Extracts Of Homogenates Research Articles

In vitro β-hematin formation assays with plasma of mice infected with Plasmodium yoelii and other parasite preparations : Comparative inhibition with quinoline and endoperoxide antimalarials

Formation of β-hematin in vitro could be catalyzed in the presence of various preparations related to the malaria parasite viz., the cell free homogenate of Plasmodium yoelii, lipid extract of the parasite homogenate, purified malarial hemozoin and synthetic β-hematin. Plasma from mice infected with P. yoelii also catalyzed in vitro β-hematin formation with highly significant efficiency. The plasma based β-hematin formation assay was highly sensitive, as the background absorbance was almost negligible due to absence of any preformed hemozoin. The plasma β-hematin synthesizing activity was recovered in the lipid extract. The quinoline and endoperoxide antimalarials act by inhibiting hemozoin biosynthesis in the malaria parasite. Therefore, the in vitro β-hematin formation assay is useful for the screening and identification of blood schizontocidal antimalarials acting through interruption of heme detoxification in the parasite. Quinoline and endoperoxide antimalarials showed about three fold greater inhibition of β-hematin synthesizing activity in the plasma-based assays as compared to that of P. yoelii homogenate-based assays. The specificity of the inhibition was similar in both preparations. The plasma-based assay therefore provides a better alternative than the parasite homogenate-based assay for in vitro screening and identification of novel inhibitors of hemozoin biosynthesis as potential blood schizontocidal antimalarials.

Evidence of coprostanol estrogenicity to the freshwater mussel Elliptio complanata

Coprostanol (5β(H)-cholestan-3βol) is a reduced metabolite of cholesterol produced by micro-organisms found in the intestinal tract of mammals. This substance abounds in urban effluents and is accumulated by organisms living in the vicinity of municipal effluent outfalls. In an earlier study, freshwater mussels exposed to contaminated river water for 62 days accumulated large quantities of coprostanol (Cop) in their soft tissues (16 μg/g dry wt.). Moreover, these mussels were found to have elevated levels of vitellin in their hemolymphs, suggesting estrogenic effects. Although municipal wastewaters are known to contain other estrogenic compounds capable of inducing Vn synthesis in mussels, the estrogenic potential of coprostanol was singled out for examination. To this end, mussels were first injected with concentrations of coprostanol via the abductor muscle route, and allowed to stand in aerated water for 72 h at 15°C. The levels of Vn in mussel hemolymph were assayed using the organic alkali-labile phosphate method. A competitive estradiol-binding assay was then devised to measure the ability of coprostanol to compete in the binding of fluorescein-labeled estradiol-albumin to cytosolic proteins. Coprostanol partially reversed the binding of labeled estradiol-albumin to cytosolic proteins with an EC 50 of 1 mM. In addition, injections of coprostanol and estradiol-17β led to increased levels of vitellins in the hemolymph of treated mussels. Moreover, incubation of cop in gonad homogenate extracts in the presence of NADPH led to the formation of two compounds, as determined by high-performance thin-layer chromatography. One of these compounds appears to be the C17 oxidation product of coprostanol, whose polarity is similar to that of estradiol. The results present evidence that coprostanol is estrogenic to freshwater mussels.

PCR-based detection of an emerging avian pneumovirus in US turkey flocks.

Avian pneumovirus (APV) or turkey rhinotracheitis virus (TRTV) is an important respiratory pathogen of domesticated poultry in many countries in Europe, Africa, and Asia. Until recently, the United States was considered free of APV. In late 1996, an atypical upper respiratory tract infection appeared in turkey flocks in Colorado and shortly thereafter in turkey flocks in Minnesota. An avian pneumovirus (APV-US) that was serologically distinct from the previously described TRTV was isolated as the primary cause of the new syndrome. The nucleotide sequence of a fragment of the APV-US fusion gene was determined and used to develop a polymerase chain reaction-based assay that specifically detects APV-US viral nucleic acid sequences in RNA extracts of tracheal swabs and turbinate homogenates. The assay is highly sensitive in that it can detect <0.01 TCID50 of APV. The availability of this assay enables the rapid and accurate determination of APV-US in infected poultry flocks.

Thyroid hormone receptors in neonatal, prepubertal, and adult rat testis.

