Biomedical subjects

H Paulus

Publications and source records attributed to H Paulus.

At least 37 records · Page 2Linked to original sources

PubMed1998

When are bioavailability studies required? A German proposal.

The Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM), the German drug regulation authority, issued guidelines for determining whether bioavailability/bioequivalence studies are required for certain drugs. This decision tree is based on pharmacodynamic, pharmacokinetic, and physicochemical criteria. Details of this decision tree were worked out by an expert panel, the Bioavailability Commission at the BfArM. The decision tree has been in use by German regulatory authorities for more than 10 years. In the meantime, its essentials were adopted by the European Committee for Proprietary Medicinal Products (CPMP) and by the World Health Organization (WHO) for their "Guidelines on interchangeability of multisource pharmaceutical products." This article reviews the original decision tree of the BfArM and provides examples of drugs that have been assessed according to its rules. The current procedure of the German regulatory authorities for judging the necessity of bioavailability trials, which reflects the status quo of regulatory practice in Germany, is also discussed.

Biological Availability↗

PubMed1998

Costs attributable to osteoarthritis.

OBJECTIVE: To estimate charges attributable to osteoarthritis (OA) in a managed care organization. METHODS: Longitudinal study based on insurance claims incurred and filed between 1991 and 1993 in a national managed care organization. Patients with claims for OA were randomly sampled to yield 20,000 study subjects. Charges per person-year were determined for these patients and compared to those of comparison subjects matched for age, sex, and insurance plan without claims for OA. RESULTS: Total charges per patient-year adjusted to 1993 dollars for patients with OA <65 and > or =65 years of age were $5,294 and $5,704, respectively, while charges for controls were $2,467 and $3,741, respectively. Thus, charges due to OA were $2,827 and $1,963, accounting for 5% of total plan charges. CONCLUSION: Patients with symptomatic OA incur charges for medical care at about twice the rate of plan enrollees without claims for OA and account for a substantial proportion of total charges in a managed care plan.

Aged↗

PubMed1997

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Affinity Labels↗

PubMed1997

Identification of ribosomal protein S1 as a poly(A) binding protein in Escherichia coli.

To elucidate the metabolic function of mRNA polyadenylation in Escherichia coli. we searched for a polyadenylate-binding protein as a potential mediator of the function of the poly(A) moiety. Using a nitrocellulose filter-binding assay and a Northwestern blot technique, a protein in the ribosomal supernatant fraction of E coli was identified and purified to homogeneity. N-terminal sequence analysis yielded a 25-residue sequence which corresponded to the 25 N-terminal amino acids of protein S1, one of the proteins of the E coli 30S ribosomal subunit. Poly(A) binding to S1 protein was inhibited by Mg2+ and Mn2+ and by ATP and stimulated 8-fold by 100 mM KCl. The binding of S1 to poly(A) occurred with an association constant of 3 x 10(6) M-1 and seemed to be only mildly cooperative. Competition studies of the binding of poly(A) and poly(C) to purified S1 protein were consistent with the presence of two polynucleotide binding sites, of which one binds poly(A) five times more strongly than poly(C), whereas the other binds poly(C) 50 times more strongly than poly(A). Poly(A) bound to 30S ribosomal subunits but not to 50S ribosomes. To study possible association of S1 with the poly(A) tracts of E coli mRNA in the process of translation, poly(A) RNA was isolated from polysomes by oligo(dT) cellulose chromatography and the poly(A) RNA with bound protein was eluted either directly or after digestion with RNase T1 and A. When subjected to Western blot analysis with antibody to S1, both poly(A) RNA and isolated poly(A) tracts revealed bound S1 protein. The implications of these results for the possible interaction of poly(A) tracts of mRNA and the translational machinery of E coli are discussed.

Bacterial Proteins↗

PubMed1997

Protein splicing and autoproteolysis mechanisms.

It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.

Animals↗

PubMed1997

Protein splicing: estimation of the rate of O-N and S-N acyl rearrangements, the last step of the splicing process.

The last step in the sequence of reactions that lead to protein splicing is the intramolecular O-N or S-N acyl rearrangement of the ester or thioester linkage, respectively, between the two exteins and hydrolysis of the aminosuccinimide residue at the C-terminus of intein. This paper presents data on the rates of O-N and S-N acyl rearrangements of two model depsipeptides as a function of pH and temperature. The rates of rearrangement of both the oxygen ester and the thioester depsipeptide increased strikingly with pH, with the thioester being about 10(3) times more reactive at pH 5.5, and had a relatively low dependence on temperature, indicative of a low activating energy. The rates of O-N and S-N acyl rearrangement of these two model depsipeptides greatly exceed the rate of protein splicing, explaining why the last step of protein splicing can occur without catalysis by the intein.

