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Biomedical subjects

R Metz

Publications and source records attributed to R Metz.

At least 19 recordsLinked to original sources
PubMed2002

The potential of pathogens as biological control of parthenium weed (Parthenium hysterophorus L.) in Ethiopia.

P. hsyterophorus is an exotic invasive annual weed now causing severe infestation in Ethiopia. Studies on diagnosis, incidence and distribution of pathogens associated with parthenium weed in Ethiopia were carried out from 1998-2002. Several fungal isolates were obtained from seed and other parts of parthenium plants. Among them were putative pathogenic fungal species of the genus Helminthosporium, Phoma, Curvularia, Chaetomium, Alternaria, and Fusarium. However, pathogenecity test of the isolates obtained showed no or non-specific symptoms. It was concluded that these pathogens could be opportunistic with insignificant potential for biological control of parthenium. Two most important diseases associated with parthenium were a rust disease, caused by Puccinia abrupta var. partheniicola, and a phyllody disease, caused by a phytoplasma of fababean phyllody (PBP) phytoplasma group. The rust was commonly found in cool mid altitude (1500-2500 m) areas while phyllody was observed in low to mid altitude regions (900-2500 m) of Ethiopia, with a disease incidence up to 100% and 75%, respectively, in some locations. Study of the individual effects of the rust and phyllody diseases under field conditions showed a reduction on weed morphological parameters (plant height, leaf area, and dry matter yield). Parthenium seed production was reduced by 42% and 85% due to rust and phyllody, respectively. Phyllody and rust diseases of parthenium showed significant potential for classical biological control of parthenium after further confirmation of insect vectors that transmit phyllody and host range of phyllody disease to the related economic plants in Ethiopia.

Alternaria↗
PubMed2001

Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair.

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.

Animals↗
PubMed2000

A plausible mechanism for gene correction by chimeric oligonucleotides.

Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.

Cell-Free System↗
PubMed1999

Gene repair using chimeric RNA/DNA oligonucleotides.

An experimental strategy has been developed for the site-specific alteration of genomic DNA. The approach is based on the observation that oligonucleotides containing complementary RNA/DNA hybrid regions are more active than duplex DNA in homologous pairing reactions in vitro. The chimeric molecules are designed with a homologous targeting sequence comprised of a DNA region flanked by blocks of 2'-O-methyl RNA residues (the chimeric strand), its complementary all-DNA strand, thymidine hairpin caps, a single-strand break, and a double-stranded clamp region. The oligonucleotide can align in perfect register with a genomic target except for the designed single base pair mismatch, which is recognized and corrected by harnessing the cell's endogenous DNA repair system. The mechanism of repair has been studied using mammalian cell-free extracts and bacterial systems and has revealed a mismatch correction pathway distinct from homologous recombination. The chimeric molecules have been demonstrated to be effective in the alteration of single nucleotides in episomal and genomic DNA in cell culture, as well as genomic DNA of cells in situ. This is a potentially powerful strategy for gene repair for the myriad hepatic genetic diseases caused by point mutations.

Animals↗
PubMed1998

Sensitive high-performance liquid chromatographic determination of arbidol, a new antiviral compound, in human plasma.

A highly sensitive and selective HPLC method was developed and validated for the determination of arbidol in human plasma. The method involves the liquid-liquid extraction of drug and internal standard from plasma with tert.-butyl methyl ether followed by evaporation and reconstitution in mobile phase. UV detection was done at 315 nm. The limit of quantification for arbidol in plasma was 0.005 microgram/ml. Linearity in plasma was proven over the whole calibration range (10.2-0.005 micrograms/ml). The method was validated according to GLP guidelines and its suitability was demonstrated by analysis of samples from a pharmacokinetic study.

Antiviral Agents↗
PubMed1998

Post cardiac surgery phrenic nerve palsy: value of plication and potential for recovery.

