Laboratory lotto? Are you gambling with your success?
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Communication↗
Biomedical subjects
B Keller
Publications and source records attributed to B Keller.
Overproduction of phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 in Escherichia coli.
The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus was overproduced in Escherichia coli. Three different promoter systems were used for the overexpression of the pheST genes: the tac, araB, and T7 promoters. Despite several attempts for improvement, the overproduction of the FRS was lower than that found with most of the other T. thermophilus genes. Nevertheless, enzyme amounts sufficient for biochemical and biophysical studies could be obtained more easily from the overproducing E. coli than from T. thermophilus, since at least fivefold higher specific FRS activity was present in the overproducing cells than in T. thermophilus. Also, a simple purification procedure was established. After heat treatment at 70 degrees C to remove thermolabile E. coli proteins, only three chromatographic steps, i.e., Q-Sepharose FF, hydroxyl apatite, and heparin-Sepharose chromatography, were necessary to obtain apparently homogeneous FRS. With a different plasmid construction we introduced six histidine residues at the N terminus of the alpha subunit. Thus, affinity chromatography on a nickel-chelate matrix can be used for the purification of FRS as well as for its mutant variants, which may be less stable than the native FRS and cannot be purified with heat treatment. We also cloned the pheST genes in a phagemid, which will enable mutagenesis studies and overexpression in a one-vector system without any subcloning steps.
Amino Acid Sequence↗
Common occurrence of homologues of petunia glycine-rich protein-1 among plants.
The presence of specific glycine-rich proteins (GRP) related to petunia GRP1 (ptGRP1) was examined in three species of monocots (wheat, barley and maize) and five species of dicots (rape, turnip, soybean, crabapple and tomato). Protein blot analysis showed that anti-ptGRP1 antibody cross-reacted with a single different polypeptide in all species except maize. The molecular mass of these polypeptides ranged from 14 to 55 kDa. Tissue-print immunoblots of rape petioles and stems showed that the rape ptGRP1 homologue, like ptGRP1, is primarily located in the vascular tissue, and that its expression decreases with developmental age of the tissue. In barley, the ptGRP1 homologue is found in leaf vascular bundles, and may also be present in the surrounding bundle sheaths. Unlike the dicots examined, expression of the protein did not appear to decrease significantly with developmental age.
Blotting, Western↗
In vitro binding of the tomato bZIP transcriptional activator VSF-1 to a regulatory element that controls xylem-specific gene expression.
The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs-1) specifically activated both the -82 CaMV 35S and the -76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro. To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro. This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal -82 CaMV 35S promoter in vivo. Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the -76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.
Amino Acid Sequence↗
Streamlining documentation: an opportunity to reduce costs and increase nurse clinicians' time with patients.
Clinical documentation can take up to 25 percent of a nurse's time in an acute care setting, not to mention the time spent by other clinicians and ancillary caregivers on documentation. Institutions can reduce documentation time by systematically determining what information is crucial to the provision of patient care across all disciplines and then using a redesign process that mirrors work redesign. By following a structured process that challenges the philosophy and function of the documentation system, a hospital's multidisciplinary staff members can use their expertise to design alternatives that achieve documentation's original intent-to document diagnoses and treatment accurately-in a more efficient and useful way.
Cost Control↗
Genetic and physical characterization of the LR1 leaf rust resistance locus in wheat (Triticum aestivum L.).
The objective of this study was to characterize the leaf rust resistance locus Lr1 in wheat. Restriction fragment length polymorphism (RFLP) analysis was performed on the resistant line Lr1/6* Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F2 populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism between Lr1/6* Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs) Lr1/6* Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F2 populations. One probe (pTAG621) showed very tight linkage to Lr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing the Lr1 resistance gene in different genetic backgrounds showed the same band as Lr1/6* Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant line Lr1/6* Thatcher. This STS, specific for the Lr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked to Lr1 should ultimately provide the basis for positional cloning of the gene.
Alleles↗
[Endorectal advancement flap-plasty vs. transperineal closure in surgical treatment of rectovaginal fistulas. A prospective long-term study of 88 patients].