Acylation↗

PubMed1996

Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.

Amino Acid Sequence↗

PubMed1996

Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process.

Protein splicing involves the self-catalyzed formation of a branched intermediate, which then resolves into the excised intervening sequence and the spliced protein. A possible mechanism for branched intermediate formation is an N-O rearrangement of the peptide bond involving the amino group of the conserved serine/cysteine residue at the upstream splice junction to yield a linear peptide ester intermediate. This possibility was examined in using an in vitro splicing system involving the intervening sequence from the DNA polymerase of the extremely thermophilic archeon, Pyrococcus sp. GB-D. Because thioesters react much more rapidly with nitrogen nucleophiles at neutral pH than do oxygen esters, protein-splicing precursors in which the serine residue of interest was replaced by cysteine were constructed and purified. In the presence of 0.25 M hydroxylamine or 0.1 M ethylene diamine at pH 6 or higher, these constructs underwent rapid cleavage at the upstream splice junction, consistent with the aminolysis of a thioester. The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage products. Comparison of the C-terminal peptide hydroxamate with the synthetic peptide hydroxamates with respect to chromatographic mobility, colorimetric assay, amino acid composition, and high-resolution mass spectrometry showed that the hydroxylamine-sensitive site in the splicing precursor was the peptide bond adjacent to the serine residue at the upstream splice junction. These results provide evidence that the peptide bond at the upstream splice junction can undergo a self-catalyzed N-O or N-S acyl rearrangement to yield a linear polypeptide ester intermediate and suggest that this kind of rearrangement constitutes the first step in protein splicing.

Amino Acid Sequence↗

PubMed1996

Lysine-induced premature transcription termination in the lysC operon of Bacillus subtilis.

The expression of the Bacillus subtilis lysC operon, which encodes the first specific enzyme of lysine biosynthesis, is controlled by the availability of the end product, lysine. The question of whether lysine exerts its control by inducing premature termination of transcription was addressed using Northern blot analysis. Whereas lys-C-specific RNA from lysine-starved B. subtilis consisted primarily of the expected full-length mRNA (1.6 kb), that from bacteria grown with an excess of lysine consisted of a truncated 0.27 kb RNA in place of the full-length 1.6 kb transcript. On the other hand, a B. subtilis aecA mutant, in which the lysC operon was derepressed owing to a single nucleotide substitution in the region corresponding to the lysC leader transcript, produced full-length lysC mRNA, but no 0.27 kb RNA, even during growth with excess lysine. Mapping of the truncated 0.27 kb lysC RNA by hybridization with oligonucleotide probes showed that it corresponded to the upstream portion of the lysC leader transcript, extending from the transcription initiation site to a putative rho-independent terminator element. Quantitative transcript analysis by hybridization with specific oligonucleotides showed that lysine did not affect the number of lysC-specific RNA molecules but promoted the stoichiometric replacement of full-length mRNA with truncated 0.27 kb molecules. These results indicate that lysine regulates the expression of the lysC operon by effecting the premature termination of transcription at a rho-independent terminator site in the lysC leader region and that the site of the aecA mutation, far upstream of the putative terminator element, must play an essential role in premature transcription termination by a mechanism which is not yet understood.

Bacillus subtilis↗

PubMed1996

The relationship between socioeconomic status and recently diagnosed rheumatoid arthritis.

OBJECTIVE: To examine the role of socioeconomic status (SES) in physical functioning, pain, and depressive symptoms among newly diagnosed rheumatoid arthritis (RA) patients with severe disease. METHODS: Data are from 118 non-Hispanic patients of European origin at baseline of a longitudinal study of early, severe RA. Outcome measures with the Health Assessment Questionnaire (HAQ) functional disability index, the HAQ visual analog pain scale, and the Center for Epidemiologic Studies Depression Scale. Hierarchical regression analyses were conducted using a health status block (disease activity and comorbidities), a non-SES related social structure block (age, sex and Lubben Social Network Scale), and indicators of SES (income and education). RESULTS: Non-SES related social structure and SES were important independent determinants of functional disability and depressive symptoms, but both they and the health status variables were unrelated to pain. Further, neither income nor education was related to disease activity or comorbidities. CONCLUSION: It cannot be argued from these data that poorer health status explains the link between SES and disability and depressive symptoms. Although, at baseline, the psychosocial effects of early RA are more severe for those with SES, the disease does not appear to be more severe. It may be that the biologic impact of status differentials will become clearer as the effects of treatment and the course of the disease unfold over time.