OBJECTIVES: Evaluation of an aggressive policy for the treatment of phrenic nerve palsy (PNP), following cardiac operations, with emphasis on early diaphragmatic plication. Attention was given to the incidence and predisposing factors for PNP and the potential for recovery following plication. METHODS: From 1 June 1991 to 1 January 1996 we prospectively screened patients for PNP following cardiac surgery. The diagnosis was suspected if difficulty was experienced in weaning the child from the ventilator. If abnormal elevation of the hemidiaphragm was present diaphragmatic plication was performed. Echocardiography was used to assess subsequent return of diaphragmatic function. RESULTS: Seventeen children (nine boys, eight girls), out of 867 (1.9%) children younger than 16 years of age, undergoing cardiac operations were found to have PNP. The mean age was 66 days (range 1-17 months) with 16 patients below 1 year out of a total of 285 patients (incidence 5.6%) and one patient 17 months old. The incidence following open procedures was 11/190, following closed procedures 2/95 and following reoperation 4/83. PNP was diagnosed from 2 to 44 days (mean 14 days) following surgery. It was present on the right side in seven cases, the left in nine and was bilateral in one patient. Two patients were extubated at the time of diagnosis, one patient could be extubated shortly thereafter. Fourteen children underwent diaphragmatic plication, at a median 5 days post diagnosis. Extubation was possible 1-60 days (mean 4 days) after plication. Mean follow-up was 19 +/- 5 months. Subsequent recovery of diaphragmatic movement was documented in seven (41%) children. Time to recovery following plication was 16 months, without plication 38 months. CONCLUSION: Prospective screening for PNP revealed an incidence in children younger than 1 year of 6%. Early plication substantially reduces the duration of ventilation, with its associated reduced morbidity and ICU stay.

Algorithms↗
PubMed1998

Investigation on the bioequivalence of 2 oral preparations containing spironolactone and furosemide.

The bioequivalence of 2 formulations containing spironolactone and furosemide was determined. The test preparation was Spironolacton 50 plus Heumann tablets, a new generic spironolactone preparation, developed by Heumann Pharma GmbH, the reference preparation was Osyrol 50-Lasix capsules, Hoechst AG. The study was designed as a randomized 2-period, 2-sequence, crossover study. A daily dose of 50 mg spironolactone and 20 mg furosemide was administered over 5 days to 24 healthy volunteers in the fasting state. Plasma samples were assayed for spironolactone, its 2 active metabolites canrenone and 7alpha-thiomethylspirolactone, and furosemide by HPLC. Statistical analysis was performed by ANOVA and by nonparametric methods. Because spironolactone was rapidly eliminated from plasma, its pharmacokinetics could only be evaluated with regard to maximum plasma levels. This parameter did slightly miss the criteria for bioequivalence. For canrenone and 7alpha-thiomethylspirolactone bioequivalence was given. For furosemide the test formulation was found to be equivalent concerning the extent of bioavailability. Bioequivalence with regard to maximum concentrations could not be shown. However, from the point of view of pharmacodynamics, this finding may not necessarily be of clinical relevance.

Adult↗
PubMed1997

Analysis of metabolites--a new approach to bioequivalence studies of spironolactone formulations.

The aldosterone antagonist spironolactone undergoes extensive and complex biotransformation. For investigation of bioequivalence of 2 oral spironolactone formulations, Spironolacton 50 Heumann and Aldactone 50, the pharmacokinetics and bioequivalence of the parent drug and 2 predominant active metabolites, canrenone and 7 alpha-thiomethylspirolactone, were determined in a 2-way crossover study in 24 young healthy male volunteers after multiple oral dosing of 100 mg once daily. Plasma samples were measured by a newly developed HPLC assay and individual pharmacokinetic parameters of the 3 compounds were calculated by use of noncompartmental techniques. Statistical analysis was performed by ANOVA and nonparametric methods. Spironolactone was rapidly cleared from plasma. Therefore, only Css,max and tss,max were determined. Concerning Css,max bioequivalence was found with 90% classical shortest confidence interval ranging from 80.7-112.4%. The intrasubject variability for Css,max was determined to be 28.1%. Higher and persisting concentrations were observed for the metabolites. For canrenone 90% classical shortest confidence intervals were calculated as 95.4-105.0% for AUCss,tau, as 92.9-105.8% for Css,max, and as 89.1-106.3% for peak trough fluctuation (PTF). In the case of 7 alpha-thiomethylspirolactone the values were 84.2-103.0% for AUCss,tau, 77.0-98.6% for Css,max, and 85.0-100.4% for PTF. For tss,max nonparametric 90% confidence intervals were determined as 0.00 to 1.50 h for spironolactone and canrenone and as -0.50 to 1.00 h for 7 alpha-thiomethylspirolactone. The intraindividual variability was below 30% for all pharmacokinetic parameters in the case of the metabolites. Thus, bioequivalence of the test and the reference formulation can be concluded. The study suggests the inclusion of parent compound and metabolites for bioequivalence testing of spironolactone formulations. Intraindividual subject variability was clearly diminished by investigating bioequivalence under steady-state conditions.

Administration, Oral↗
PubMed1997

Protein phosphatase-2A associates with the cytoskeleton to maintain cell spreading and reduced motility of nonmetastatic Lewis lung carcinoma cells: the loss of this regulatory control in metastatic cells.

