A highly conserved domain of the maize activator transposase is involved in dimerization.

Previous studies have presented indirect evidence that the transposase of the maize transposable element Activator (TPase) is active as an oligomer and forms inactive macromolecular complexes expressed in large amounts. Here, we have identified and characterized a dimerization domain at the C terminus of the protein. This domain is the most highly conserved region in the transposases of elements belonging to the Activator superfamily (hAT element superfamily) and contains a characteristic signature motif. The isolated dimerization domain forms extremely stable dimers in vitro. Interestingly, mutations in five of the six conserved residues of the signature motif do not affect in vitro dimerization, whereas mutations in other, less strictly conserved residues of the signature motif do. Loss of dimerization in vitro correlates with loss of TPase activity in vivo. As revealed by in situ immunofluorescence staining of mutant TPase proteins, the dimerization domain also is involved in forming inactive macromolecular aggregates when overexpressed, and the TPase contains one or more additional interaction functions.

Biochemical characterization of the catalytic domain of membrane-type 4 matrix metalloproteinase.

A C-terminal truncated form of membrane-type 4 matrix metalloproteinase (MT4-MMP; MMP 17), lacking the hemopexin-like and transmembrane domain, was expressed in Escherichia coli. The catalytic domain was produced by tryptic activation of the recombinant proenzyme and proved to be catalytically active towards the fluorogenic substrate for matrix metalloproteinases (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu(3-(2,4-dinitrophenyl)-L-2,3-diaminopro-p ionyl)-Ala-Arg-NH2. In contrast to the other three MT-MMPs (MT1-, MT2-, and MT3-MMP), the catalytic domain of MT4-MMP does not activate progelatinase A, nor does it hydrolyze one of the offered extracellular matrix (ECM) proteins, such as collagen types I, II, III, IV, and V, gelatin, fibronectin, laminin or decorin. TIMP-1, a poor inhibitor of MT1-, MT2- and MT3-MMP, suppresses MT4-MMP activity effectively. The progelatinase A/TIMP-2 complex that usually reacts like TIMP-2 also inhibits MT4-MMP. TIMP-2, a strong inhibitor of other MT-MMPS, inhibits MT4-MMP at low concentrations. With increasing TIMP-2 concentration, however, activity passes through a minimum and then increases until at high TIMP-2 concentration the activity is the same as in the absence of TIMP-2. TIMP-1 or the progelatinase A/TIMP-2 complex do not prevent reactivation of MT4-MMP catalytic domain at high TIMP-2 concentrations.

Single- and multiple-dose kinetics of ofloxacin in patients on continuous ambulatory peritoneal dialysis (CAPD).

To evaluate the pharmacokinetics of ofloxacin, a novel quinolone antibiotic, in patients with end-stage renal disease (ESRD) on continuous ambulatory peritoneal dialysis (CAPD), we investigated 6 patients in a single-dose study and 9 patients in a multiple-dose study, all without peritonitis. In the single-dose study, patients received 200 mg ofloxacin orally. Serum concentrations (Cmax) peaked at 3.1 +/- 0.3 mg/L (mean +/- SEM), 1.6 +/- 0.5 h after p.o. administration of the drug. Elimination half-life (t1/2) was 26.8 +/- 2.5 h. Peritoneal clearance accounted for 10% of the total body clearance. After 5-h dwell time, ofloxacin concentrations in the dialysate were 1.5 +/- 0.2 mg/L, which is above the MIC90 for most bacteria responsible for peritonitis in patients on CAPD. In the multiple dose study, 200 mg ofloxacin were administered twice, with a time interval of 12 h, followed by 200 mg for 9 days every morning. Mean trough serum levels were 2.6 +/- 1.0 mg/L, mean peak concentrations were 4.1 +/- 1.7 mg/L. Mean ofloxacin concentrations in the peritoneal effluent were 1.9 +/- 0.9 mg/L. It is concluded that an oral loading dose of 400 mg on the first day and a maintenance dose of 200 mg ofloxacin/day does not lead to significant accumulation, even though the elimination by the peritoneal route is only small. The proposed dosing regimen could be an adequate therapy of peritonitis and exit-site infections in patients on CAPD since levels reached in the dialysate effluent are bactericidal. The clinical usefulness in the treatment of peritonitis has to be proven in further studies.