UNLABELLED: A prospective study was carried out on 88 patients with rectovaginal fistulae to evaluate the value of two sphincter-saving techniques: primary occlusion of the intraanal ostium and endorectal advancement flap (n = 37) or transperineal repair with levator interposition (n = 34). Causes were Crohn's disease 35, obstetric injury 31, proctological-gynecological operation 11, cryptoglandular 11. Perineal group: 11 patients underwent concomitant anterior sphincter plication. Crohn group (n = 35): endorectal advancement flap was performed in 8 patients only, and 10 with intra- or supraanal stenosis were treated by transperineal approach, 12 (34%) with extended perianal fistula complaints required primary proctectomy, and operative therapy was not possible in 5 with persistent rectal inflammation. No deaths occurred. Postoperatively 12 cases (17%) of suture leakage occurred (flap group (FG): 16.2%, transperineal group (TPG): 17.6%). Persistent or recurrent fistula occurred in 8 patients (11%), 5.4% FG, 17.6% TPG. Disturbance of continence was observed in one patient after endorectal approach. Postoperatively there were no significant changes in the resting anal pressure and maximum voluntary contraction pressure. A complete primary healing with no further recurrence (follow-up 3 months to 9.5 years) was noted in 78.4% FG and 64.7% TPG. One patient with postoperative incontinence after the endorectal flap, had undergone anterior levator plication with perineal body reconstruction. CONCLUSIONS: Endorectal advancement flap allows preservation of the sphincter and is an effective method for repair of rectovaginal fistulae. The endorectal advancement flap proved to result in a better primary healing rate with 85% than the mucosal advancement flap with 65%. Perineal procedures are indicated in selected patients with simultaneous sphincter plication and in Crohn's fistulae associated to intra- or supraanal stenosis.
Colostomy↗
Human liver cytochrome P4503A biotransformation of the cyclosporin derivative SDZ IMM 125.
In humans, cytochrome P4503A (CYP3A) is the major cytochrome P450 gene family that metabolizes SDZ IMM 125 (IMM) to its primary metabolites. Human liver microsomes could be used for this study, because the metabolite profile matched that found in human blood. The apparent affinity (KM) of IMM for the cytochrome P450 proteins (5.1 +/- 1.8 microM) is similar to that of cyclosporin A (CSA). CSA competitively inhibited the metabolism of IMM, increasing the KM 2- and 4.6-fold in the presence of 4 and 10 microM CSA, respectively (Ki 3.8 +/- 1.1 microM). Ketoconazole exhibited competitive inhibition kinetics toward IMM biotransformation, increasing the KM of IMM 1.8-fold at 0.5 microM ketoconazole and 3.5-fold at 1 microM ketoconazole, with no effect on Vmax (Ki of 0.5 +/- 0.4 microM). These results indicate that both CSA and ketoconazole would cause drug interactions, interfering with the biotransformation of IMM. The metabolism of IMM was also greatly inhibited (approximately 80%) by the CYP3A suicide substrate triacetyloleandomycin and a CYP3A inhibitory antibody, indicating the involvement of CYP3A proteins in the biotransformation of IMM. Confirmation of CYP3A4 involvement in the formation of the three primary IMM metabolites was demonstrated with recombinant cells expressing human CYP3A4. Therefore, compounds interacting with CYP3A proteins are expected to cause drug-drug interactions (i.e. the antimycotics ketoconazole and clotrimazole, the steroids ethinylestradiol and testosterone, the ergots, the calcium channel blocker nifedipine, and the immunosuppressants FK-506 and rapamycin).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Sites of biotransformation for the cyclosporin derivative SDZ IMM 125 using human liver and kidney slices and intestine. Comparison with rat liver slices and cyclosporin A metabolism.