Metastatic Lewis lung carcinoma (LLC-LN7) variants have previously been shown to have reduced levels of protein phosphatase-2A (PP-2A) activity as compared to the nonmetastatic LLC-C8 cells. The present study showed that inhibition of PP-2A in the nonmetastatic LLC-C8 cells caused a rapid change from a spread to a rounded morphology and increased their in vitro invasiveness through laminin. In contrast, the metastatic LLC-LN7 cells were rounded and invasive, which was not affected by inhibition of PP-2A. To determine whether these differences could be attributed to alterations in PP-2A association with the cytoskeleton, the extent of PP-2A colocalization with microtubules was tested. Immunostaining for tubulin showed prominent filamentous fibers in nonmetastatic LLC-C8 cells and small foci of PP-2A immunostaining along these microtubules. In contrast, the tubulin staining was diffuse throughout the metastatic LLC-LN7 cells and there was little evidence of association with PP-2A. Western blot analyses showed that this reduced level of PP-2A association with microtubules in metastatic LLC-LN7 cells was not due to differences in levels of the PP-2A subunits. Instead, it may be due to the reduced association of the subunits into the heterotrimeric form of the PP-2A holoenzyme. These studies show the importance of PP-2A in maintaining a spread morphology and in restricting invasiveness, and a loss of this regulatory control in metastatic cells. This loss of PP-2A regulatory control in metastatic cells may be due to a reduction in the trimeric form of the PP-2A holoenzyme.

Animals↗
PubMed1996

Sensitive determination of nitrofurantoin in human plasma and urine by high-performance liquid chromatography.

A highly sensitive and selective HPLC method was developed and validated for the determination of nitrofurantoin in human plasma and urine. The method involves the liquid-liquid extraction of drug and internal standard from plasma with ethyl acetate followed by evaporation and reconstitution in mobile phase. Urine samples were simply diluted with purified water. UV detection was done at 370 nm. The limit of quantification for nitrofurantoin in plasma was 0.010 micrograms/ml. In urine nitrofurantoin could be quantified down to 0.380 microgram/ml. Linearity was proven over the whole calibration range in plasma (2.48-0.0100 microgram/ml) as well as in urine (187 micrograms/ml-0.380 microgram/ml). The method was validated according to Good Laboratory Practice guidelines and its suitability was demonstrated by analysis of samples from a pharmacokinetic study.

Anti-Infective Agents, Urinary↗
PubMed1996

Improved high-performance liquid chromatographic determination of amoxicillin in human plasma by means of column switching.

A highly sensitive and selective HPLC method was developed for the determination of amoxicillin in human plasma. After addition of buffer and internal standard, the sample was ultrafiltered and injected on to a precolumn to remove polar plasma interferences. Detection was effected with a UV detector set at 230 nm. The limit of quantification for amoxicillin was 50.1 ng/ml with an imprecision of 4.2% using 0.25 ml of plasma. Linearity was confirmed over the whole calibration range (25.4-0.0501 micrograms/ml) and the inter-day variation ranged from 2.0 to 4.5%. The method was validated according to GLP guidelines and its suitability was demonstrated by the analysis of several hundred samples in a bioequivalence study. The method can be used to determine pharmacokinetic parameters of amoxicillin in humans after a single oral dose of 500 mg.

Amoxicillin↗
PubMed1996

Improved determination of sulfadiazine in human plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of sulfadiazine in human plasma and human urine was developed and validated. The method involves the acid extraction of drug and internal standard from plasma with ethyl acetate followed by evaporation and reconstitution in mobile phase. Urine samples were simply diluted with purified water. Recovery, linearity, intra- and inter-day variation of sulfadiazine were tested and found appropriate. The quantitation range was 0.0299-15.2 micrograms/ml for plasma samples and 0.578-148.8 micrograms/ml for urine samples. The method is suitable for the quantitation of sulfadiazine from pharmacokinetic studies.

Anti-Infective Agents↗
PubMed1996

Adjuvant treatment of node-positive breast cancer with cyclophosphamide, doxorubicin, fluorouracil, and vincristine versus cyclophosphamide, methotrexate, and fluorouracil: final report after a 16-year median follow-up duration.

