Interstitial lung disease as the first manifestation of systemic sclerosis.

We describe three patients with progressive fibrosing interstitial lung disease (ILD) as the first and only manifestation of systemic sclerosis. In one patient the presence of anti-Scl-70 autoantibodies suggested systemic sclerosis to be the underlying cause of the disease. In the two other subjects, however, anti-Scl-70 antibodies were negative. In these patients the lung disease preceded other manifestations of systemic sclerosis by several years. Diagnosis, prognosis and treatment of systemic sclerosisassociated ILD is discussed.

[Two patients with emphysematous pyelonephritis]. / Twee patiënten met lucht in het pyelum.

A 55-year-old man with diabetes mellitus was sick with shivering, abdominal pain and Escherichia coli in blood and urine cultures; a 40-year-old woman with asthmatic bronchitis had abdominal pain and anaemia. Both had renal dysfunction and the CT scan showed hydronephrosis with a perirenal inflammatory infiltrate and gas in the right and left renal pelvis, respectively. The man had a difficult recovery after treatment with antibiotics and percutaneous drainage. In the woman, a calculus obstructed the pyelo-ureteral passage; the resected kidney contained a squamous cell carcinoma. She was operated, received chemotherapy and recovered. Emphysematous pyelonephritis is a rare, severe disease. Percutaneous drainage and, if necessary, nephrectomy are paramount in the treatment. The condition is observed mostly in patients with diabetes mellitus or obstruction of the urinary tract.

Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis.

The antiviral activity of surfactin, a cyclic lipopeptide antibiotic and biosurfactant produced by Bacillus subtilis, was determined for a broad spectrum of different viruses, Semliki Forest virus (SFV), herpes simplex virus (HSV-1, HSV-2), suid herpes virus (SHV-1), vesicular stomatitis virus (VSV), simian immunodeficiency virus (SIV), feline calicivirus (FCV), murine encephalomyocarditis virus (EMCV). In vitro experiments showed biphasic virus inactivation kinetics for enveloped viruses during treatment. Inactivation of enveloped viruses, especially herpes- and retroviruses, was much more efficient than that of non-enveloped viruses. For those viruses susceptible to its action, surfactin was active at 25 microM in medium containing 5% fetal calf serum (FCS). Concentrations up to 80 microM of surfactin led to a titre reduction of >4.4 log10 CCID50/ml for HSV-1 in 15 min and for SIV and VSV in 60 min. The inactivation rate increased linearly with the incubation temperature by a factor 2.4/10 degrees C and logarithmically with the concentration. Serum components, probably proteins and/or lipids, influence the effective surfactin concentration. A disruption of the viral lipid membrane and partially of the capsid was observed by electron microscopy. These findings suggest that the antiviral action, postulated also in other investigations, seems to be due to a physicochemical interaction of the membrane-active surfactant with the virus lipid membrane. Surfactin may be useful for application in virus safety enhancement of biotechnological and pharmaceutical products.

Analysis of surfactin synthetase subunits in srfA mutants of Bacillus subtilis OKB105.

The srfA operon of Bacillus subtilis functions in the biosynthesis of the lipopeptide antibiotic surfactin. On the basis of nucleotide sequence and genetic analysis, it is believed to encode three enzymes (E1A, E1B, and E2) that catalyze the incorporation of the surfactin substrate amino acids. Insertion, deletion, and amino acid substitution mutations of srfA were analyzed for subunit composition and activity as determined by assays of both amino acid-dependent ATP-PPi exchange and aminoacyl thioester formation. Insertion mutations in srfAA (encoding E1A, the subunit that incorporates Glu, Leu, and D-Leu) eliminated production and activity of all three enzymes. Deletions within srfAA and extending from srfAA to srfAB (encoding E1B, which incorporates Val, Asp, and D-Leu) abolished the activity and production of all three enzymes. Insertions between srfAA and srfAB and within srfAB eliminate the production and activity of E1B and E2. An insertion mutation in srfAC (encoding E2, which incorporates Leu) abolished the activity of E2 only. Mutations of the active serine in the putative 4'-phosphopantetheine-binding motif of the second and third domains of E1A eliminated thioester formation and severely reduced the ATP-PPi exchange activity of the two domains. However, the same mutation in the first domain of E1B had little effect on Val-dependent ATP-PPi exchange activity but abolished thioester formation. These results indicate that the coding assignments of the srfA genes are srfAA (E1A), srfAB (E1B), and srfAC (E2).