SDZ IMM 125 (IMM), the hydroxyethyl derivative of cyclosporin A (CSA), is metabolized by human liver slices to analogous primary metabolites, hydroxylated IMM1 and IMM9 and N-demethylated IMM4N, as for CSA (M17/AM1, M1/AM9, and M21/AM4N), but the rate and extent of IMM biotransformation is less than for CSA. Initial rates of IMM metabolite formation in the human liver slice cultures are 6.6 +/- 2.8 nmol/hr/g liver at 1 microM IMM and 24.3 +/- 22.9 nmol/hr/g liver at 10 microM IMM, whereas the rate of CSA metabolite formation is 1.8-fold faster at both concentrations. The percentage of unchanged IMM is 73% at 1 microM and 80% at 10 microM after 24 hr, reflecting the lower extent of IMM metabolism, about one-third (1 microM) and one-half (10 microM) that of CSA. In rat liver slices, IMM is metabolized to the same primary metabolites as in human liver slices, but more slowly and remains 90% unchanged at 24 hr. Human jejunum formed the same primary metabolites of IMM and CSA as in liver. Upscaling the slice rate of biotransformation revealed that human jejunum would contribute considerably to the first-pass of IMM and CSA, being approximately 2 to 3-fold slower than the rate in liver. The inhibition of both IMM and CSA biotransformation by triacetyloleandomycin implicates the involvement of cytochrome P4503A proteins. Human kidney cortex slices metabolized IMM to IMM1 and IMM9, accounting for approximately 75% of the total metabolites. Total metabolite formation represented approximately 64% of liver metabolite formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Cloning and sequencing of the pheU gene for tRNA(Phe) of Thermus thermophilus HB8, and genomic mapping of the pheU and pheST genes.
The gene pheU for tRNA(Phe) from the extreme thermophile Thermus thermophilus HB8 was cloned and sequenced. It differed from the published tRNA(Phe) sequence by one nucleotide. Both the pheU gene and the pheST genes encoding the alpha and beta subunits of phenylalanyl-tRNA synthetase were located on the physical map of the T. thermophilus chromosome, where pheU was present on a hitherto unrecognized HpaI DNA fragment.
Base Sequence↗
Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence.
The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco. Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems. A 169 bp fragment (-205 to -36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (-205 to -64) strongly activated these minimal promoters both in vascular and cortical cells. These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between -64 and -36. The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations. A 24 bp sequence (bp -119 to -96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (-98 to -73, corresponding to the RSE regulatory element) induced vascular-specific expression. Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression.
Base Sequence↗
Anti-thyroid peroxidase (anti-TPO) antibodies in thyroid diseases, non-thyroidal illness and controls. Clinical validity of a new commercial method for detection of anti-TPO (thyroid microsomal) autoantibodies.
The identification of the thyroid peroxidase (TPO) as the main antigen of the thyroid microsomal fraction has enabled the development of a sensitive and specific assay for detection of the corresponding autoantibodies. We evaluated the diagnostic validity of the anti-TPO assay in 303 patients with different types of thyroid disease and in controls. Clearly elevated anti-TPO values (anti-TPO > 500 units/ml) were found in 59% of patients with thyroiditis but in none of the controls or the patients with non-thyroidal illness. The mean anti-TPO levels in these two control groups were 26 +/- 31 units/ml (mean +/- S.D.) and 39 +/- 34 units/ml, respectively. The highest frequency of positive results (88%) was obtained in patients with auto-immune hypothyroidism (clinical diagnosis: Hashimoto's thyroiditis) followed by patients with Graves' disease (53%). With a cut-off point of 200 units/ml, a sensitivity of 96% was obtained for Hashimoto's thyroiditis and of 59% for Graves' disease with a specificity of 100% (50 cases). The new method (anti-TPO, Dynotest) was compared with three conventional methods (35 samples). The results for all measurements were in general agreement. In two cases the results were clearly discordant: one sample contained high anti-thyroglobulin antibody concentrations, the other was obtained from a patient with non-thyroidal illness. In both instances the 'classical' assays yielded false-positive results. Treatment of autoimmune hyperthyroidism resulted in a median decrease in anti-TPO levels of over 50% after reaching the euthyroid state (P < 0.05), whereas in persistent hyperthyroidism no consistent changes were observed. In autoimmune hypothyroidism a marked variability in anti-TPO levels was noted. Some patients showed a clear decrease in anti-TPO levels during T4 substitution whereas in others no consistent changes were observed.
