were obtained, and mutations were always found in the regions with more advanced dysplasia in these adenomas. Thirteen of the 36 adenomas with focal carcinoma showed heterogeneity of mutations between the adenoma region and the focal carcinoma. Seven of which had mutations only in the adenoma region. These findings indicated that the K-ras gene mutations occur during the late stage of adenoma progression and may confer a more advanced morphological phenotype of adenoma, but these mutations are not mainly involved in malignant transformation from adenoma to carcinoma.     

Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases

It is demonstrated that in vivo irradiation with artificial UV-B for several hours significantly reduces the amount of large DNA extractable from immobilized Euglena in comparison with non-irradiated controls. This UV-B effect can be eliminated by a drastic reduction of the divalent ion concentration in the extracellular medium, i.e. the substitution of the culture medium by Tris-buffered agarose. Moreover, in vitro degradation of large DNA is demonstrated for crude protein extracts isolated from non-irradiated or UV-B-irradiated Euglena. The nuclease activity is shown for both crude protein extracts and purified nucleases; in both cases, two protein bands possessing nuclease activity are obtained with apparent molecular masses of 26 and 40 kDa and their activity is inhibited by specific nuclease inhibitors, i.e. aurintricarboxylic acid and ATP, applied at a concentration as low as 10(-8) M. Moreover, in vitro, nuclease activity clearly depends on the pH, with an optimum around pH 4.5, and on the ion composition of the extracellular medium. A strong stimulating effect is shown for Ca2+ with an optimum around 10(-4) M; this effect is potentiated by Zn2+ and Mn2+, but strongly counteracted by Mg2+ and the calmodulin inhibitors trifluoperazine and N- (6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W5). These results favour the concept which explains the lethal UV-B effect on Euglena as arising from a change in the general metabolic state of the cell and an activation of a DNA-degrading system, i.e. activation of metal-dependent nucleases (U.K. Tirlapur, D.-P. H?der and R. Scheuerlein, UV-B mediated damage in the photosynthetic flagellate, Euglena gracilis, studied by image analysis, Beitr. Biol. Pflanzen, 67 (1992) 305-317).     

Rhodopsin phosphorylation and its role in photoreceptor function

Light-stimulated phosphorylation of rhodopsin was first described 25 years ago. This paper reviews the progress that has been made towards (i) understanding the nature of the enzymes that phosphorylate and dephosphorylate rhodopsin (ii) identifying the sites of phosphorylation on rhodopsin and (iii) understanding the physiological importance of rhodopsin phosphorylation. Many important questions related to rhodopsin phosphorylation remain unanswered and new strategies and methods are needed to address issues such as the roles of Ca2+ and recoverin. We present one such method that uses mass spectrometry to quantitate rhodopsin phosphorylation in intact mouse retinas.     

A novel Euglena gracilis chloroplast operon encoding four ATP synthase subunits and two ribosomal proteins contains 17 introns

Electromyographic activity of anterior temporal and masseter muscles was measured in 92 young healthy men and women with sound dentitions during rest position, contact in centric occlusion and clench. Male and female mean potentials were similar except in clench, where males had higher electromyographic levels. Mean pooled electromyographic potentials were 1.9 microV (TA) and 1.4 microV (MM) during rest position, 6.5 microV (TA) and 2.8 microV (MM) during contact in centric occlusion. Mean maximum voluntary clench potentials were 181.9 microV (TA) and 216.2 microV (MM) in men, 161.7 microV (TA) and 156.8 microV (MM) in women. Examined muscles were more asymmetric at low electromyographic activity (rest and centric occlusion) with the temporal muscle less asymmetrical than the masseter. In females temporal muscle activity tended to dominate at every contraction level, while in males masseter activity was stronger in clench, and temporal activity in centric occlusion and in rest position.     

Transplantation of the M. gracilis in the reconstruction of hand function

Free muscle transplantation with motor innervation offers the possibility to add contractile elements to upper extremities with extensive loss of musculature due to direct trauma or untreated compartment syndrome (Volkmann's contracture). The functional cross section area and the mean resting fiber length determine the maximum power and the contracting amplitude of the donor muscle. Although considerably weaker than the finger flexors to be replaced, the gracilis muscle was the preferred donor muscle because of its neurovascular pedicle and the minimal donor site morbidity. In a series of 15 gracilis transplantations, all 13 muscles that survived regained function. Finger motion was dependent on the preoperative condition of tendons and joints. Even after complete loss of the flexor and extensor compartment a useful upper extremity could be restored, which was preferable to the only alternative-amputation.     

Current concept of structure and function of the Golgi apparatus. On the 100-anniversary of the discovery by Camillo Golgi

Early pregnancy factor (EPF) has been identified as a homologue of chaperonin 10 (cpn10) with immunosuppressive and growth factor properties. As a homologue of cpn10, it belongs to the heat shock family of proteins (hsp) but, unlike other members of this family, EPF is detected extracellularly. Early pregnancy factor was first discovered in pregnancy serum by the rosette inhibition test, and the novelty of its discovery was that its presence could diagnose pregnancy within 6-24 h of a fertile mating. As well as being a monitor of the presence of a viable embryo, it is necessary for embryonic survival. In this capacity it acts as both an immunosuppressant and growth factor. Early pregnancy factor is also a product of proliferating primary and neoplastic cells and functions as an autocrine growth factor both in vivo and in vitro. It has a modifying effect on the outcome of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Early pregnancy factor is considered to be one of the major factors involved in the modification of multiple sclerosis observed during pregnancy.     

Effect of zidovudine (AZT) on the structure and function of rat skeletal muscle

The role of the anti-HIV agent zidovudine (ZDV = AZT) in the generation of mitochondrial myopathies and subsequent skeletal muscle contractile deficiencies was evaluated in male rats given ZDV in drinking water (1 mg/mL). After 6 weeks, there was no difference in treadmill run time between experimental (n = 6) and control (n = 6) rats. ZDV did not affect tension output by the gastrocnemius-plantaris-soleus muscle group when stimulated in situ at frequencies of 15, 30, 45, and 75 tetani/min, nor did the drug affect the cytochrome oxidase activity of fast glycolytic, fast oxidative glycolytic, or slow oxidative fiber types after 6 or 15 weeks of treatment. A group of female rats, similarly evaluated after 6 weeks of ZDV at 1 (n = 4) or 2 (n = 4) mg/mL, also did not display any discernable deficiencies. However, when the data from all 10 control rats were compared with those of the 19 ZDV rats, the cytochrome oxidase activity of fast oxidative glycolytic muscle of the ZDV rats was significantly higher (35.0 +/- 1.36 versus 40.7 +/- 1.14 mumol.min-1.g-1; p < 0.05). No ultrastructural abnormalities were observed in 15-week ZDV-treated cardiac muscle or in any of the three skeletal muscle fiber types. These results suggest that ZDV-related myopathies observed in AIDS patients may be due to interactions between the drug and complications associated with HIV infection.