An automated high-performance liquid chromatographic method for the determination of aminoglycosides in serum using pre-column sample clean-up and derivatization.

An automated high-performance liquid chromatographic method for the determination of the aminoglycosides amikacin, dibekacin, gentamicin, netilmicin, sisomicin and tobramycin is described. The procedure involves sample clean-up by adsorption of the aminoglycosides on a pre-column, subsequent derivatization with o-phthalaldehyde and on-line separation of derivatives by column switching. A short cation-exchange column serving concurrently as a guard column in combination with a reversed-phase column was used for separation. Except for the determination of netilmicin an internal standard consisting of an aminoglycoside was used in each assay. The signals of the aminoglycosides determined were linear within the range of 1-16 mg/l serum. The inter-assay imprecision (n = 10) calculated as coefficient of variation was less than 6%. The results were obtained within 20 min after injection of the serum sample. Easy performance and flexibility make the procedure feasible for therapeutic drug monitoring.

Comparison of a simplified liquid chromatographic assay of gentamicin in serum with enzyme immunoassay and bioassay.

A sensitive and reproducible high-performance liquid chromatographic (HPLC) method is described for assay of gentamicin in serum using ultrafiltration of serum samples and pre-column derivatization with o-phthalaldehyde. The procedure was compared with a bioassay and an enzyme immunoassay. Coefficients of correlation were 0.98 for HPLC vs. bioassay and 0.97 for HPLC vs. immunoassay. The between-day imprecision (n = 5) was estimated from the coefficients of variation at a concentration of 6 mg/l. Values were 9.4% for the bioassay, 6.5% for the immunoassay and 2.4% for the chromatographic method. The major advantage of the method described is simplified sample preparation and optimal serum extraction. The latter is important in routine use because it prolongs column life and thus reduces costs.

Pharmacokinetics of ticarcillin during cisplatin chemotherapy.

Cancer chemotherapeutic agents and antimicrobial antibiotics are often given concomitantly. Cisplatin, which has become increasingly important in cancer treatment, has shown nephrotoxicity as a dose-limiting feature. The alpha-carboxy-penicillin ticarcillin (tc) has a wide spectrum of antimicrobial activity, especially against Pseudomonas. The plasma half-life of tc is correlated with renal function. The combined use of cisplatin (20 mg/m2, days 1-5) and tc (3 X 5 g/24 h, days 1-5) with renal protection by vigorous hydration (2400 ml of 0.9% NaCl/24 h continuous infusion, days 1-5) in a group of 12 cancer patients did not alter the BUN and creatinine serum levels. The mean serum concentration of tc, which was monitored in a patient 15, 30, and 45 min after injection of 5 g on 4 consecutive days together with cisplatin, did not differ from the levels of tc reported when used without concomitant cisplatin therapy. Thus these preliminary data show that the pharmacology of tc may not be altered significantly when applied together with cisplatin and that cumulative nephrotoxicity must not be expected with this combination when sufficient hydration is used.

Comparison of the conventional methods and high-performance liquid chromatography for the determination of trimethoprim, sulfamethoxazole and its metabolite in serum.

