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P Köhler

Publications and source records attributed to P Köhler.

At least 37 records · Page 2Linked to original sources
PubMed1996

Comparison of genetic groups determined by molecular and immunological analyses of Giardia isolated from animals and humans in Switzerland and Australia.

Nine axenic isolates of Giardia originating from four different host species in Switzerland were subjected to genetic analysis using the polymerase chain reaction (PCR) to amplify segments of genes encoding different trophozoite variant surface proteins (VSPs). Three genotypes were identified on the basis of product yield, size, and restriction-fragment-length polymorphisms (RFLPs). Five G. duodenalis isolates (O1, B1, B2, B3-1A1 and C1--from a sheep, three calves and a dog, respectively) were classified as belonging to genetic group I of Andrews et al. (1989). DNA amplified from the VSP genes tsp11, tsa417 and vsp1267 of these isolates was indistinguishable in size and restriction characteristics from that amplified from group-I Giardia isolated from humans in Australia. One human-derived Swiss isolate (H2-17A1), typed as belonging to genetic group II, yielded a vsp1267-specific PCR product that was indistinguishable by size or restriction sites from the equivalent 1.6-kb product amplified from human-derived Australian group-II organisms. This isolate also yielded 1.8-kb tsp11 and 0.52-kb tsa417/tsp11-like PCR products possessing RFLPs typical of group-II organisms. Three isolates (O2-4A1, O3 and H3-15K2--originating from two sheep and a human, respectively) represent a novel genotype that is closely related to genetic groups I and II. These three isolates exhibited identical RFLPs in their tsp11 PCR products and failed to yield a vsp1267 PCR product. An antiserum specific for the 90-kDa VSP of the sheep-derived clone O2-4A1 reacted strongly by immunofluorescence and on Western blots with surface proteins from the O2, O3 and H3 isolates only--consistent with the genetic classification determined above. The data provide no evidence for the occurrence of host-specific genotypes.

Animals↗
PubMed1996

Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis.

This paper reports the cloning and molecular characterization of the gene encoding pyruvate phosphate dikinase (PPDK) from Giardia. The ORF is 2652 nucleotide residues in length and not interrupted by introns. The gene appears to exist as a single copy in the genome and predicts a 97629 Da protein containing 884 amino acid residues. Comparison of the deduced Giardia PPDK sequence with those of homologous enzymes from other organisms revealed high sequence similarities and the presence of various conserved domains known to be essential for substrate binding and catalysis. Analysis of the ppdk gene and 19 other protein-coding genes from the protist revealed no typical TATA boxes, positioned at around -30, but the presence of two novel consensus sequence motifs in the 5' flanking regions. One is an AT-rich element immediately preceding the translation initiation codon and the other a 14-bp box centered at -30. These shared consensus sequence patterns present in the 5' flanking region of Giardia genes are suggested to play a role in the control of transcription initiation.

Animals↗
PubMed1996

[PCR for detection and characterization of parasites (Leishmania, Echinococcus, Microsporodia, Giardia)].

The application of PCR for the diagnosis and the characterisation of parasites is discussed using four examples. (1) A PCR assay that was developed for the detection of Leishmania was shown to be as highly sensitive as in vitro cultivation of the parasites from lymph node aspirates and bone marrow biopsies. (2) Single eggs of Echinococcus multilocularis can principally be detected and identified by PCR. However, a cumbersome sample preparation is inevitable in order to remove PCR-inhibitory substances present in fox faeces. (3) Analysis of the amplified SSU rRNA gene of Encephalitozoon-like isolates from humans, rabbits and farm foxes confirmed for the first time that E. cuniculi-isolates from humans and animals are indistinguishable and that they are of importance as opportunistic parasites in HIV-infected patients. (4) Swiss isolates of Giardia originating from humans, calves, sheep and a dog could be allocated into three distinct genetic groups by analysing several PCR-amplified genes that encode for variant-specific surface proteins. There was no evidence for the presence of host-specific genotypes.

