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K Reuter

Publications and source records attributed to K Reuter.

At least 37 records · Page 2Linked to original sources
PubMed1999

Overproduction, purification, crystallization and preliminary x-ray diffraction studies of the human spliceosomal protein U5-15kD.

The gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding. The crystals belong to the orthorhombic space group P21212 with a = 62.3, b = 65.7, c = 37.1 A. They diffract to at least 3.0 A and contain one molecule in the asymmetric unit. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection.

Cloning, Molecular↗
PubMed1999

Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases.

The Bacillus subtilis Sfp protein is required for the non-ribosomal biosynthesis of the lipoheptapeptide antibiotic surfactin. It converts seven peptidyl carrier protein (PCP) domains of the surfactin synthetase SfrA-(A-C) to their active holo-forms by 4'-phosphopantetheinylation. The B. subtilis sfp gene was overexpressed in Escherichia coli and its gene product was purified to homogeneity and crystallized. Well diffracting single crystals were obtained from Sfp as well as from a selenomethionyl derivative, using sodium formate as a precipitant. The crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = b = 65.3, c = 150.5 A. They diffract beyond 2.8 A and contain one molecule in the asymmetric unit.

Bacillus subtilis↗
PubMed1997

Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori.

The aroB gene from Helicobacter pylori strain P1 was cloned and further characterized by sequence analysis and by functional complementation of the aroB mutation in Escherichia coli. The aroB gene encodes the enzyme 3-dehydroquinate synthase which catalyzes one of the early steps in the shikimate pathway. This pathway, which creates aromatic molecules from sugar precursors, is present in prokaryotes, fungi and plants but is absent from mammalian cells. The predicted amino acid sequence of the H. pylori aroB gene product showed significant homology (30-40% identity and 50-60% similarity) to 3-dehydroquinate synthases from various other prokaryotes and eukaryotes. The single gene on a plasmid was biologically active in E. coli. It suppressed the specific phenotype of aroB mutants by restoring the shikimate pathway-dependent synthesis of aromatic amino acids and the production of the siderophore enterobactin. Two other reading frames were found adjacent to the aroB gene. The first, designated as orf1, had no significant homology to proteins and genes present in databases, whereas the second was found to share a significant degree of homology with the tgt gene encoding tRNA-guanine transglycosylase from a variety of other bacteria (40-50% identity and 60-70% similarity). The function of the tgt gene was confirmed by heterologous complementation. The gene on a plasmid was shown to complement the queuosine biosynthesis defect in a genetically defined tgt- strain of E. coli. The presence of the aroB gene and the putative tgt homologue in unrelated H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers.

Amino Acid Sequence↗
PubMed1996

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile.

Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray structures of Zymomonas mobilis TGT and of its complex with preQ1 [Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850-2857] have revealed a specific preQ1 binding pocket and allowed a proposal for tRNA binding and recognition. We have used band-shift experiments in denaturing conditions to study the enzymatic reaction performed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate. Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation of this residue into alanine by site-directed mutagenesis leads to an inactive mutant unable to form a covalent intermediate with tRNA, proving that aspartate 102 is the active site nucleophile in TGT. To investigate the recognition of the wobble guanine in the preQ1 binding pocket, we mutated aspartate 156, the major recognition element for preQ1, into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent intermediate with tRNA in the first step of the reaction mechanism in comparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ1 binding pocket is required for the cleavage of the glycosidic bond. The three mutants were crystallized and their X-ray structures determined. The mutants display only subtle changes to the wild-type protein, confirming that the observed biochemical results are due to the chemical substitutions rather than structural rearrangements.

Aspartic Acid↗
PubMed1996

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.

Amino Acid Sequence↗
PubMed1996

Purification, crystallization, and preliminary x-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis.

The tRNA modifying enzyme tRNA-gnanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the quenine precursor 7-aminomethyl-7-deazagnanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for X-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 A, b = 65.1 A, c = 71.9 A, and beta = 97.5 degrees and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 A resolution.

Chromatography, Ion Exchange↗
PubMed1996

[Memory performance of Parkinson patients with and without sleep apnea syndrome].

