Biomedical subjects

R Kreutzer

Publications and source records attributed to R Kreutzer.

At least 19 recordsLinked to original sources

PubMed2001

Correlation between processing efficiency for ribonuclease P minimal substrates and conformation of the nucleotide -1 at the cleavage position.

It is demonstrated that acceptor stem duplexes derived from native tRNAs which contain a three-nucleotide extension at the 5'-terminus of mature tRNA are minimal substrates for ribonuclease P from both Escherichia coli and Bacillus subtilis. Variants with a cytidine at position -1 are most efficiently processed whereas the G -1 variant represents a comparatively poor substrate. An A -1 acceptor stem variant is a slightly better substrate than the G -1 variant though generally distinctly less efficient than the C -1 duplex. This is in qualitative agreement with the frequency of the occurrence of the corresponding nucleotides at position -1 in natural substrates, which is highest for pyrimidines and least for G. NMR analyses of the corresponding acceptor stems reveal that the conformation of the nucleotides at position -1 correlates with the substrate preferences of Ribonuclease P: Whereas C -1 adopts a conformation characterized by a glycosidic angle in the anti range (close to high-anti), the G -1 is clearly in syn conformation, and that of A -1 is intermediate between high-anti and syn. The riboses of nucleotides -1 are in all cases predominantly 2'-endo puckered.

Adenosine↗

PubMed2001

DNA-binding of phenylalanyl-tRNA synthetase is accompanied by loop formation of the double-stranded DNA.

The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus has previously been shown to bind DNA. We demonstrate that the "winged" helix-turn-helix motifs in the duplicate domains B5 are the relevant structural elements for this DNA-binding property. By altering particular amino acids in the "wing", the affinity of the FRS to DNA was significantly reduced. Based on experimental data, which indicate that the FRS prefers a certain DNA structure rather than a particular consensus sequence, we propose a novel loop model for the DNA-binding mode of the FRS. In our model we assume that two segments of the same DNA molecule are bound simultaneously by both B5 domains and are aligned in parallel, while the intervening DNA forms a loop. Due to the limited flexibility of the DNA, loop formation is only possible if the respective intervening DNA stretch exceeds a certain length. Several lines of evidence support this model. (1) We demonstrate by gel retardation assays that the DNA requires a minimal number of ca 80 base-pairs to be bound by the FRS. (2) In the presence of the FRS, DNA longer than ca 80 base-pairs has a significantly increased DNase I accessibility. This agrees well with its known preferential cleavage at positions where the minor grove is on the outside of looped-out DNA molecules. (3) The initial cleavage by DNase I of >80 bp long DNA occurs in the middle of the fragment. In a looped molecule this is the position with the highest accessibility to DNase I. The function of the FRS related to DNA binding is still unknown. Since the FRS exists in the nucleus of rapidly growing mammalian cells, and protein-induced DNA bending or looping contributes to several transcription, replication, and recombination systems in both prokaryotes and eukaryotes, it is likely that the FRS, in addition to its aminoacylation function, influences common cellular processes via DNA binding.

Acylation↗

PubMed2001

NMR spectroscopic evidence for Mn(2+)(Mg(2+)) binding to a precursor-tRNA microhelix near the potential RNase P cleavage site.

The binding of Mg(2+)/Mn(2+) to acceptor stem microhelices as minimal models for precursor-tRNA(Gly) is demonstrated by NMR spectroscopy. From the evaluation of COSY and NOESY spectra, binding sites for Mg(2+)/Mn(2+) can be inferred. In particular, one binding site exists near the ribose moiety of nucleotide -1 at the position of cleavage by RNase P. From comparison with a variant possessing a deoxynucleotide at this position, it is concluded that the 2'-OH group of this nucleotide is indispensable for coordinating the divalent metal ion. Hence, this catalytically important metal ion is "pre-bound" to the precursor-tRNA before complexation with RNase P.

