Capsid protein gene sequences of four tobacco mosaic virus strains defective for virus assembly.
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Amino Acid Sequence↗
Biomedical subjects
W Rohde
Publications and source records attributed to W Rohde.
Mutational analysis of the nucleic acid-binding 17 kDa phosphoprotein of potato leafroll luteovirus identifies an amphipathic alpha-helix as the domain for protein/protein interactions.
The 17 kDa protein (pr17) of potato leafroll luteovirus is translated from a subgenomic PLRV RNA by internal translation initiation and binds to single-stranded nucleic acids (E. Tacke, D. Prüfer, J. Schmitz, and E. Rohde, 1991, J. Gen. Virol. 72, 2035-2038). Chemical crosslinking of in vitro expressed pr17 provided evidence for the preferential formation of pr17 homodimers which were also detected in PLRV-infected potato plants and isolated from potato lines expressing the PLRV pr17 transgene. Mutation analysis identified an amphipathic alpha-helix within the acidic amino-terminus of pr17 which acts as the domain for protein/protein interactions. Pr17 was predominantly associated with subcellular fractions enriched for nuclei, chloroplasts, mitochondria, and membranous structures. In addition it was shown that pr17 was phosphorylated in planta and that this modification did not inhibit binding of the protein to nucleic acids.
Amino Acid Sequence↗
Myb genes from Hordeum vulgare: tissue-specific expression of chimeric Myb promoter/Gus genes in transgenic tobacco.
The structures of the three Myb-related genes Hv1, Hv5 and Hv33 from barley were determined. They contain a single intron located in the second repeat unit of the Myb-related domain. By analogy to the animal MYB oncoproteins this conserved region of the gene product was shown by filter-binding experiments to exhibit nucleic acid-binding activity. Tobacco plants transgenic for chimeric Myb promoter/Gus genes express the enzyme in a developmentally controlled and tissue-specific manner. During germination and early stages of plant growth, GUS activity is seen in the root cap and adjacent meristematic tissue. At later stages of plant development, GUS activity is predominantly observed in the shoot apical meristem, the roots and the nodal regions of the stem. Within the stem at stages of secondary growth, Myb promoters are active in defined cell types. In the internode low GUS activity is displayed by the innermost cell layer of the cortex, the starch sheath, that surrounds the vascular cylinder of secondary xylem and phloem tissue, as well as in pith rays originating from vascular cambium initials. In the nodal region Myb promoter-controlled Gus expression is mainly confined to the abaxial starch sheath of the leaf trace, to the branch traces and to internal strands of primary phloem. It is suggested that in addition to their activity in meristematically active plant tissues Myb genes are expressed in conductive tissues that are closely associated with vascular bundles.
Amino Acid Sequence↗
Molecular cloning and expression patterns of three alleles of the Deficiens-homologous gene St-Deficiens from Solanum tuberosum.
Three alleles of the Deficiens-homologous potato gene St-Deficiens (St-Def) present in the genome of a tetraploid Solanum tuberosum variety were isolated and characterized. For one allele (St-Def pD13) the complete molecular structure was determined by sequence analysis and comparison with its cDNA, while for the other two alleles (pD10, pD12) only partial sequences of regulatory and coding regions were obtained. All three alleles showed (except for one amino acid exchange in pD10) identical sequences in the coding region. While sequence variation was observed within the respective promoters starting some 300 nt upstream of the putative transcriptional start site, the 3' terminal promoter sequences were highly conserved. Within this region, a sequence of 81 nt was identified which showed 73% sequence identity to a corresponding sequence in the Deficiens promoter. This region which contains a putative serum response element was previously shown to regulate the expression of the Deficiens gene in Antirrhinum majus. Expression patterns for the three alleles in transgenic potato lines expressing St-Def promoter/Gus constructs were identical. GUS activity was predominantly located in petals and stamens as expected for the activity of a Def-homologous gene, but a significant level of expression was also detected in the ovary wall and in the vascular bundles supporting anthers and petals. The promoters were also active in abscission structures at the junction of flower stem and pedicel, as well as in anther stomium.