We investigated the effect of SAS on the long term memory in PD patients and compared them with patients with brain infarction or cataract (control group), respectively. PD patients develop SAS and cognitive impairments more often then healthy people. Since SAS leads to a fragmentation of the sleep structure it interferes with memory. Therefore SAS may be a pathogenic factor of cognitive impairment in PD. We studied 14 patients (7 with and 7 without SAS) with PD, brain infarction or cataract using the LGT-3, which measures the verbal, numeric and figural long term memory. All patients answered subtest 7 of the Wechsler-Memory-Scale at night and in the morning. The saving between the evening and morning measure was calculated. An analysis of variance was performed using the SAS-condition and the different groups as independent variable. A significant difference (p < or = 0.005) of all memory measurements between patients with and without SAS but not between the different groups has been proved with a superiority of the patients without SAS. A significant difference of the influence of the SAS for verbal memory score on patients with PD (pp < or = 0.05) and brain infarction (pp < or = 0.005) respectively and the control group could be demonstrated. Therefore we found a quantitative difference of memory consolidation in PD patients with SAS. The influence of SAS on memory in patients with neurological disease is more pronounced compared to the control group.

Cataract↗
PubMed1995

Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme.

tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration.

Amino Acid Sequence↗
PubMed1994

Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli.

An Escherichia coli mutant described by Noguchi et al. [Noguchi, S., et al. (1982) J. Biol. Chem. 275, 6544-6550] contains tRNA lacking the hypermodified wobble nucleoside queuosine (Q) due to an inactive tRNA-guanine transglycosylase (TGT). TGT catalyzes the posttranscriptional base exchange of the Q precursor preQ1 with the genetically encoded guanine in tRNA(Asp,Asn,His,Tyr). The mutant tgt gene was cloned and sequenced; it contained a single point mutation resulting in the change of serine 90 to phenylalanine. Overexpression of the mutant gene yielded TGT(S90F) that showed a reduced solubility and did not purify in the same fashion as the wild-type enzyme. TGT(S90F) has no detectable enzymic activity. To determine whether serine 90 performs a catalytic role in the TGT reaction or whether the loss of activity was caused solely by a conformational change of the enzyme, we used site-specific mutagenesis to construct serine-to-alanine (S90A) and serine-to-cysteine (S90C) mutants. Both S90A and S90C mutants were purified in a manner identical to that used for the wild-type enzyme. SDS-PAGE of dimethyl suberimidate-cross-linked mutants showed a pattern identical to that of the wild-type TGT, indicative of a trimeric quaternary structure. Native PAGE of wild-type and mutant TGTs in the absence and presence of substrate tRNA exhibited band shifts indicating that both mutants retain the ability to bind tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Maternal umbilicus: ultrasound window to the gravid uterus.

Transabdominal sonograms of 68 consecutive pregnant women were reviewed retrospectively. Images of fetal parts obtained through the umbilical window (UW) were compared with those obtained through the paraumbilical window (PUW) as to (1) thickness of tissue to amniotic cavity, (2) clarity of the part imaged, and (3) noise produced. The paraumbilical tissue thickness had a mean of 18 mm; the umbilical tissue thickness, a mean of 11 mm. In patients where the difference in thickness of their paraumbilical tissue and umbilical tissue was only 0 mm to 6 mm (group 1), the UW improved clarity in 10 of 34 (29%), reduced clarity in 3 of 34 (9%), and did not change clarity in 21 of 34 (62%). Improvement of images with use of the UW was not statistically significant. However, in the group of patients with the difference in tissue thickness between the paraumbilical and umbilical areas of > 6 mm (group 2), clarity was improved in 19 of 32 (60%), reduced in 2 of 32 (6%), and was unaltered in 11 of 32 (34%). In this group the image improvement with the UW was statistically significant. Images through the UW had relatively less noise. In group 1, noise was reduced in 12 of 35 (34%). The effect of the UW on noise reduction was more significant in group 2 (tissue thickness difference > 6 mm): 24 of 31 (77%) of cases.

Female↗
PubMed1991

Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes.

The plasmid pPR20 contains the gene tgt, which encodes tRNA guanine transglycosylase (Tgt), on a 33-kbp DNA insert from a region around 9 min on the Escherichia coli linkage map. The plasmid was subcloned to determine the sequence and organization of the tgt gene. Tgt is a unique enzyme that exchanges the guanine residue with 7-aminomethyl-7-deazaguanine in tRNAs with GU(N) anticodons. After this exchange, a cyclopentendiol moiety is attached to the 7-aminomethyl group of 7-deazaguanine, resulting in the hypermodified nucleoside queuosine (Q). Here we give the complete sequence of a 3,545-bp StuI-BamHI DNA fragment where we found the tgt gene and three previously unknown genes encoding proteins with calculated molecular masses of 42.5 (Tgt), 14, 39, and 12 kDa. The gene products were characterized on sodium dodecyl sulfate gels after synthesis in a combined transcription-translation system. The mRNA start sites of the open reading frames (ORFs) were determined by primer extension analysis. Plasmids containing the ORF encoding the 39-kDa protein (ORF 39) complemented a mutation in Q biosynthesis after the Tgt step. This gene was designated queA. The genes are arranged in the following order: ORF 14 (transcribed in the counterclockwise direction), queA, tgt, and ORF 12 (all transcribed in the clockwise direction). The organization of the promoter sequences and the termination sites suggests that queA, tgt, and ORF 12 are localized on a putative operon together with the genes secD and secF.