AIDS-Related Opportunistic Infections↗
PubMed1995

Purification and characterization of phospho enol pyruvate carboxykinase from Trypanosoma brucei.

ATP-dependent phospho enol pyruvate carboxykinase (EC 4.1.1.49; PEPCK, ATP) was purified from glycosomes of cultured procyclic Trypanosoma brucei to electrophoretic homogeneity. The purified enzyme exhibited a mean specific activity of 83 units mg-1, as measured in the carboxylation direction at 30 degrees C. A similar activity was obtained for the decarboxylation reaction. The enzyme was shown to be a homodimer in solution with a subunit molecular mass of 59 kDa. Amino acid sequence analysis suggested that the PEPCK (ATP) is identical to the trypanosomal protein p60, the sequence of which was previously predicted from the corresponding nucleotide sequence by other investigators. The basic nature of the enzyme was indicated by a high isoelectric point (pH 8.9). The enzyme was found to be strictly dependent on adenosine nucleotides for activity, as well as on the presence of Mn2+. Mg2+ was found to be ineffective as activator of the trypanosomal enzyme, but a combination of subsaturating (< or = 300 microM) concentrations of Mn2+ and high concentrations of Mg2+ caused a synergistic effect on the carboxylation activity, indicating a dual cation requirement. Mn2+ is necessary to activate the enzyme and Mn2+ or Mg2+ most likely forms the cation-nucleotide complex as the active form of the substrate. Relatively high (5 mM) levels of ATP were required to produce a significant inhibition of the carboxylation reaction. Quinolinic acid, a structural analogue of oxaloacetate, completely inhibited the decarboxylation reaction at a 1 mM concentration. The apparent Michaelis constants of the enzyme were 490 microM for PEP, 37 microM for oxaloacetate, 40 microM for ADP, 10.3 microM for ATP, 970 microM for Mn2+ and 26 mM for HCO3-. Endogenous substrate concentrations were found to be 327 nmol PEP, 1486 nmol ADP, 4200 nmol ATP and 11.5 nmol Mn2+ (ml cell volume)-1. Our kinetic data suggest that under physiological conditions PEPCK (ATP) in T. brucei is bidirectional and that its activity is regulated primarily by mass action. The physiological relevance of the enzyme in procyclic T. brucei is discussed.

Amino Acid Sequence↗
PubMed1995

Disulphide bonds in wheat gluten: cystine peptides derived from gluten proteins following peptic and thermolytic digestion.

Gluten from the wheat variety Rektor was extracted with 70% aqueous ethanol. The insoluble portion (whole glutenin) was partially hydrolysed with trypsin at pH 6.5 and separated on a Sephadex G25 column. The high molecular weight fraction 1 was further hydrolysed with pepsin at pH 2.0. To remove low molecular weight proteins, a portion of whole glutenin was extracted with dilute acetic acid. The residue (enriched glutenin), which contained mostly LMW and HMW subunits of glutenin, was hydrolysed with thermolysin at pH 6.5. The peptic and tryptic hydrolysates were separated on a Sephadex G25 column and the peptide fractions with the highest cystine content were separated further by reversed-phase high-performance liquid chromatography (RP-HPLC). Cystine peptides were detected by differential chromatography (RP-HPLC prior to and after reduction of disulphide bonds) and then isolated by preparative RP-HPLC. After reduction, cysteine peptides were alkylated and analysed for their amino acid sequence. Altogether, 19 cystine peptides were characterized and assigned to known sequences of gluten proteins; 16 peptides confirmed the positions of disulphide bonds present in LMW subunits and gamma-gliadins, as described previously. For the first time, a cystine peptide has been isolated, representing an intermolecular disulphide bond between the y-type of HMW and LMW subunits. Furthermore, a cystine peptide was assigned to gamma-gliadins; thus, all cysteine residues of gamma-gliadins are documented by at least one cystine peptide. One peptide analysed came from the alpha-amylase inhibitor CM 16. Altogether the results indicate that the intramolecular linkages of gluten proteins are not formed at random, but are strongly directed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Studies on compounds promoting the in vitro transformation of Trypanosoma brucei from bloodstream to procyclic forms.