Alleles↗
Short- and long-term effects of perinatal interleukin-1 beta-application in rats.
Nervous, endocrine and immune systems are regarded as a complex functional unit, interacting by their specific chemical messengers-neurotransmitters, systemic hormones and hormone-like mediators of immune cells (cytokines). Cytokines are known to affect several endocrine axes. Interleukin-1 beta (IL) was administered in rats intraperitoneally twice daily from day 17 to 21 of pregnancy. Some of the IL-treated mothers were rapidly decapitated 1.5 h after the last injection. The fetuses were delivered by cesarean section. Maternal plasma CRF, ACTH and corticosterone were found to be significantly elevated. Fetal adrenal and thymus weights were lower, and plasma corticosterone did not differ from controls. Fetal plasma testosterone was decreased in males, androstenedione was increased in females. Open-field testing revealed a higher total locomotor activity of IL offspring than of controls. IL offspring showed worse results in Skinner box learning than controls. Sexual behavior was only affected in males, showing a higher percentage of female-type lordosis behavior after castration and estrogen treatment compared to controls. At the age of 6 months responsiveness to 'novel-environment stress' of IL groups was significantly lower than that of controls in terms of plasma corticosterone. These results indicate that prenatal treatment with IL-1 beta results in long-lasting alterations in psychomotor development, behavior as well as in the neuroendocrine system.
Adrenal Glands↗
[Permanent cardiac pacing in children. Personal experience].
Pacemaker implantation was performed in 26 children aged from 5 months to 15 years (mean 4.6). The indications for pacemaker implantation were: sick sinus syndrome in 4 patients, III degrees idiopathic A-V block in 2 both with congenital heart disease and III degrees post-op. A-V block in 20 patients. 34 pacemakers produced by seven different companies were implanted. Epicardial electrode was used in all but one patient in whom endocardial electrode was introduced. Demand for impulse voltage was 2.4-5.2 V (mean 4.4 V), impulse duration was from 0.25 ms to 1.65 ms (mean 0.62 ms). Three out of 26 pts. (11.5%) died (1-intraoperative bleeding, 1-ventricular rhythm disturbances, 1-sudden death). In seven children first exchange of the pacemaker (one for mechanic damage and 6 for exhausted batteries) was performed mean 4.3 years after previous implantation. In two children second battery exchange was necessary mean 7.5 years after previous (both exhausted batteries). In five children a damaged epicardial electrodes were exchanged (21%).
Adolescent↗
Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement.
Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.
Biotin↗
Somatomedin-C/IGF-I, insulin and prolactin levels in Ullrich-Turner's syndrome.
To clarify the pathogenesis of growth retardation in patients with Ullrich-Turner's syndrome (TS) we have investigated basal SmC/IGF-I, insulin and prolactin concentrations. Compared with 56 age matched healthy controls basal SmC/IGF-I concentration in 51 patients with TS older than 9-11 years was significantly lower (age group 13-14 years; TS 273 +/- 47 and controls 479 +/- 114 ng/ml). Mean basal prolactin level in 43 patients with TS (406 +/- 211 microU/ml) was significantly higher (p less than 0.01) than in 192 female controls (age 3-11 years; 264 +/- 176 microU/ml). Basal insulin concentration in 28 TS patients in comparison to 20 healthy children of a control group was significant higher (TS 18 +/- 8 microU/ml; controls 9 +/- 4 microU/ml; p less than 0.01). It seems that neither insulin nor prolactin are relevant stimulators of Smc/IGF-I in man, especially in patients with gonadal dysgenesis. Considering these results we speculate that despite higher prolactin and higher insulin levels in TS, the lower SmC/IGF-I concentrations may be predominantly related to the abnormal sex steroid secretion.
Adolescent↗
Lifelong enhanced diabetes susceptibility and obesity after temporary intrahypothalamic hyperinsulinism during brain organization.