Structural and functional organization of the surfactin synthetase multienzyme system.

By gel filtration of a crude extract of Bacillus subtilis ATCC 21332 and OKB 105, the multienzyme system that forms the lipoheptapeptide surfactin was separated into three enzyme fractions, E1, E2, and E3. E1, which appeared near the exclusion limit of the column, activates all amino acid components of surfactin as aminoacyladenylates and thioesters according to the thioester mechanism. In addition, a leucine-activating enzyme (E2) and an acyltransferase (E3) were detected that show molecular masses of approximately 160 and 40 kDa, respectively. The surfactin synthetase multienzyme system was reconstituted by complementation of all three enzyme fractions, yielding high rates of lipopeptide formation. E1 is composed of two multifunctional polypeptides (E1A and E1B) with molecular masses of 460 and 435 kDa, respectively, that can be separated by high-resolution anion-exchange chromatography on Pharmacia Mono Q. E1A binds L-Glu and L-Leu in a molar ratio of 1:2, whereas E1B incorporates L-Val, L-Asp, and L-Leu in a molar ratio of 1:1:1. The hydroxy fatty acid moiety is contributed by the acyltransferase accepting the hydroxy fatty acid coenzyme A thioester as substrate. The transfer of the hydroxy fatty acid to E1A and the formation of the hydroxyacyl-L-glutamate intermediate are the initiation steps in the biosynthesis of surfactin. The amino acid-activating enzyme components E1A, E1B, and E2 have been highly purified and partially characterized.

[Plasma- and tissue concentrations following intramuscular administration of etofenamat. Pharmacokinetics of etofenamat and flufenamic acid in plasma, synovium, and tissues of patients with chronic polyarthritis after administration of an oily solution of etofenamat]. / Plasma- und Gewebespiegel-Studien nach intramuskulärer Applikation von Etofenamat. Pharmakokinetik von Etofenamat und Flufenaminsäure in Plasma, Synovia und Geweben von Patienten mit chronischer Polyarthritis nach Applikation öliger Lösung von Etofenamat.

Studies on Plasma and Tissue Concentrations of Etofenamate following Intramuscular Application/Pharmacokinetics of etofenamate and flutenamic acid in plasma, synovia and tissues of patients with chronic polyarthritis after application of oily etofenamat solution Pharmacokinetics of etofenamate (ETO, CAS 30544-47-9; Rheumon i.m.) and flufenamic acid (FLU, CAS 530-78-9) were investigated in plasma, synovial fluid, and tissues after single intramuscular application of etofenamate to patients with rheumatoid arthritis. 62 patients with indicated operative procedure in the knee-joint received a single dose of etofenamate dissolved in oil before operation. At definite times between 1.5 and 48 h post injectionem samples from 6 patients of each time group were collected. Samples of plasma, synovial fluid, synovial membrane, muscle, bone, hyaline cartilage, and fat tissue and in some cases meniscus cartilage were taken. Concentrations of ETO and its active metabolite, FLU, were determined by HPTLC. In all tissues investigated, concentration/time courses of ETO and FLU were observed. ETO and FLU were measured first in all matrices 1.5 h at the latest 3 h post injectionem. Pharmacokinetics in tissues follows that in plasma. Rate-limiting step is the liberation of drug from the oil depot. For a long period pharmacokinetics of ETO and FLU is mainly determined by the constant liberation from the oil depot (zero order kinetics of liberation). Zero order kinetics is deduced from the linear ascent of the cumulated AUC (in percent) vs. time plot. It is directly related to the liberation of drug from the galenical formulation.(ABSTRACT TRUNCATED AT 250 WORDS)

[Plasma level studies in volunteers after intramuscular injection of various doses of etofenamate in an oily solution]. / Plasmaspiegel-Studien an Probanden nach intramuskulärer Applikation verschiedener Dosen Etofenamat in öliger Lösung.

