Immunodetection of cytoskeletal structures and the Eg5 motor protein on deep-etch replicas of Xenopus egg cortices isolated during the cortical rotation

Summary— We have developed a new method for immunogold detection on deep-etch replicas of isolated Xenopus egg cortices in order to examine the interactions of different cortical elements in three dimensions at high resolution. We have applied this technique to vegetal cortices isolated during the second half of the first cell cycle. The vegetal cortical region at this time is the site of cellular machinery responsible for the ‘cortical rotation’. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone between cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved amongst these aligned microtubules were extensive sheets of ER lying in layers parallel to the egg surface. Cytokeratin filaments were found to associate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules. We propose revised models for the mechanism of cortical rotation based on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.     

Presence of unique repeated insertion sequences in nodulation genes of Rhizobium ‘hedysari’

Cloning and sequencing of DNA from a symbiotic large plasmid in Rhizobium hedysari strain IS 123 required for its nodulation of the mediterranean legume crop Hedysarum coronarium (sulla) and complementation studies of nod^- mutant derivatives led to the characterization of a 30-kb region containing common and host-specific nod genes. This DNA region also contained at least six copies of a novel insertion sequence-like structure, some of which appeared to have suffered deletions. This 0.8 kb novel element carries two 17-bp flanking inverted repeats and an open reading frame showing homology with a transposase from Staphylococcus aureus. Hybridization studies revealed that several strains of Rhizobium hedysari carry this element in various copy number. The six copies in strain IS 123 appear clustered specifically within the pSym nod region.The significance of this IS element in rhizobia and its possible use as a probe for taxonomic and phylogenetic studies of Rhizobiaceae is addressed.     

Cloning and expression of mouse 60 kDa ribonucleoprotein SS-A/Ro

Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5′ RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kindney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.     

Alternating bolus and continuous infusion 5-fluorouracil: a strategy to overcome resistance to this fluoropyrimidine in advanced colorectal cancer patients

Focusing our effort on the importance of FUra scheduling we have tested the hypothesis that pulse and continuous infusion (CI) of the fluoropyrimidine have different mechanisms of cytotoxicity. Our initial approach was to compare the mechanism of resistance of a cell line resistant to a short term exposure to FUra (HCT-8/FU4hR) to that of a cell line resistant to a prolonged exposure to the fluoropyrimidine (HCT-8/FU7dR). Cytotoxicity studies showed that HCT-8/FU4hR cells were still sensitive to FUra given as a 7-d exposure, suggesting different mechanisms of resistance. Indeed, rapid recovery of TS activity after drug removal was evident in the HTC-8/FU7dR cell line while HCT-8/FU4hR cells were similar to the parental cell line with regard to both the degree of in situ TS inhibition by FUra and duration of inhibition after FUra removal. In contrast, labelling studies with [³H-6] FUra (4 h exposure, 100 M) showed that the incorporation of the fluoropyrimidine into RNA is significantly decreased in HCT-8/FU4hR cells as compared to parental HCT-8 cells.Given the lack of cross resistance between the two schedulesin vitro, a pilot trial was done on patients with colorectal cancer refractory to bolus FUra. On 15 patients failing after FUra+LV or FUra alone 1 PR, 3 MR, 3 SD and 8 P were observed, confirmng a certain degree of activity of CI FUra in patients clinically resistant to bolus FUra.Based on this rationale, a phase II trial of schedule-oriented biochemical modulation of FUra in advanced colorectal cancer patients was conducted, employing a hybrid regimen of 2 biweekly cycles of FUra bolus (600 mg/sqm), preceeded by (24 h interval) methotrexate, 200 mg/sqm (in order to maximize the RNA effect of the drug) alternating with FUra continuous infusion, 200 mg/sqm daily for 3 weeks, modulated by leucovorin, 20 mg/sqm weekly bolus (in order to maximize the DNA effect).Thirty-three consecutive patients (median ECOG PS 1) with advanced measurable colorectal cancer and no prior therapy for metastatic disease entered the study, from February 1992 to August 1993. Three complete and 13 partial responses were obtained among these 33 patients (RR=48%, 95% confidence limis, 31–66%). After a median follow-up time of 23 months, 16 patients are still alive. The median progression free survival and overall survival were 9.6 and 20.8 months, respectively. No toxic deaths or grade 4 toxicity occurred. The incidence of grade 3 toxicity per patient in any cycle was: mucositis 6%, diarrhea 3% and vomiting 3% for the bolus part and 21%, 3% and 6% respectively, for the continuous infusion part of the regimen. Hand-foot syndrome occurred in 27% of the patients treated with the continuous infusion regimen.In conclusion, this experimental and clinical project has generated a novel regimen of schedule oriented biochemical modulation that is twice as active and half as toxic compared to bolus FU+LV given with either the daily x 5 or the weekly schedule. This high clinical activity is very encouraging, especially considering that 1) consecutive patients were entered, 2) the responses were independently reviewed, 3) the progression free survival and survival were much longer than those actually reported for this disease, 4) the toxicity of the program, in particular the bolus regimen, was relatively low allowing further intensification.     