Newborn male Wistar-rats received bilateral intrahypothalamic insulin-agar-implants on the 2nd or 8th day of life. In male control animals only the insulin-free indifferent agar-vehicle was implanted at the same age. In both experimental groups with temporary intrahypothalamic hyperinsulinism during brain organization the following results were obtained: 1) Higher body weight gain starting at the end of the hypothalamic differentiation period and continuing during juvenile life until adulthood, resulting in increased relative body weight as a sign of obesity; 2) A tendency to basal hyperinsulinaemia in juvenile and adult age; 3) Impaired glucose tolerance in adulthood; 4) Increased diabetes susceptibility to a single "subdiabetogenic" dose of streptozotocin in adult age. In view of these and previous observations a teratogenetic role of high insulin concentrations during the organization of glucoregulatory hypothalamic structures is hypothesized and the possible relevance of such hyperinsulinism as a predisposing factor for a lifelong enhanced diabetes and/or obesity risk is suggested.
Animals↗
Obesity and enhanced diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding.
To investigate possible permanent consequences of an early postnatal overfeeding, the following experimental model was used: Male Wistar rats were divided into three groups after birth: (1) Small litters with 3-4 newborns (overnutrition), (2) normal litters with 12 animals (normonutrition), and (3) large litters with 20-24 newborn rats (undernutrition). After weaning all animals had free access to tap water and standard pellet diet. The serum insulin level of animals from small litters on day 15 of life was highly significantly increased as compared to the other groups. These overfed hyperinsulinaemic rats showed a higher body weight gain during the suckling period trough juvenile life until adulthood, associated with enhanced mean food intake and resulting in an increased relative body weight (per body length) as a sign of obesity. The obesity was found to be correlated with basal hyperinsulinaemia and increased systolic blood pressure in the small-litter-adults. Moreover, the early postnatally overnourished animals developed an increased type I-like diabetes susceptibility to a "subdiabetogenic" dose of streptozotocin in adulthood. These results suggest once more that hyperinsulinism during brain differentiation, in the present experiment induced by early postnatal overnutrition, may represent a predisposing factor for the development of obesity, of increased diabetes susceptibility and also of increased cardiovascular risk in later life.
Animals↗
Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.
The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.
Amino Acid Sequence↗
Structure of a chalcone synthase gene from Hordeum vulgare.
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Acyltransferases↗
Structure of the Hordeum vulgare gene encoding dihydroflavonol-4-reductase and molecular analysis of ant18 mutants blocked in flavonoid synthesis.
A full-length cDNA clone encoding barley dihydroflavonol-4-reductase was isolated from a kernel-specific cDNA library by screening with the cDNA of the structural gene (A1) for this enzyme from maize. Subsequently, the gene corresponding to the barley dihydroflavonol-4-reductase cDNA was cloned and sequenced. The gene contains three introns at the same positions as in the Zea mays gene, corresponding to the positions of the first three of the five introns present in the genes of Petunia hybrida and Antirrhinum majus. In vitro transcription and translation of the Hordeum vulgare cDNA clone yielded a protein which converts dihydroquercetin into 2,3-trans-3,4-cis-leucocyanidin with NADPH as cofactor. The protein has a deduced amino acid sequence of 354 residues and a molecular weight of 38,400 daltons. Dihydroflavonol reductases of barley, maize, petunia and snapdragon are highly polymorphic in the NH2- and C-terminal parts of the polypeptide chain while a central region of 324 residues contains 51% identical amino acids. This identity increases to 81% when only the barley and maize enzymes are compared. Recessive mutants in the Ant18 gene tested so far lack dihydroflavonol-4-reductase activity and accumulate small amounts of dihydroquercetin but have retained activity for at least two other enzymes in the flavonoid pathway. In testa-pericarp tissue of mutants ant18-159, ant18-162 and ant18-164, wild-type levels of steady state mRNA for dihydroflavonol reductase have been measured, while mRNA for this enzyme is not transcribed in mutant ant18-161. These data are consistent with the proposal that the Ant18 locus carries the structural gene for dihydroflavonol-4-reductase of barley.
Alcohol Oxidoreductases↗
Nitrate-induced hypothyroidism is associated with a reduced concentration of growth hormone-releasing factor in hypothalamic tissue of rats.