Plasma level Studies on Volunteers after Intramuscular Application of Different Doses of Etofenamate in Oily Solution. After i.m. injections of etofenamate (active substance of Rheumon i.m.) in oily solution to 12 volunteers, courses of plasma levels of etofenamate, flufenamic acid and fenamate (sum of etofenamate and flufenamic acid) were measured by HPTLC. Maximum levels of etofenamate, flufenamic acid and fenamate, as well as areas under the plasma level time curve (AUC) after 250, 500 and 1000 mg etofenamate respectively are proportional to dose. Maxima of fenamate plasma levels are reached after 6.3, 6.2 and 5.4 h respectively, half maximal levels are present already after 2 h. The mean residence time is 21.8, 18.8 and 15.7 h. These values obtained from different doses are not statistically different from each other. Pharmacokinetics are therefore linear and dose independent. The courses of fenamate levels can be described by a two compartment model. The elimination half lives after 250, 500 and 1000 mg are 2.1, 2.3 and 1.9 h, the invasion half-lives (dominant half-life) 8.8, 7.8 and 6.8 h. Terminal half-lives are 50.3, 63.7 and 35.4 h. Since plasma levels have decreased to 2% of the maximum level after one terminal half-life, they have no practical importance for the duration of activity or for accumulation. No sex related differences are found for dose dependent and independent parameters. From the data it can be derived that after i.m. injection of etofenamate in oily solution a prolongation of the dominant half-life occurs by a factor of 4-5 (as compared to oral data) which is caused by prolonged liberation from the oily depot. This long lasting liberation of etofenamate leads to a prolonged residence time after a fast increase, at the same time avoiding unnecessary high peak levels. Therefore it is guaranteed that even after i.m. administration of 1000 mg etofenamate in oily solution plasma levels of fenamate do not exceed those after 300 mg given orally. According to pharmacokinetic data a fast onset of action, good tolerability and a therapeutic action over a period of 24 h can be expected.

[Animal experimental evidence of the long-lasting efficacy of etofenamate by prolongation of the half-life after intramuscular application]. / Tierexperimenteller Nachweis langanhaltender Freisetzung von Etofenamat unter Verlängerung der Halbwertszeit nach intramuskulärer Applikation.

Animal Experimental Evidence of Long-lasting Liberation of Etofenamate by Half-life Prolongation after Intramuscular Application. The purpose of this investigation was to show in animal experiments that by i.m. injection of etofenamate (active substance of Rheumon i.m.) in oily solution the following effects could be obtained: a fast onset of action (gain of therapeutically relevant drug levels shortly after injection) a long-lasting efficacy (prolonged liberation from the oil depot) and better tolerability as compared to other intramuscularly applicable antiinflammatory drugs (avoidance of high plasma spikes). Etofenamate in rats is liberated with a half-life of 1.29 days from the place of application (cutaneous half-life 8.5 h). Flufenamic acid in muscles is found only in traces. After i.m. administration of etofenamate to dogs maximum plasma levels of etofenamate and flufenamic acid were reached within 2 and 4 h, resp. The mean half-lives of plasma elimination are 14 h for etofenamate and 23.2 h for flufenamic acid formed esterolytically from etofenamate (flufenamic acid oral half-life 2-4 h). Maximum plasma levels after etofenamate are only 6.5-11.8% of the maximum levels after equivalent amounts of flufenamic acid administered orally. According to these data etofenamate i.m. is a drug formulation with fast increasing plasma levels, prolonged half-life and lower maximum plasma levels as compared to orally administered preparations. The results are confirmed in animals (pharmacodynamics, toxicology and tolerability) and man (kinetics, clinical studies).

[Renal elimination and metabolism of etofenamate in volunteers after administration of various doses]. / Renale Elimination und Metabolismus von Etofenamat bei Probanden nach intramuskulärer Applikation verschiedener Dosen.