In vitro differentiation of pleomorphic blood-stream forms of Trypanosoma brucei to procyclic culture forms occurred rapidly and at high rates at 27 degrees C in a culture medium containing 1 mM cis-aconitate as the transformation-inducing agent. Citrate was required at a much higher concentration (10 mM) to produce a similar transformation rate. The highest percentage of transformed cells was obtained when bloodstream-form trypanosomes were treated with pronase in the absence of a feeder-cell layer. However, under these conditions, the amount of procyclic forms obtained after 72 h was lower than that obtained in the presence of cis-aconitate. Trypsin was also capable of inducing transformation in the absence of a feeder-cell layer, but this treatment again resulted in low numbers of transformed cells. Blood-stream-form trypanosomes were incapable of taking up citrate to any significant extent and the citrate content of these stages was negligible. After 72 h of exposure to citrate (3 mM), intracellular levels of this compound remained very low (< 1 nmol (10(9) cells)-1), increasing in established procyclic stages to approximately 1.7 nmol (10(9) cells)-1. These observations suggest that the tricarboxylic acid (TCA)-cycle metabolite-dependent transformation may be initiated externally to the trypanosome cell membrane. The ability of both citrate and cis-aconitate to bind calcium and, thus, to reduce the concentration of this cation in the culture medium was found not to be responsible for the triggering effect on trypanosome transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitic Acid↗
PubMed1994

Separation of a cysteine-rich surface antigen-expressing variant from a cloned Giardia isolate by fluorescence-activated cell sorting.

Clonally derived trophozoites (clone O2-4A1, from a sheep) of the morphologically defined group of Giardia duodenalis change their cysteine-rich surface proteins (CRISPs) spontaneously during in vitro culture. This phenomenon constitutes a serious obstacle for studies that rely on a pure population of cells bearing a particular variant CRISP. We describe herein the successful separation and quantitation of a trophozoite subpopulation expressing a 90-kDa major surface antigen (CRISP-90) from a heterologous population using fluorescence-activated cell sorting (FACS) on the basis of fluorescein-tagged antibodies directed specifically against O2-4A1 CRISP-90. During subsequent in vitro culture, the purified cell population exhibited a progressive decline in the proportion of cells labeled by CRISP-90 specific antibodies. After 46 generations, only one-third of the total trophozoite population reacted with the anti-CRISP-90 antibodies.

Animals↗
PubMed1994

[Not Available].

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Botany↗
PubMed1993

Disulphide bonds in wheat gluten: further cystine peptides from high molecular weight (HMW) and low molecular weight (LMW) subunits of glutenin and from gamma-gliadins.

Glutenin was prepared from gluten of the wheat variety Rektor by extraction of gliadin with aqueous ethanol. It was cleaved successively into soluble peptides by the enzymes trypsin and thermolysin. Separation of the peptide mixtures was performed by gel permeation chromatography (GPC) on Sephadex G25 and reversed phase high performance liquid chromatography (RP-HPLC) on ODS-Hypersil. Cystine peptides were detected by differential chromatography of the samples prior to and after reduction. After isolation by multi-step RP-HPLC, the cystine peptides were reduced. The resulting cysteine peptides were alkylated with 4-vinylpyridine, separated by RP-HPLC and sequenced by means of the Edman degradation. The isolated cystine peptides represented a considerable portion of the total cysteine in glutenin: four out of seven cysteine residues of HMW subunits, and eight out of nine cysteine residues of LMW subunits are documented by at least one cystine peptide. Most of the peptides corresponded to known sequences of gluten protein components. From the structures of some tryptic peptides, inter- and intramolecular disulphide bonds for HMW subunits of glutenin have been proven. Cystine peptides from the thermolytic digest have been assigned to LMW subunits of glutenin and to gamma-gliadins. Other peptides have been closely related to partial sequences of these protein components. The results have allowed several conclusions about the arrangement of intra- and intermolecular disulphide bridges in gluten proteins.