Base Pairing↗

PubMed2001

Assessment of commercial laboratories performing hair mineral analysis.

CONTEXT: Hair mineral analysis is being used by health care practitioners and promoted by laboratories as a clinical assessment tool and to identify toxic exposures, despite a 1985 study that found poor reliability for this test. OBJECTIVE: To assess whether the reliability of data from commercial laboratories advertising multimineral hair analyses for nutritional or toxicity assessment has improved since the 1985 study. DESIGN, SETTING, AND PARTICIPANTS: A split hair sample taken from near the scalp of a single healthy volunteer was submitted for analysis to 6 commercial US laboratories, which analyze 90% of samples submitted for mineral analysis in the United States. MAIN OUTCOME MEASURES: Agreement of test results for each analyte, laboratory reference ranges, laboratory characteristics, and interpretation of health implications. RESULTS: Laboratory differences in highest and lowest reported mineral concentrations for the split sample exceeded 10-fold for 12 minerals, and statistically significant (P<.05) extreme values were reported for 14 of the 31 minerals that were analyzed by 3 or more laboratories. Variations also were found in laboratory sample preparation methods and calibration standards. Laboratory designations of normal reference ranges varied greatly, resulting in conflicting classifications (high, normal, or low) of nearly all analyzed minerals. Laboratories also provided conflicting dietary and nutritional supplement recommendations based on their results. CONCLUSIONS: Hair mineral analysis from these laboratories was unreliable, and we recommend that health care practitioners refrain from using such analyses to assess individual nutritional status or suspected environmental exposures. Problems with the regulation and certification of these laboratories also should be addressed.

Biomarkers↗

PubMed1999

Prevalence of people reporting sensitivities to chemicals in a population-based survey.

To describe the prevalence and correlates of reports about sensitivities to chemicals, questions about chemical sensitivities were added to the 1995 California Behavior Risk Factor Survey (BRFS). The survey was administered by telephone to 4,046 subjects. Of all respondents, 253 (6.3%) reported doctor-diagnosed "environmental illness" or "multiple chemical sensitivity" (MCS) and 643 (15.9%) reported being "allergic or unusually sensitive to everyday chemicals." Sensitivity to more than one type of chemical was described by 11.9% of the total sample population. Logistic regression models were constructed. Hispanic ethnicity was associated with physician-diagnosed MCS (adjusted odds ratio (OR) = 1.82, 95% confidence interval (CI) 1.21-2.73). Female gender was associated with individual self-reports of sensitivity (adjusted OR = 1.63, 95% CI 1.23-2.17). Marital status, employment, education, geographic location, and income were not predictive of reported chemical sensitivities or reported doctor diagnosis. Surprising numbers of people believed they were sensitive to chemicals and made sick by common chemical exposures. The homogeneity of responses across race-ethnicity, geography, education, and marital status is compatible with a physiologic response or with widespread societal apprehensions in regard to chemical exposure.

Adolescent↗

PubMed1999

Asthma hospitalization trends in California, 1983-1996.

Annual asthma hospitalization rates were calculated for California's ethnically diverse population from 1983 through 1996. Trends were examined for four race/ethnicity groups: Hispanics, African-Americans, non-Hispanic Caucasians, and Asians/Pacific Islanders. The overall rate decreased by 30% during the time period. African-Americans had the highest rate, more that three times greater than the rate for Caucasians. Among children, the rates for Caucasians decreased by one-third, while rates increased for Hispanics and Asians. The rate for African-American children remained generally constant and was four times higher than the rate for Caucasians. Data from 1996 were assessed for repeat admissions, age and sex differences in rates, costs, and progress toward national goals.

Adolescent↗

PubMed1998

The phenylalanyl-tRNA synthetase specifically binds DNA.