Developmental changes in the brain-stem serotonergic nuclei of teleost fish and neural plasticity

Summary 1. During early ontogeny, the serotonergic neurons in the brain stem of the three-spined stickleback shows a temporal and spatial developmental pattern that closely resembles that of amniotes.2. However, in the adult fish, only the midline nuclei of the rostral group (dorsal and median raphe nuclei) and the dorsal lateral tegmental nucleus are consistently serotonin-immunoreactive (5-HTir), whereas the groups of the upper and lower rhombencephalon (raphe pontis, raphe magnus, and raphe pallidus/obscurus nuclei) are variable and, when present, contain relatively small numbers of 5-HTir neurons.3. Using specific antisera against tryptophan 5-hydroxylase and aromaticl-amino acid decarboxylase, we have shown that the lateral B9 group and the groups of the upper and lower rhombencephalon are consistently present in adult sticklebacks. The results are discussed in relation to other known instances of neurotransmitter plasticity or transient neurotransmitter expression in teleost fish.4. While there are several instances of transient expression of neurotransmitter markers by discrete neuronal populations, there is so far no evidence of changes from one neurotransmitter phenotype to another in the brain of teleost fish. However, there are indications of plasticity of expression of catecholamines and indoleamines, and their respective synthesizing enzymes, as reflected in age-dependent changes and variation between individuals of different physiological status.5. As the brain grows continuously in teleost fish, and new neurons are added from proliferative regions, synaptic connections may be expected to undergo remodeling in all brain regions throughout life. Thus, the teleostean brain may be considered a suitable model for experimental studies of different aspects of neural plasticity.     

An improved method for the measurement of total lipid-bound sialic acids after cleavage of α2,8 sialic acid linkage withVibrio cholerae sialidase in the presence of cholic acid,SDS and Ca2+

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD₃ and GD₂) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca²⁺ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994).     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

γ-Glutamyl conjugation of 5-hydroxytryptamine (serotonin) in the earthworm (Lumbricus terrestris)

The conversion of 5-hydroxytryptamine to several potential metabolites was examined in the annelid earthworm (Lumbricus terrestris). 5-hydroxytryptamine and some related amines were found to be present in several tissues of the earthworm. Injection of 5-hydroxytryptamine into the body cavity of the earthworm resulted in the production of a -glutamyl conjugate of 5-hydroxytryptamine. Incubations of the anterior nerve cord of the earthworm resulted in the accumlation of considerable amounts of 5-hydroxytryptamine and -glutamyl 5-hydroxytryptamine in the incubation medium. The earthworm did not produce any N-acetyl 5-hydroxytryptamine and only very little 5-hydroxyindoleacetic acid. Experiments involving the injection of radiolabeled 5-hydroxytryptamine or coninjection of radiolabeled glutamic acid with unlabeled 5-hydroxytryptamine into the earthworm resulted in the production of radiolabeled -glutamyl 5-hydroxytryptamine. This work demonstrates that the enzymatic conversion of 5-HT in the earthworm is markedly different from that of vertebrates and insects.