Thyroid hormone (TH) is involved in the differentiation and development of rat testis, whereas its role in adult testis function is still undefined. The aim of our work has been to further analyze the presence in the testis of rats of various ages of messenger RNA (mRNA) coding the different TH receptor (TR) subtypes using a sensitive assay, such as reverse transcriptase-polymerase chain reaction (RT-PCR). To rule out the possibility of an "illegitimate transcription," we have analyzed both T3-binding capacity of adult rat testis and the presence in the same organ of TR proteins by immunohistochemistry, using specific antibodies directed against the various TR isoforms. Messenger RNA coding for TR alpha1 and alpha2 isoforms was clearly visible in gels prepared from RT-PCR samples obtained from the testis of rats of all ages, including adults, whereas mRNA for the TR beta1-beta2 was absent. The T3 maximal binding capacity (Cmax) by nuclear extracts of testicular homogenates gradually decreased from birth to adulthood, still remaining significantly detectable in adult testis, and represented approximately 1% of the Cmax observed in the liver. The immunostaining technique revealed an intense nuclear staining along the basement membrane of testicular tubules prepared from rats of all ages and incubated with an antipeptide antibody specific for TR alpha1 (alpha1-403). Staining with an antipeptide antibody specific for TR beta1 (beta-62) was never present. Our data show that mRNAs coding for the functional TR alpha1, and also for the still undefined alpha2, are present in the testis of rats of all ages. T3-binding activity and immunohistochemical studies confirmed that the message is translated into proteins. The transcriptional activity clearly decreased from birth to adulthood, but it still remained significantly present. The presence of a TR alpha1 message indicates that the adult rat testis may be directly responsive to T3 and, therefore, suggests an action of TH on rat testis that is not only developmental, but also metabolic.

Phosphorylation of prolidase increases the enzyme activity.

Prolidase [EC 3.4.13.9] is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline-containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. Although, the increase in the enzyme activity is correlated with increased rate of collagen turnover, the mechanism by which prolidase is regulated remain largely unknown. In the present study we found that phosphorylation of fibroblast's prolidase may be an underlying mechanism for up regulation of the enzyme activity. Supporting evidence comes from the following observations: (1) immunoprecipitated prolidase was detected as a phosphotyrosine protein as shown by western immunoblot analysis, (2) tyrosine kinase inhibitor-erbstatin induced (in a dose dependent manner) a decrease in prolidase activity in cultured human skin fibroblasts, (3) anti-phosphotyrosine antibody reduced and phosphotyrosine phosphatase 1B antibody (anti-PTP 1B) increased (in a dose dependent manner) the prolidase activity in extract of fibroblast's homogenate, (4) decrease in prolidase activity from collagenase treated or serum starved fibroblasts can be partially prevented by incubating fibroblast's homogenate extract with anti-PTP 1B antibody. These results provide evidence that prolidase is phosphotyrosine enzyme and suggest that the activity of prolidase may be up regulated by the enzyme phosphorylation.

Novel Lymphocyte Growth-Inhibiting Tripeptide: N-Acetyl-Glu-Ser-GlyNH2

The limited and predetermined number of cells that constitutes an organ or specialized cell population is to all appearances regulated according to a negative feedback principle involving growth inhibitors with sufficient tissue specificity. To find growth-inhibiting factors in lymphoid cells, we followed established purification procedures and assays. We found a single-peak fraction in water extracts of dog spleen homogenates that inhibited proliferation of Molt (T cell) lymphoma cells at low concentrations in vitro, with no significant effect on a B cell lymphoma cell line (Ramos). C-terminal amino acid sequencing and MS analysis showed the factor to be a tripeptide: N-acetyl-Glu-Ser-GlyNH2. Treatment with a synthetic tripeptide with the structure N-acetyl-Glu-Ser-GlyNH2 decreased the number of cell doublings of Molt cells. The peptide also delayed cell flux at the G2-M transition of the cell cycle, while incorporation of tritiated thymidine was not altered at the examined time points in this cell line. However, DNA synthesis in PPD-stimulated normal human lymphocytes was significantly inhibited and with a bell-shaped dose-response curve.

Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay.

A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of P. brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally P. brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven P. brasiliensis isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to the gp43 gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 x 10(3) to 1.3 x 10(7) CFU of P. brasiliensis per g of lung. Uninfected BALB/c mice and H. capsulatum-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of P. brasiliensis in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.

Inhibition of Methyl-n-Amylnitrosamine Hydroxylation by Diallyl Sulfide and Phenethylisothiocyanate in the Rat