The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus is modularly composed of several different domains, some of which are not required for aminoacylation. In particular, the enzyme has the structural prerequisites for a DNA-binding protein. We demonstrate by gel retardation and competition experiments that the FRS specifically binds certain DNA sequences of the T. thermophilus genomic DNA. Although the implication of this finding is not yet understood, increasing evidence indicates an alternative function of this enzyme not related to aminoacylation. This might be a fundamental cellular process involved in cell proliferation which is related in bacteria and in humans.

Acylation↗

PubMed1998

Glycyl-tRNA synthetase from Thermus thermophilus--wide structural divergence with other prokaryotic glycyl-tRNA synthetases and functional inter-relation with prokaryotic and eukaryotic glycylation systems.

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes. Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly-tRNA synthetase from T. thermophilus acylates eukaryotic tRNA(Gly) while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNA(Gly). A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1-C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10-Y25)-G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set. These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.

Amino Acid Sequence↗

PubMed1997

Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation.

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA(Phe) was studied. Removal of domain 8 of the beta subunit resulted in drastic reduction of the dissociation constant of the FRS x tRNA(Phe) complex. Neither the removal of arginine 2 of the beta subunit, which makes the only major contact between domains beta1-5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1-5 of the beta subunit may not be essential for efficient aminoacylation of tRNA(Phe) by the FRS and rather be involved in other functions.

Acylation↗

PubMed1997

Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes.

Two aspartyl-tRNA synthetases (AspRSs) were isolated from Thermus thermophilus HB8. Both are alpha2 dimers but differ in the length of their polypeptide chains (AspRS1, 68 kDa; and AspRS2, 51 kDa). Both chains start with Met and are deprived of common sequences to a significant extent. This rules out the possibility that AspRS2 is derived from AspRS1 by proteolysis, in agreement with specific recognition of each AspRS by the homologous antibodies. DNA probes derived from N-terminal amino acid sequences hybridize specifically to different genomic DNA fragments, revealing that the two AspRSs are encoded by distinct genes. Both enzymes are present in various strains from T. thermophilus and along the growth cycle of the bacteria, suggesting that they are constitutive. Kinetic investigations show that the two enzymes are specific for aspartic acid activation and tRNAAsp charging. tRNA aspartylation by the thermostable AspRSs is governed by thermodynamic parameters which values are similar to those measured for mesophilic aspartylation systems. Both thermophilic AspRSs are deprived of species specificity for tRNA aspartylation and exhibit N-terminal sequence signatures found in other AspRSs, suggesting that they are evolutionarily related to AspRSs from mesophilic prokaryotes and eukaryotes. Comparison of the efficiency of tRNA aspartylation by each enzyme under conditions approaching the physiological ones suggests that in vivo tRNAAsp charging is essentially ensured by AspRS1, although AspRS2 is the major species. The physiological significance of the two different AspRSs in T. thermophilus is discussed.

Amino Acid Sequence↗

PubMed1997

A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix.

The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition.

Alanine-tRNA Ligase↗

PubMed1997

Organization of the Thermus thermophilus nusA/infB operon and overexpression of the infB gene in Escherichia coli.

The structural gene for translation initiation factor IF2 from Thermus thermophilus was identified on the basis of the N-terminal amino acid sequence of intact T thermophilus IF2 and an internal 25 kDa IF2 fragment. A total of 5135 bp was cloned and sequenced, comprising the open reading frames for p15A, NusA, p10A, IF2, p10B and SecD, which may form an operon. There are pronounced similarities between the operon arrangement and primary sequence of the T thermophilus genes and proteins, respectively, and their counterparts from other organisms. The T thermophilus infB gene was expressed to a high level in E coli. Four hundred milligrams of homogenous T thermophilus IF2 were prepared from 60 g of overproducing cells.

Amino Acid Sequence↗

PubMed1997

Biological monitoring for mercury within a community with soil and fish contamination.