Activities of Daily Living↗

PubMed1995

Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence.

Protein splicing is a self-catalyzed, posttranslational process which converts a precursor polypeptide into two new proteins by the excision of an internal polypeptide segment and the ligation of the flanking polypeptides. Evidence has been presented that protein splicing involves a branched intermediate, which is resolved into the two protein products by the cyclization of an asparagine residue to aminosuccinimide [Xu, M. Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., & Perler, F. (1994) EMBO J. 13, 5517-5522]. This report describes the chemical synthesis of a peptide with a C-terminal aminosuccinimide residue, corresponding to the putative C-terminus of the excised intervening sequence (intein) derived from the thermostable DNA polymerase of Pyrococcus species GB-D. The synthetic aminosuccinimide peptide was compared with the C-terminal cyanogen bromide peptide of the excised intein and found to be indistinguishable in terms of its chromatographic properties, high-resolution mass spectrum, and colorimetric assay involving reaction with hydroxylamine. This establishes definitively that protein splicing is accompanied by the cyclization of asparagine to yield an aminosuccinimide residue at the C-terminus of the excised intein and that this unusual residue is therefore a natural constituent of spliced proteins. The effects of pH and temperature on the stability of the synthetic aminosuccinimide peptide are described.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

American College of Rheumatology. Preliminary definition of improvement in rheumatoid arthritis.

OBJECTIVE: Trials of rheumatoid arthritis (RA) treatments report the average response in multiple outcome measures for treated patients. It is more clinically relevant to test whether individual patients improve with treatment, and this identifies a single primary efficacy measure. Multiple definitions of improvement are currently in use in different trials. The goal of this study was to promulgate a single definition for use in RA trials. METHODS: Using the American College of Rheumatology (ACR) core set of outcome measures for RA trials, we tested 40 different definitions of improvement, using a 3-step process. First, we performed a survey of rheumatologists, using actual patient cases from trials, to evaluate which definitions corresponded best to rheumatologists' impressions of improvement, eliminating most candidate definitions of improvement. Second, we tested 20 remaining definitions to determine which maximally discriminated effective treatment from placebo treatment and also minimized placebo response rates. With 8 candidate definitions of improvement remaining, we tested to see which were easiest to use and were best in accord with rheumatologists' impressions of improvement. RESULTS: The following definition of improvement was selected: 20% improvement in tender and swollen joint counts and 20% improvement in 3 of the 5 remaining ACR core set measures: patient and physician global assessments, pain, disability, and an acute-phase reactant. Additional validation of this definition was carried out in a comparative trial, and the results suggest that the definition is statistically powerful and does not identify a large percentage of placebo-treated patients as being improved. CONCLUSION: We present a definition of improvement which we hope will be used widely in RA trials.

Adult↗

PubMed1995

Applications of SNMS in archaeometry.

The recently developed High Frequency Mode HFM of electron gas SNMS allows investigations on insulating samples with the well known advantages of the SNMS Direct Bombardment Mode DBM for the analysis of conducting samples. HFM has been applied to analyses of different historic ceramic and glass samples in order to demonstrate the possibilities of SNMS in this field. It is shown that manufacturing places of ceramic samples could be distinguished by SNMS mass spectra ("fingerprints"). Furthermore questions of the constituents of colour remains on a painted ceramic ("Kaisermedaillon") could be answered by our SNMS analyses. SNMS investigations have been also applied to corrosion phenomena on different glass samples.

Journal Article↗

PubMed1994

Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C-terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali-labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.

Alkalies↗

PubMed1994

Deoxyribonucleic acid replication in fetal cells.

OBJECTIVES: Our purpose was to develop a sensitive method for assessing the replication time of specific human genes in cultured fetal cells and for detecting potential replication defects. STUDY DESIGN: Synchronous progression of diploid human fetal lung cells through S phase was achieved by releasing from serum restriction with minimum essential medium alpha modification plus 10% fetal bovine serum, followed by hydroxyurea blockage at the G1/S boundary. Deoxyribonucleic acid replication was studied in permeabilized cells using mercurated nucleotides to label nascent deoxyribonucleic acid. RESULTS: A high degree of synchrony in traversal of S phase was indicated by flow cytometry and a well-defined 7-hour period of deoxyribonucleic acid synthesis. The replication of the topoisomerase II gene occurred in a narrow time span 3 hours after entry into S phase. CONCLUSIONS: Fetal cells have been highly synchronized at the beginning of S phase, and the replication time of a specific gene can be defined within a narrow time window.