Renal Elimination and Metabolism of Etofenamate after Intramuscular Administration of Different Doses to volunteers. Renal elimination of etofenamate (active substance of Rheumon i.m.) after i.m. injection of oily solution of etofenamate to volunteers was investigated by HPTLC and GC. After injection of 250, 500 and 1000 mg etofenamate, free and conjugated flufenamic acid (flu), 5-hydroxy- and 4'-hydroxy flufenamic acid (5-OH-flu, 4'-OH-flu) were found as main metabolites in urine. Besides that several minor metabolites were identified. The ratio of free to conjugated metabolites was 1:10 to 1:25. From the doses administered 30% were eliminated as main metabolites. Overall amounts (in mg) of the eliminated metabolites and the doses correlated with each other (r = 0.9334), whereas the percent ratio of 5-OH-flu and of 4'-OH-flu increased with dose. Half lives of renal elimination for flu, 5-OH-flu and 4'-OH-flu are largely independent of dose. The half life of flufenamic acid corresponds roughly to data from plasma levels (7-9 h), the two hydroxy derivatives are eliminated into urine with half lives from 15 to 24 h. The results show, that i.m. injection of an oily etofenamate solution follows a linear dose independent kinetic, while the amounts absorbed and renally eliminated are proportional to dose. The results correspond to plasma level studies.

Coordinated synthesis of leghemoglobin and root protein R18 in yellow lupin.

Uninfected roots of yellow lupin contain an abundant 18 kDa protein (referred to as R18), absent in the mature nodules. Some properties of this polypeptide are apparently similar to those of lupin leghemoglobins. However, the lack of any immuno crossreaction between R18 and leghemoglobin and differences in N-terminal amino acid sequences indicate that these proteins are coded by different genes. The decrease in the content of R18 protein in developing nodule is associated with the increased synthesis of leghemoglobin. This implies coordination of both events.

Cross-linked amino acids in the protein pair S13-S19 and sequence analysis of protein S13 of Bacillus stearothermophilus ribosomes.

The 30S ribosomal subunits from Bacillus stearothermophilus were cross-linked under native conditions with the bifunctional reagent diepoxybutane. The dominant protein-protein cross-link in the 30S ribosomal subunit between proteins S13 and S19 [Brockmöller, J., & Kamp, R.M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935] was isolated on a preparative scale. The presence of a single cross-link site between cysteine-83 of protein S13 and histidine-68 of protein S19 was established by microsequence analysis of isolated cross-linked peptides. This cross-link site was further confirmed by different analytical methods including fast atom bombardment mass spectrometry of the cross-linked peptide. The cross-linking site is located in the highly conserved C-terminal regions of proteins S13 and S19. In addition, the complete amino acid sequence of protein S13 from B. stearothermophilus is determined. Sequence comparison with the homologous Escherichia coli protein S13 revealed 58% identical amino acid residues.

Primary structure of the archaebacterial Methanococcus vannielii ribosomal protein L12. Amino acid sequence determination, oligonucleotide hybridization, and sequencing of the gene.

The primary structure of ribosomal protein L12 from Methanococcus vannielii has been determined by direct amino acid sequence analysis with automated liquid phase Edman degradation of the entire protein and manual 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate sequencing of fragments obtained by enzymatic digestion and by partial acid hydrolysis. The knowledge of the amino acid sequences of these various fragments allowed the synthesis of two oligonucleotide probes complementary to the 5'- and the 3'-end of the gene, and they were used for hybridization with digested M. vannielii chromosomal DNA. Both oligonucleotide probes gave similar and clear hybridization signals. The plasmid pMvaX1 containing the entire gene of protein L12 was obtained. The nucleotide sequence complemented the partial amino acid sequence, and it is in full agreement with the protein sequence and the amino acid analysis. Comparison of secondary structural elements and hydrophobicity plots of the M. vannielii protein L12 with the known L12 sequences derived from other archaebacterial and eukaryotic sources show strong homologies among these sequences. They contain an exceptional highly conserved hydrophilic sequence area in the C-terminal part of the proteins. In comparison with eubacterial L12 proteins, the conservation is reduced to single amino acid residues. However, the eubacterial L12 proteins have hydrophilic regions similar to those of L12 from M. vannielii. These regions are predicted to be located at the surface of the proteins, as has been proven to be the case in crystallized Escherichia coli L12 protein. It is possible that the strongly conserved hydrophilic sequence regions form part of the factor-binding domain.