Amino Acid Sequence↗
PubMed1993

Variant cysteine-rich surface proteins of Giardia isolates from human and animal sources.

Cloned Giardia isolates obtained from a sheep, a calf, and a human possessed a major membrane protein that showed marked intraspecific variations in size as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following surface biotinylation and radioiodination. Metabolic labeling with [35S] cysteine and electrophoretic analysis also revealed for each cloned isolate a predominant protein that corresponded in size to the major surface protein demonstrated by surface labeling techniques. Immunoprecipitation studies with a polyclonal antiserum specifically directed against the 90-kDa major cysteine-rich protein purified from a subclone of the sheep isolate (O2-4A1) showed that the cysteine-rich protein and the major surface protein are identical. The surface location of the antigen was further corroborated by the reaction of fluorescence-labeled antibodies raised against the 90-kDa O2-4A1 cysteine-rich protein with the entire surface of live trophozoites of the homologous clone. The ability of the cloned Giardia isolates to undergo variations of their cysteine-rich surface protein (CRISP) was demonstrated by the spontaneous appearance of new CRISPs in clonally derived populations during prolonged in vitro culturing and in cultures of the O2-4A1 clone that had survived treatment with the cytotoxic anti-90-kDa CRISP antiserum specific for the surface antigen of this clone. The surviving progeny were devoid of the original CRISP, as judged by resistance to the immune serum. Subsequent cysteine metabolic labeling of the recloned surviving trophozoites demonstrated a large number of new variants, each expressing a single CRISP that varied significantly in molecular weight from those in the different cloned lines. These studies suggest that the presence of CRISPs and their variations are not restricted to Giardia isolates obtained from humans but are universal phenomena among the Giardia duodenalis types of organisms.

Animals↗
PubMed1993

Chondrosarcoma treated by reimplantation of resected bone after autoclaving and supplementation with allogeneic bone matrix. A case report.

An extensive intramedullary lesion of the femur was diagnosed as a Grade I chondrosarcoma in 1986. At definitive surgery, four fifths of the femoral diaphysis were resected, autoclaved, and reimplanted. The reconstruction, after stabilization with AO plates, was supplemented with allogeneic bone matrix to induce new bone formation. No autografts were used. Despite subsequent infection and sequestration of the autoclaved tumorous segment, the reconstructed area was gradually enveloped by new bone. Three years after primary surgery, the infection persisted. The AO-plated and autoclaved sequestered bone was excised. Six years after the operation, the patient walks bearing full weight and works full time. There are no signs of tumor recurrence.

Adult↗
PubMed1992

Activity, mechanism of action and pharmacokinetics of 2-tert-butylbenzothiazole and CGP 6140 (amocarzine) antifilarial drugs.

A variety of recently developed drugs, designed to be used in antifilarial chemotherapy, contain a thiocarbonylamide group as a common structural element. One group of these compounds is based on a 2-tert-butylbenzothiazole ring in which the carbonylamide linkage is present as an isothiocyanate, dithiocarbamic acid ester or thiourea derivative. The single representative of another series is an N-methylpiperazine adduct of amoscanate (CGP 6140 or amocarzine). CGP 6140 is currently undergoing clinical trials in patients suffering from onchocercosis. All of the drugs with antifilarial activity affect the motility of filarial worms in vitro. The primary site of action of most of these compounds is the mitochondrion. The drugs result in the swelling of this organelle and also the inhibition of respiration and other associated metabolic functions. The dithiocarbamic acid esters (e.g., CGP 20376) are devoid of intrinsic antifilarial activity. Activity of these compounds requires conversion to the corresponding isothiocyanates. This occurs spontaneously in aqueous solution at physiological pH. The thiourea compounds were found to inhibit acetylcholinesterase activity by competing with its substrate. There is also evidence that the latter drugs are metabolized by a host-derived enzyme to their isothiocyanate analogs. CGP 6140 affects both mitochondrial function and acetylcholinesterase activity. The biochemical effects of the antifilarial compounds were not found to be significantly different between mammalian and parasite test systems. Biochemical and pharmacokinetic studies suggest that the selective toxicity of the new series of drugs towards filarial parasites is most likely preferential drug uptake by the pathogen. The lack of a unique target for these compounds in the parasite may explain the side-effects seen upon their administration to humans.