Formation of the stable 2-, 3-, and 4-hydroxy derivatives of methyl-n-amylnitrosamine (MNAN) probably reflects cytochrome P-450-catalyzed activation of MNAN by 1-hydroxylation. Here we studied inhibition of the oxidation of MNAN to hydroxy-MNANs (HO-MNANs) by freshly excised tissues from MRC-Wistar rats treated with the vegetable-derived chemicals diallyl sulfide (DAS) and phenethylisothiocyanate (PEITC). Rats were gavaged with DAS (200 mg/kg), PEITC (163 mg/kg), or vehicle (corn oil) alone. After various times, the rats were killed, the esophagus, nasal mucosa, and liver were removed, and the tissues/tissue slices were incubated for two hours with 23 μM MNAN. HO-MNAN formation was measured by gas chromatography-thermal energy analysis. Significant (p < 0.01) 72-75%, 40%, and 44% inhibitions of total HO-MNAN formation were observed for nasal mucosa removed at 3-18 hours, for esophagus at 18 hours, and for liver at 3 hours, respectively, after gavage of DAS. Significant (p < 0.03) 46-75% inhibition of HO-MNAN formations was observed for the esophagus at 2-24 hours after gavage of PEITC. In disposition studies, rats were treated with DAS (200 mg/kg) in corn oil and sacrificed after various intervals. DAS was determined by gas chromatography of tissue homogenate extracts. After gavage of DAS, its total recovery from all tissues studied was 27% of the dose after 45 minutes and 15-19% after 90 and 180 minutes, with >80% of the recovered DAS in the stomach contents. Up to 2% per tissue of the recovered DAS was found in the stomach wall, liver, and blood. After intraperitoneal injection of DAS, ≤2% of the dose was recovered in the blood and ≤0.7% in the liver. Hence, gavage of DAS and PEITC significantly inhibited HO-MNAN formation for up to 18 and 24 hours, respectively, whereas DAS was >80% metabolized 90 minutes after its gavage. These findings suggest that long-lasting inhibitors or their metabolites, or inactivation of P-450 enzymes, were responsible for the persistence of inhibition of MNAN metabolism.

FTIR spectroscopic and HPLC chromatographic studies of carbon tetrachloride induced acute hepatitis in rats: damage in liver phospholipid membrane.

Carbon tetrachloride (CCl4) induced rat hepatitis was studied by observing an FTIR spectrum of the liver microsomal or homogenate extract compared with those of model compounds. The microsomal extract from the liver of healthy control rats showed almost the same spectrum as a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 by weight). Intraperitoneal injection of CCl4 decreased the absorption intensity due to th --C--H in the--C==H at 3012 cm(-1) in the microsomal extract, and it developed a new 1,2-diacylglycerol band at 1070 cm(-1) in the homogenate extract. An HPLC study was added to assign the 1070 cm(-1) band to 1,2-diacylglycerol. These findings were interpreted from the peroxidation of the microsomal membrane and the regenerative proliferation of the damaged cell.

Studies on neurosteroids X. Determination of pregnenolone and dehydroepiandrosterone in rat brains using gas chromatography-mass spectrometry-mass spectrometry.

An assay method for pregnenolone and dehydroepiandrosterone in rat brains is developed using gas chromatography (GC)-electron ionization-mass spectrometry (MS)-MS. The extract of the rat brain homogenate containing deuterated internal standard with organic solvent is purified by silica gel minicolumn chromatography. The obtained fraction is derivatized into methyloxime, treated with dimethylisopropylsilylimidazole, and then subjected to GC-MS-MS. The method is applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which are divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increases more than three times after stress, dehydroepiandrosterone in both regions is not so clearly influenced by stress.

Elements of the kallikrein–kinin system are present in rat seminiferous epithelium

Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein–kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein–kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.

Gas chromatography-mass spectrometry assay for determination of ketamine in brain

A sensitive and precise gas chromatography–mass spectrometry method with selected ion monitoring has been developed for determination of ketamine in the brain using chlorpheniramine as an internal standard. The assay is based on the acid extraction of brain homogenate with hexane and ethyl ether with subsequent alkaline ethyl ether extraction. The analytical procedure has a coefficient of variation of 3.0–5.3% and from 3.8 to 6.1% for extraction from water or spiked brain samples, respectively. The lowest detectable level of ketamine was 1 ng in any brain region. This level of detection was used to measure the ketamine concentrations in cerebellum, brain stem, midbrain, hypothalamus, and cortex of C57B1/6 mice at awakening following intraperitoneal injection of a hypnotic dose. The ketamine concentrations in mouse brain were in the range from 41.6 to 48.6 ng/mg of tissue.

Metabolism of the new liposomal anticancer drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine in mice.