To assess the impact of elevated levels of inorganic mercury in soil and dust and organic mercury in fish, biological monitoring was conducted among Native Americans living next to an inactive mercury mine in Clear Lake, California. Of resident tribal members, 46% (n = 56) participated in biomonitoring. Urine mercury levels are equivalent to background, indicating that soil and dust exposures among study participants are not substantial. The average blood organic mercury level among study participants is 15.6 +/- 8.8 micrograms/l (n = 44), which is higher than levels reported by others among those who do not consume fish (2 micrograms/l). Consistent with results from other studies, a correlation between fish consumption and blood organic mercury is observed (p = 0.03). The margin between observed and established adverse effect levels for adults is examined for blood organic mercury and found to be less than 10-fold for 20% of the study population. Protective public health efforts for the study population and other similarly exposed populations, notably those who consume commercial fish products, are considered.

Adult↗

PubMed1997

Rice burning and asthma hospitalizations, Butte County, California, 1983-1992.

We investigated the association between rice burning and daily asthma hospitalizations in Butte County, California, from 1983 to 1992. Eighty-two percent of planted rice was burned, with a mean of 555 acres burned on days when burning was permitted. For 60% of the days during this period, no rice burning occurred. Peak burning occurred in fall and spring but was not correlated with criteria pollutants. Asthma admissions averaged 0.65/day and peaked in March. In the basic Poisson model with daily asthma hospitalizations as the outcome of interest, burn acreage showed a small but statistically significant elevation of risk for hospitalization per acre of rice burned [relative risk (RR) = 1.0001; 95% confidence interval (CI), 1. 00004-1.0002], after adjusting for maximum daily temperature, seasonal factors, and yearly population. In this model, burn acreage showed a dose-response effect as acreage burned increased. Days with the greatest acreage burned (>499 acres) had the largest risk of hospitalization (RR = 1.23; CI, 1.09-1.39), and days with moderate burning (between 100 and 499 acres) had a slightly lower risk of admission (RR = 1.2; CI, 1.05-1.37). Elevations of air pollutants were not associated with days of increased rice burning; however, rice burn acreage was shown to have a small but statistically significant effect on asthma morbidity in Butte County. This evidence suggests that further limitations on the daily amount of rice straw permitted to be burned should be considered to reduce pulmonary morbidity related to asthma.

Adolescent↗

PubMed1997

Pavlovian conditioning and multiple chemical sensitivity.

Pavlovian conditioning processes may contribute to some symptoms of multiple chemical sensitivity (MCS). This review summarizes the potential relevance of the literature on conditional taste and olfactory aversions, conditional sensitization, and conditional immunomodulation to understanding MCS. A conditioning-based perspective on MCS suggests novel research and treatment strategies.

Animals↗

PubMed1996

Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu.

The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis. The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu. EF-Tu that was cleaved at the Arg59-Gly60 peptide bond [EF-Tu-(1-59)/EF-Tu-(60-405)] bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis. However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T. thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu. EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405). By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T. thermophilus EF-Tu. These amino acid substitutions did not substantially affect either the affinity of EF-Tu. GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity. The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.

Amino Acid Sequence↗

PubMed1996

Elongation factor Ts from Thermus thermophilus-- overproduction in Escherichia coli, quaternary structure and interaction with elongation factor Tu.

The gene encoding the elongation factor Ts from Thermus thermophilus was sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF-Ts from E. coli with 282 amino acid residues, EF-Ts from T. thermophilus is considerably shorter, differing by 86 amino acids. EF-Ts from the thermophile is stable at high temperatures, which facilitates its separation from E. coli proteins. Purified T. thermophilus EF-Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190. The modification of Cys19O by iodoacetamide affects neither the dimerization nor the ability of EF-Ts to facilitate the nucleotide exchange of elongation factor Tu. The disulfide bridge was detected only in purified EF-TS, but not in protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF-TU . EF-Ts)2 complex, a ternary EF-TU . EF-Ts2 complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capable of forming homodimers.

Amino Acid Sequence↗

PubMed1996

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↗