Animals↗
PubMed1991

Disulphide bonds in wheat gluten: isolation of a cystine peptide from glutenin.

Gluten from the wheat variety Rektor was extracted with 70% aqueous ethanol. The residual protein (glutenin) was hydrolysed with trypsin. The partial hydrolysate was separated into seven fractions by gel permeation chromatography on Sephadex G 25. Four cystine-containing peptides were isolated from fraction 5 by reversed-phase high-performance liquid chromatography on ODS-Hypersil. The cystine peptides were detected by differential chromatography of the non-reduced and the reduced samples. The primary structure of the peptides was solved by the Edman degradation reaction and by partial hydrolysis with thermolysin. Three peptides derive from the alpha 2- and beta-purothionins. The structure of the fourth peptide was determined as (Formula; see text) This sequence corresponds to positions 44-48 of known sequences of the high molecular weight (HMW) subunits 9, 10, and 12. Since Rektor contains the HMW subunits 9 and 10, it could be concluded that two HMW subunits 9 or 10, or one subunit 9 and one subunit 10 were linked parallel via two disulphide bridges.

Amino Acid Sequence↗
PubMed1991

Critical comparison of Giardia duodenalis from Australia and Switzerland using isoenzyme electrophoresis.

Isoenzyme electrophoresis using 13 enzyme systems was applied to 31 Australian and 7 Swiss isolates of Giardia of human, cat, cattle, dog, sheep and rat origin. The Portland (ATCC No. 30888) reference strain was also included. The 39 isolates were divided into 22 different zymodemes. These consisted of 19 zymodemes containing the P1 and Australian isolates and three zymodemes containing Swiss isolates only. Differences in enzyme profiles between zymodemes was measured by euclidean distance and it was found that Australian isolates of Giardia exhibited more variation than the Swiss isolates. Relationships between zymodemes determined by clustering analysis are discussed with particular reference to the zoonotic potential of Giardia.

Animals↗
PubMed1991

Alterations in Krebs cycle enzyme activities and carbohydrate catabolism in two strains of Trypanosoma brucei during in vitro differentiation of their bloodstream to procyclic stages.

A rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of Trypanosoma brucei STIB348 and EATRO1244 bloodstream to procyclic trypomastigotes. In accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of CO2, succinate and glycerol. During differentiation pyruvate excretion decreased within 48 h to the low levels produced by 28-day procyclic stages. Concomitant with the decline in pyruvate formation, acetate appeared as a new product and the rates of respiratory CO2 increased considerably. The amount of carbon released with these compounds could account for nearly all of the glucose carbon consumed. Rates of glucose utilization and formation of acetate and CO2 in cells differentiated for 48 h were essentially the same as those found in 28-day procyclics. Succinate and glycerol excretion remained low during the entire transformation process, and no significant difference in the pattern and quantities of end products were found between the two trypanosome strains. During trypanosome differentiation the changes in metabolism were associated with marked alterations in enzyme activity levels. Activities of the tricarboxylic acid (TCA) cycle enzymes citrate synthase, isocitrate dehydrogenase (NAD+), succinate dehydrogenase and fumarase were not detectable in bloodstream trypomastigotes but appeared upon differentiation for 24 h. An exception was citrate synthase whose activity was not demonstrable until 48 h postinoculation into culture. After 48 h the majority of the TCA cycle enzyme activities continued to increase steadily until day 28. Pyruvate kinase activity decreased in differentiating cells after 48 h to about 25% of the level found in bloodstream trypomastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitic Acid↗