PURPOSE: To determine the long-term impact on disease-free survival (DFS) and overall survival (OS) of adjuvant anthracycline-based chemotherapy, when prospectively compared by random allocation with standard cyclophosphamide, methotrexate, and fluorouracil (CMF) in node-positive (N+) breast cancer patients. PATIENTS AND METHODS: Two hundred forty-nine patients with N+ breast cancer, recruited from eight French cancer centers, were randomized to receive 12 monthly cycles of adjuvant chemotherapy, either CMF (n = 112) or doxorubicin, vincristine, cyclophosphamide, and fluorouracil (AVCF) (n = 136). All had a negative metastatic work-up before inclusion, which was stratified by accrual center, tumor stage (International Union Against Cancer [UICC]), and menopausal status. RESULTS: No severe adverse effect related to grade 4 (World Health Organization [WHO]) toxicity was observed. There was no difference in second primary tumor incidence between the two arms. The treatment given was 88% of planned for AVCF and 75% for CMF in both premenopausal and menopausal patients. With a median follow-up time of 16 years (range, 13 to 17), the OS and DFS rates are significantly longer in the AVCF arm (56% v 41% [P = .01] for OS, and 53% v 36% [P = .006] for DFS). These differences are significant, irrespective of tumor stage (T1 to T2 v T3 to T4), and remain positive in patients with or without postoperative locoregional radiotherapy (55% of cohort). When analyzed according to menopausal status, the differences remain significant only for premenopausal patients. CONCLUSION: This set of mature controlled data confirms the added value of anthracycline-based combination adjuvant therapy for N+ breast cancer patients when compared with CMF, with both regimens given for 1 year.

Adult↗
PubMed1995

Pharmacokinetics and bioavailability of different formulations of aciclovir.

The pharmacokinetics and bioavailability of aciclovir (CAS 59277-89-3) were examined after administration of newly developed 200 mg and 400 mg tablets. Two studies, each with 24 subjects of either sex, were performed. In the three-way study I, two different tablets containing 200 mg of aciclovir (test and reference products) and a short infusion of 250 mg aciclovir were compared. In the two-way study II, the bioequivalence of a newly developed 400 mg aciclovir tablet was tested against a standard product. Irrespective of dose, the peak plasma aciclovir levels were obtained 1.5 h after administration of the tablets. In the case of the 200 mg tablets, the mean Cmax-values were 454 ng/ml (pilot test formulation, T) and 525 ng/ml (reference formulation, R), whereas the mean Cmax-values after administration of the 400 mg tablets were 779 (T) and 800 (R) ng/ml for test and reference formulation, respectively. The mean AUC calculated to the time of the last measurement in each instance was in the order of 2290 (T) and 2275 (R) ng/ml x h (200 mg tablets) or 3726 (T) and 3855 (R) ng/ml x h (400 mg tablets). The amount of the dose renally excreted as unchanged aciclovir was measured at 20.8 (T) - 21.8 (R)% with the 200 mg tablets and 14.3 (T) - 15.1% (R) with the 400 mg tablets.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyclovir↗
PubMed1995

Expression and characterization of mouse cFos protein using the baculovirus expression system: ability to form functional AP-1 complex with coexpressed cJun protein.

The products of the proto-oncogenes c-fos and c-jun play important roles in cell growth control, differentiation, and malignant transformation. Purified oncogenic proteins are essential tools in cell growth control studies. Here we describe the production of mouse cFos and cJun oncoproteins in Sf9 insect cells using the baculovirus expression system. The mouse c-fos cDNA was subcloned into two different baculovirus expression vectors, namely pVL1392 and pVLMH6, to generate, respectively, nonfusion and His-fusion cFos oncoproteins in insect cells. The products were characterized by immunofluorescence, immunoblotting, immunoprecipitation, and ability to bind to the in vitro translated JunB protein to form the AP-1 complex. A His-cJun fusion protein was also produced in insect cells using the pVLMH6 baculovirus vector. Coexpression of cFos and cJun in insect cells yielded functional AP-1 complexes as judged from gel retardation assays. The results point to the possibility of using insect cells for structural and functional studies of different Fos/Jun AP-1 complexes.

Animals↗
PubMed1994

A C-terminal domain in FosB, absent in FosB/SF and Fra-1, which is able to interact with the TATA binding protein, is required for altered cell growth.

Transcriptional regulation in eukaryotes is thought to occur through interactions between specific transcription factors and the general transcription machinery. We show that the regulatory protein FosB, but not FosB/SF or Fra-1, specifically and stably associates with the TATA box binding protein (TBP) and the multiprotein complex TFIID. The binding to TBP is specified by the last 55 C-terminal amino acids of FosB, requiring a small amino acid sequence, termed the 'TBP binding motif' (TBM). Deletion of the TBM affects transcriptional activity slightly, but it is adjacent to a proline-rich sequence which constitutes the major transactivation domain. However, both regions are required for the transformation of Rat-1A cells by FosB. Transfection experiments demonstrate that inhibition of transactivation due to excess levels of Gal4-FosB (squelching) can be partially relieved by the co-expression of TBP, which establishes that TFIID is a functional target of FosB. Since TBP binding is not exhibited by FosB/SF or Fra-1, we suggest that the activity mediated by the TBP interaction is one differentiating characteristic that distinguishes the FosB functions from those of FosB/SF and Fra-1.

Amino Acid Sequence↗