A modified high-performance liquid chromatographic (HPLC) method for sensitive and rapid determination of trimethoprim, sulfamethoxazole and its metabolite N4-acetylsulfamethoxazole has been compared with the bioassay for trimethoprim and a colorimetric procedure for sulfonamides. The sensitivity of the (HPLC) method has been increased by ultrafiltration of the sample. Thus, the sample dilution was markedly reduced compared to the values obtained with precipitation procedures. The recovery was 102.7 +/- 6.1% for trimethoprim, 93 +/- 5.4% for sulfamethoxazole and 90.2 +/- 7.9% for N4-acetylsulfamethoxazole. The between-day reproducibility was 5% (n = 5). The coefficients of correlation for HPLC and reference methods were 0.993 (bioassay) and 0.995 (colorimetric assay).

Rapid and reliable identification of Staphylococcus aureus by a latex agglutination test.

A latex slide agglutination test detecting clumping factor and protein A simultaneously is recommended for rapid and reliable routine identification of Staphylococcus aureus. Strains (836) of staphylococci isolated from clinical specimens were examined, all S. aureus strains identified by conventional methods were correctly differentiated by the latex test, and no false-positive results occurred with other staphylococci. The reagent is easy to prepare since plasma is the coating material.

[Protein-A-hemagglutination test, a reliable method for rapid identification of S. aureus (author's transl)]. / Protein-A-Hämagglutinationstest, eine zurverlässige Methode zur schnellen Identifizierung von S. aureus.

369 staphylococcal strains isolated from clinical material were examined for tubeagglutination with sensitized sheep red cells in a standardized assay to study its reliability for routine identification of S. aureus. Colonies isolated from blood agar plates correlated in 99.5% with the (optimized) coagulase reaction. The test is easily to perform and results can be read after 2 hours, whereas the reference methods coagulase, hyaluronidase and deoxyribonuclease took as much as 24 h. The reliability of these tests is discussed.

[Isolation of a carbon dioxide-dependent strain of E. coli from the urine of a patient with chronic pyelonephritis (author's transl)]. / Isolierung einer CO2-bedürftigen E. coli-Mutante aus Urin.

Since anaerobic culture techniques were introduced into bacteriological routine laboratories reports increase about Enterobacteria, which doesn't grow under normal culture conditions. We recently isolated a carbon-dioxide dependent strain of E. coli, which didn't grow aerobically even on optimized culture mediums. Though the species was at first isolated in an anaerobic jar further investigation showed optimal growth in an atmosphere containing 20 per cent oxygen enriched with (10 per cent) carbon dioxide. There are some indications that the mutant developed under chemotherapy. A serological grouping wasn't possible.

Evidence for the incorporation of molecular oxygen, a pathway in biosynthesis of N-glycolylmuramic acid in Mycobacterium phlei.

The hypothesis that the biosynthesis of the glycolyl group of muramic acid in the peptidoglycan of Mycobacterium phlei is catalyzed by a N-acetyl hydroxylase is strongly supported by the experiments reported in this paper. 18O is incorporated into the N-substituent of muramic acid isolated from the peptidoglycan of M. phlei grown under pure oxygen enriched with the 18O isotope.

[Experiences with the API 20A system in routine species identification of anaerobes (author's transl)]. / Erfahrungen mit dem API 20A-Testsystem bei der Identifizierung von Anaerobiern aus täglichen Routine.

The API 20A System was tested in three modifications: a) The microtubes were inoculated with the API anaerobe basal medium, filled up completely with sterile mineral oil and incubated aerobically. b) The test strips were inoculated with the basal medium and incubated in an anaerobic chamber. c) The strips were inoculated with a modified Viande-Levure medium containing Tween 80, vitamin K3 and hemin. The microtubes were covered with sterile mineral oil and incubated in an anaerobic chamber. Each procedure was compared with the conventional method (PRAS) of the Virginia Polytechnic Institute. The overall agreement between the three modifications of the API System and the conventional method was 83.2, 91.7, and 98.5% related to the number of tests performed. The advantage of the modified medium was also demonstrated by measuring the growth rate of some anaerobes in thioglycolate broth, API basal medium and VL-medium, modified as mentioned above, nephelometrically. So the micromethod is more accurate and reliable when inoculated with an improved medium.