Amino Acid Sequence↗
PubMed1989

Follicle size by ultrasound versus cervical mucus quality: normal and abnormal patterns in spontaneous cycles.

Ultrasound (US) has been demonstrated to be the method of choice for diagnosing luteinized unruptured follicle syndrome and to be a valuable adjuvant in the assessment of luteal phase defect. In this prospective study, the use of US with postcoital testing (PCT) is evaluated. Fifty control infertility patients were examined with serial US for follicle dynamics in conjunction with PCT. Standard curves for follicle dynamics versus cervical mucus quality (Insler score) were calculated. Eighteen patients referred for abnormal mucus underwent similar evaluation. Their follicle dynamics versus mucus quality were compared with those of the controls. The findings were: (1) there is a predictable relationship between follicle size and mucus quality, (2) the majority of patients with abnormal mucus have normal follicular dynamics, and (3) a minority of patients with abnormal mucus have either a narrow mucus window or abnormal follicular dynamics. In addition, US was found to be cost-effective in the overall fertility evaluation.

Cervix Mucus↗
PubMed1989

Antihypertensive contribution of sodium depletion and the sympathetic axis during chronic angiotensin II converting enzyme inhibition.

The known physiological adaptation of cardiovascular sensitivity to variations in angiotensin II (Ang II) levels would predict that the blood pressure (BP)-lowering effect of Ang II inhibition might be at least partly counterbalanced by enhanced Ang II reactivity. Therefore, factors other than Ang II inhibition per se may contribute to the antihypertensive mechanisms of angiotensin converting enzyme (ACE) inhibitors. In order to further investigate this, the body sodium-blood volume state as well as the pressor reactivity to infused Ang II or norepinephrine (NE) were assessed in 12 normal subjects and 16 patients with essential hypertension given a placebo, and after 6 weeks of intervention with enalapril (20-40 mg/day). Enalapril produced in both groups similar falls in plasma ACE activity (P less than 0.0001) and upright plasma aldosterone (P less than 0.01), and a rise in plasma renin activity (PRA; P less than 0.05). BP decreased from 156/107 +/- 3/2 (mean +/- s.e.m.) to 142/94 +/- 5/3 mmHg (P less than 0.001) in the hypertensives and from 118/84 +/- 4/2 to 111/73 +/- 4/3 mmHg (P less than 0.01) in the normal subjects. In the hypertensive patients only, the Ang II pressor reactivity relative to Ang II plasma levels during Ang II infusion was increased (P less than 0.01), while the NE pressor reactivity relative to NE plasma levels during NE infusion (P less than 0.01) as well as the exchangeable body sodium (-5%, P less than 0.001) were reduced significantly. Blood and plasma volume, levels of plasma atrial natriuretic factor and catecholamines, and the heart rate and its response to isoproterenol were unchanged in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Use of ultrasonography in the difficult postmenopausal dilation and curettage.

Six patients from our series of 21 cases are presented to illustrate the use of real-time ultrasound to complete the D&C. Thirteen of the 21 had concomitant ultrasound because of stenosis of the cervix or vagina, three because of previous vaginal procedures that distorted the anatomy, and five because of concern regarding perforation.

Aged↗
PubMed1988

XK aprosencephaly and anencephaly in sibs.

Recent studies have suggested a causal and pathogenetic relationship between holoprosencephaly and anencephaly. In support of the proposed relationship we report a sibship that includes anencephalic male twins and a female infant with a severe form of alobar holoprosencephaly, radial aplasia, and oligodactyly. The upper limb and brain malformations are considered to represent aprosencephaly syndrome. The coexistence of anencephaly and aprosencephaly within a sibship suggests that XK aprosencephaly syndrome may be an autosomal recessive disorder.

Adult↗
PubMed1987

Pharmacokinetics of amiloride in renal and hepatic disease.

The pharmacokinetics of the antikaliuretic amiloride has been studied in healthy controls and in patients with chronic renal failure or hepatitis. It was 40% bound to protein. In healthy volunteers 49% of an oral dose was recovered unchanged in the urine. The renal clearance of amiloride was about 3 times the creatinine clearance, which means that it was predominantly excreted via tubular secretion. Renal impairment reduced the clearance of amiloride, causing a prolongation of the t1/2 and drug accumulation in plasma. In hepatitis the t1/2 of amiloride was prolonged and the AUC increased. Urinary recovery (Ae) of amiloride was greater in hepatitis patients than in controls.

Adult↗