Dietary nitrate significantly inhibits the growth of male and female rats. To test the possibility that the growth hormone-releasing factor (GRF) content in hypothalamic tissue is deranged under these conditions, male and female rats were fed a diet containing 3% KNO3 for 6 weeks, compared to a normal diet (4 X 5 animals). The food intake of rats fed nitrate was reduced significantly (23 and 28% resp.). Weight gain was also decreased by 35 and 41% in male and female rats. The mean Sm-C/IGF-I concentration was 1.61 and 1.03 rU/ml in male and female control rats, whereas the concentrations in nitrate-exposed rats were 0.92 and 0.64, respectively (P less than 0.01). The GRF content of hypothalamic tissue also decreased significantly from 407 and 533 ng/g protein in controls to 174 and 229 in treated male and female rats. Nitrate exposure is characterized by hypothyroidism, food intake depression, low Sm-C/IGF-I concentrations in plasma and a decreased hypothalamic GRF content. Independent of the peripheral changes, the content of Sm-C/IGF-I in the brain remains constant. The results of the study demonstrate that thyroid hormone deficiency leads to an inhibition of GH axis already at the hypothalamic level.
Animals↗
Effect of an acute maternal stress on the fetal hypothalamo-pituitary-adrenal system in late gestational life of the rat.
We investigated in this study the response of the fetal hypothalamo-pituitary-adrenal (HPA) system during an acute maternal stress in rats, in order to find out a possible role of developing fetal hypothalamus and to correlate its function to the androgen unbalance during the critical period of sex-specific brain differentiation. Pregnant rats of days 18-22 of gestation were subjected to an acute forced immobilization, and plasma levels of corticosterone (B) and ACTH were measured in mothers and fetuses. Hypothalamic contents of CRH and beta-endorphin (EP), pituitary content of ACTH, and plasma levels of B and ACTH were measured in mothers and fetuses under the maternal stress on day 20 of gestation. By an acute exposure to the 20 minutes' stress, plasma levels of B and ACTH elevated significantly in mothers on each day of gestation. A significant increase of fetal plasma ACTH was detected from day 18 in males, and from day 20 in females. During the maternal stress on day 20 of gestation, hypothalamic contents of CRH and EP decreased significantly in male and female fetuses, when plasma levels of B and ACTH elevated significantly. These results indicate that fetal HPA axis seems to actually respond to the maternal stress during the late gestational period. Further, a release of CRH under the stress together with an activation of EP system in the fetal hypothalamus suggests a possible mechanism regulating the androgen secretion by the fetal hypothalamus via changes of the LH levels.
Adrenocorticotropic Hormone↗
The potato leafroll luteovirus 17K protein is a single-stranded nucleic acid-binding protein.
The potato leafroll luteovirus protein of Mr 17K (pr17), which is encoded by an open reading frame on the 3' half of the viral genome, was expressed by using bacterial expression vector systems. Fusion proteins were obtained for the full-length viral protein as well as its N-terminal acidic (GST/pr17N) and C-proximal (GST/pr17C) basic domains and used in nucleic acid-binding studies. Filter-bound as well as soluble pr17 bound to single-stranded RNA or DNA. The binding domain was shown to reside in the basic C-proximal part of the polypeptide, whereas the N-terminal acidic domain did not show any affinity for nucleic acid. These biochemical properties of pr17 together with its structural features suggest a regulatory role for this protein during virus replication.
Carrier Proteins↗
Nucleotide Sequence of a Chymotrypsin Inhibitor-2 Gene of Barley (Hordeum vulgare L.).
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Journal Article↗
The 12 kDa protein of potato virus M displays properties of a nucleic acid-binding regulatory protein.
The 3' terminal 1.4 kb segment of potato virus M (PVM) genomic RNA was cloned and sequenced. This part of the viral genome encodes the capsid protein CP as well as a 12 kDa protein of as yet unknown function. Both proteins were expressed in bacteria and their nucleic acid-binding properties studied. The 12 kDa protein (pr12), but not the capsid protein bound single- and double-stranded nucleic acids. This property of pr12 in conjunction with a zinc finger motif located adjacent to a basic region of the 12 kDa protein suggests that it may act as a regulatory factor during virus replication.
Amino Acid Sequence↗