Autoantibodies↗
Variance function as basis for assessment of test performance: methodological studies with two assays of prostate-specific antigen.
We evaluated the usefulness of a recently described procedure to assess the analytical performance of an assay. To demonstrate the advantages of this approach, we compared the performance of two analytical systems for determining prostate-specific antigen (PSA). Triplicate measurements of PSA with the IMx (Abbott) and the ACS 180 (Ciba Corning) were used to calculate the variance function. This function was the basis for the derivation of the critical limit (LC), the limit of detection (LD), the power of definition (PD), and the lower limit of the quantification interval. The standard deviation of the blank was extrapolated by means of the variance function. LC was calculated as the concentration at which the normal distribution of the blank intersects an adjacent normal distribution (with a defined overlap, e.g., 5%). The mean of the adjacent normal distribution represents the LD. The PD is a new mathematical approach to describe the analytical sensitivity of an assay in different ranges of the quantification interval. The procedure is statistically well defined and allows one to obtain the data on the test performance directly from patients' samples, without artificial zero controls. Therefore, use of the variance function could be a general model for the assessment of the analytical performance of an assay.
Chemistry, Clinical↗
A new look at the limits of detection (LD), quantification (LQ) and power of definition (PD)
The relationship between the concentration of the analyte and the imprecision of an analytical method can be displayed by the precision profile in which the coefficient of variation (relative standard deviation) is plotted against the concentration of the analyte. The function of the curve of the profile and its confidence limits can easily be assessed by a computer program developed by W.A. Sadler & M.H. Smith (Clin. Chem. 36 (1990), 1346-1350). For the assessment of limits of detection and of quantification the following procedure is proposed: The lower (and upper) limit of the measuring interval is defined by the point at which an acceptable CV-line intersects the confidence limit. If, in the variance function one sets the concentration to zero, the normal distribution of the random errors of the blank will result. The mean of the next adjacent normal distribution, following the variance formula and overlapping the "zero-distribution" by a defined amount, represents the limit of detection. Within the described measuring interval, or within a fraction of it, one might construct overlapping normal distributions in an analogous manner. Their number represents the "power of definition" (PD) (instead of the "analytical sensitivity"), which also depends on the concentration of the determinand according to the variance function. We tested these hypotheses by a comparison of two methods for the determination of cyclosporin A (ciclosporin, INN). Our results demonstrate that the data of the lower limits of the measuring interval and of the limit of detection agree well with data from the literature obtained in extensive interlaboratory surveys.
Algorithms↗
[10 years of rabbit autopsy--a review of frequent disease and mortality causes].
Between 1982 and 1991 3403 diseased and dead rabbits from the field were dissected. The most important findings were intestinal coccidiosis (24.1%), dysentery (20.5%), liver coccidiosis (6.9%), mucoid enteritis (5.0%), acute pasteurellosis (4.2%), pneumonia (4.0%), rhinitis (3.6%) and adiposis (3.3%). The incidence of these diseases is similar to the most important rabbit diseases in other European countries. Short recommendations on prophylactic and therapeutic measures are given.
Animals↗
[Which diagnosis do you propose? Rabbit viral hemorrhagic disease].
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Animals↗
[Diagnostic value of autoantibodies against microsomal thyroid peroxidase (anti-TPO)].
With the identification of thyroid peroxidase (TPO) as the main antigen of the thyroid microsomal fraction, the development of a sensitive and specific assay for detection of these antibodies became possible. The diagnostic value of the anti-TPO assay was evaluated in patients with different types of thyroid disease and in controls. 65% of patients with thyroiditis had elevated anti-TPO values. The frequency of elevated anti-TPO levels in patients with active autoimmune thyroiditis was 90% (clinical diagnosis Hashimoto thyroiditis) compared to 64% in patients with overt hyperthyroidism (clinical diagnosis Graves' disease). None of the controls or the patient with non-thyroidal illness showed elevated anti-TPO levels.
Autoantibodies↗
Cloning and sequence analysis of the phenylalanyl-trna synthetase genes (pheST) from Thermus thermophilus.
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Molecular Sequence Data↗