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.

Desalting and concentration of proteins in dilute solution using reversed-phase high-performance liquid chromatography.

A rapid method for desalting and concentrating dilute protein solutions using short reversed-phase columns (3-4 cm) has been described. The recovery of proteins is usually 90-100%. The method is simple and rapid and allows the desalting and concentration of protein samples simultaneously. A wide variety of proteins in the range up to 80 kDa can be desalted in microgram to milligram amounts, and volumes up to 1 liter can be concentrated to a few milliliters by a single injection.

Isolation and identification of two protein-protein crosslinks within the two subunits of Bacillus stearothermophilus ribosomes.

Bacillus stearothermophilus 30S and 50S ribosomal subunits were isolated and crosslinked with diepoxybutane. The crosslinked proteins were extracted with LiCl or with 67% acetic acid and purified by a combination of different high performance liquid chromatography techniques. The protein fractions were analysed by two-dimensional and diagonal polyacrylamide gel electrophoresis and by immunological methods. Two crosslinked protein pairs, one from the large and one from the small subunit, consisting of proteins L23-L29 and S13-S19 respectively, were isolated in milligram amounts for determination of the crosslinked amino acids.

[Retard effect of acemetacin from a commercial preparation--kinetics in humans following single and multiple administration]. / Retardeffekt von Acemetacin aus einer handelsüblichen Zubereitung--Humankinetik nach Einzel- und Mehrfachapplikation.

Acemetacin was given to 14 volunteers in a randomized cross-over arrangement as one single administration of 90 mg (1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetoxyacetic acid) (as Rantudil, Rantudil forte and Rantudil retard) and the courses of blood levels curves compared. Half-life of absorption was considerably higher with the retard formulation (2.01 instead of 0.58 h). The blood level maximum (tmax) was significantly (p less than 0.05) delayed by the retardation, blood levels were lower after 2 h (p less than 0.01), not significantly different 3-4 h after administration and higher (p less than 0.05) than the immediate release form after 6-10 h. AUC's are 5.82 +/- 4.04 (normal) and 6.75 +/- 3.24 (mumol X l-1 X h retarded). The mean residence time was prolonged from 4.1 to 6.3 h, yielding a sustained release quotient of 1.54. After multiple administration (2 retard capsules/d for 8 d) cmax corresponded to the results obtained after single application, maximum levels at 1.24 mumol X l-1 (mean) being somewhat higher than after single application (1.11 mumol X l-1), which is the usual steady-state behavior. Minimum levels in the steady-state (cmin) were 0.24-0.53 mumol X l-1. Bioavailability (AUC's between applications) was not significantly different. No accumulation was found. Levels measured during 8 days of administration of the Rantudil retard formulation corresponded to blood levels from computer simulations. Biological half-life was 4.03 h, which was similar to data obtained with Rantudil (3 X 60 mg/d) in rheumatic patients.

Quantitative determination of acemetacin and its metabolite indometacin in blood and plasma by column liquid chromatography.

A column liquid chromatographic (LC) method using UV detection for the determination of acemetacin and its metabolite indometacin in blood is described. The lower detection limit for both compounds is ca. 25 micrograms/l, the precision (coefficient of variation) is 6% for acemetacin and 10% for indometacin. The method is also suited for determination of both compounds in plasma, precisions in this case are even better than for blood, i.e. around 3% for both acemetacin and indometacin. Blood samples of three volunteers who had received 90 mg of acemetacin orally were analysed using the new method and very good agreement with results from a thin-layer chromatographic/fluorescence method was found.