Cell Cycle↗

PubMed1993

Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase.

The nucleotide sequence of a 7-kilobase segment of the Bacillus subtilis chromosome containing the entire coding regions for the enzymes catalyzing the first three steps of diaminopimelate synthesis as well as dipicolinate synthase has been determined. This group of functionally related genes, termed the dap operon, were arranged in the order orfY, orfX, asd, dapG, and dapA and were bracketed by potential rho-independent transcription terminators. The asd locus could complement the growth defect of Escherichia coli strains with an asd deletion. Disruption of the dapG locus led to the loss of aspartokinase I, with a phenotype similar to that of the temperature-sensitive dapG mutants described earlier (Roten, C. A. H., Brandt, C., and Karamata, D. (1991) J. Gen. Microbiol. 137, 951-962). The amino acid sequences of the deduced products of the asd, dapG, and dapA loci had high degrees of similarity with those of other aspartate semialdehyde dehydrogenases, aspartokinases, and dihydrodipicolinate synthases, respectively. Disruption of orfX had no effect on growth but caused a sporulation defect, characterized by low sporulation frequencies and heat-sensitive spores, which could be cured by supplementation with dipicolinate, similar to the phenotype of mutants defective in spoVF, the putative structural gene for dipicolinate synthase. Two other open reading frames, upstream of spoVF, encoded the 380 COOH-terminal residues of a protein homologous to mitochondrial processing proteases and an 85-residue polypeptide of unknown function. Transcription initiation sites associated with the orfY-orfX-asd-dapG-dapA gene cluster were mapped by primer extension. The results indicate that during vegetative growth, the three distal genes of the dap operon, asd, dapG, and dapA, are transcribed as a unit and orfY and orfX are not expressed, whereas at stage 5 of sporulation two separate transcripts are produced, one comprising all five genes, the other just the three distal genes of the operon.

Amino Acid Sequence↗

PubMed1993

The course of skin involvement in systemic sclerosis over three years in a trial of chlorambucil versus placebo.

OBJECTIVE: To describe the course of cutaneous involvement in systemic sclerosis (SSc; scleroderma) over 3 years, in the context of a placebo-controlled drug trial. METHODS: The course of skin tethering (assessed by a semiquantitative skin scoring technique) was documented annually for 3 years in 64 SSc patients with early (< 3 years duration at entry), intermediate (3-8 years duration), or late (> 8 years duration) diffuse or limited SSc. RESULTS: Mean +/- SD entry skin scores were significantly greater in the 33 diffuse SSc patients (13.1 +/- 5.0) than in the 31 limited SSc patients (4.3 +/- 2.1) (P < 0.001). In patients with diffuse SSc, the skin score remained stable for the first 12 months, but had decreased significantly by 24 months (P < 0.022) and 36 months (P < 0.004). In those with limited SSc, the skin score did not change significantly over 3 years. CONCLUSION: Trials of treatments designed to affect skin thickening/tethering should be conducted in patients who have diffuse SSc (of short, intermediate, or long duration) at entry. The best time to study therapies designed to affect skin thickening may be in the first year after entry.

Adult↗

PubMed1992

Fine-structure mapping of cis-acting control sites in the lysC operon of Bacillus subtilis.

Mutations at the aecA locus of Bacillus subtilis lead to derepression of the lysC operon, which encodes aspartokinase II, and analysis of three independent aecA mutations has shown them to be nucleotide substitutions in the lysC leader region (Y. Lu, N.Y. Chen and H. Paulus (1991) J. Gen. Microbiol. 137, 1135-1141). DNA sequence analysis of the lysC control region of nine other mutants with derepressed levels of aspartokinase II revealed each of the mutations to be associated with changes in one or a few nucleotide residues. The nucleotide substitutions were clustered at two sites in the lysC leader: in a region of imperfect dyad symmetry about 40 base pairs from the transcription start site, and in the open reading frame for a putative leader peptide, which starts about 40 residues further downstream. The effect of nucleotide substitutions at the two sites differed in that those at the upstream site gave twice the degree of derepression. A mutant with a small deletion in the leader peptide coding region potentially affecting RNA secondary structure also had a higher level of lysC derepression. These results suggest that the lysC leader region contains at least two cis-acting control sites that play important and perhaps independent roles in the repression of the lysC operon by lysine.

Amino Acid Sequence↗