Metabolism and excretion of the new antitumor drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) was investigated in mice. Mice were injected i.v. with tritium-labeled liposomal NOAC (4 micromol/mouse). Analysis of HPLC-purified extracts of liver homogenates by liquid chromatography coupled with mass spectrometry revealed only the presence of unmetabolized drug. To study the excretion of the administered drug, mice were injected with tritium-labeled liposomal NOAC or as comparison with 1-beta-D-arabinofuranosylcytosine (ara-C; 4 micromol/mouse) and housed up to 48 h in metabolic cages. Urine and feces were collected at different time points and the kinetics of excreted radioactivity were determined. After 48 h, 39% of the injected [5-3H]NOAC radioactivity was excreted in urine and 16% in feces, whereas ara-C radioactivity was only found in urine with 48% of the injected dose. Feces extracts and urine were purified by HPLC and radioactive fractions were further analyzed by liquid chromatography coupled with mass spectrometry. The radioactivity of feces extracts of NOAC-treated mice was composed of unmetabolized NOAC, hydroxylated NOAC (NOAC + OH), its sulfated derivative (NOAC + OSO3H), and unidentified metabolites, whereas in urine, the hydrophilic molecules ara-C and ara-U were found. During the period of 48 h only 2% of the injected NOAC was eliminated in its unmetabolized form, whereas 25% was identified as main metabolite ara-C. Urine collected during 48 h in ara-C-treated mice contained 33% of the injected dose as unmetabolized drug and 13% as the main metabolite ara-U. Thus, NOAC is metabolized by two major pathways, one leading to the hydrophilic metabolites ara-C and ara-U and the other to hydroxylated and sulfated NOAC.

Allium chemistry: identification of organosulfur compounds in ramp (allium tricoccum) homogenates

Supercritical fluid (SF) extracts of homogenized ramp (Allium tricoccum Ait.) were separated and characterized with liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric identification. The profiles of SF extracts of aqueous homogenates of ramp bulbs from three different seasons and growing regions revealed that the thiosulfinates were major components. In addition, some of the cepaenes (α-sulfinyldisulfides) found in extracts of onion juice, as well as allyl containing cepaenes (2-propenyl 1-(2-propenylsulfinyl)propyl disulfide), are present in the ramp extracts. The amount of allicin in ramp bulb homogenates ranged from approximately 10% to 50% of that found in extracts of aqueous garlic homogenates. The greater amount of the methyl 1-propenyl thiosulfinates in the ramp extracts relative to that found in the garlic extracts correlates with the flavor characteristics of ramp bulbs.

Studies on neurosteroids: VII. Determination of pregnenolone and its 3-stearate in rat brains using high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

An assay method for pregnenolone and its 3-stearate in rat brains has been developed using LC–atmospheric pressure chemical ionization (APCI) isotope dilution MS. The extraction of the rat brain homogenate containing deuterated internal standards (ISs) with organic solvent followed by silica gel mini-column chromatography gave two fractions containing pregnenolone and its 3-stearate together with the respective IS. Each fraction was derivatized into 3-acetate-20-methyloxime and 20-methyloxime, respectively, followed by silica gel mini-column chromatography to remove the excess reagents, and then each derivatized fraction was applied onto reversed-phase LC–APCI-MS. The method was applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which were divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increased more than five times after stress, its 3-stearate decreased after stress.

Characterization of rabbit hemorrhagic disease virus field isolates in Taiwan

Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and characterization of the causative agent. Three strains of RHD virus were isolated as the supernatants of liver homogenates reacted positively by hemagglutination (HA) assays and were infective for rabbits after second passage in animals. Following extraction of liver homogenates from animals infected with each of three isolates, each virus strain was purified by CsCl density gradient ultracentrifugation for further characterization. In negative-stained preparations, the purified virions were icosahedral, measured approximately 40 nm in diameter, and were without an envelope. Morphologically, the three isolates were identical. By immunoblotting, a protein with a molecular weight of 60 000 was identified as the major structural protein in each isolate. Furthermore, two sets of primer framed two different regions within RHD virus genome and could amplify two fragments of the expected size, respectively, from each isolate, whereas, none were obtained from uninfected control samples. The identity of the amplified products was confirmed further using different restriction endonucleases. Among three isolates of RHD virus, neither protein migration patterns of the virions nor cleavage patterns of the amplified product by restriction enzymes were found to differ.

Allium Chemistry: Supercritical Fluid Extraction and LC−APCI−MS of Thiosulfinates and Related Compounds from Homogenates of Garlic, Onion, and Ramp. Identification in Garlic and Ramp and Synthesis of 1-Propanesulfinothioic Acid S-Allyl Ester

Supercritical fluid (SF) extracts of homogenized garlic (Allium sativum L.), ramp (A. tricoccum Ait.), and onion (A. cepa) were characterized with liquid chromatography (LC) and atmospheric pressure chemical ionization mass spectrometric identification. The major thiosulfinates from garlic and ramp were readily characterized by this technique. Small quantities of ajoene, a potent antithrombotic agent, were also found in SF extracts of garlic homogenates. The profiles of onion juice extracts revealed the usual thiosulfinates, zwiebelanes, and bissulfine reported in prior studies, as well as cepaenes previously identified in extracts of onion juice through extensive isolation steps and spectroscopic methods. The presence of trace quantities of allyl compounds in onion juice and propyl compounds in garlic and ramp homogenates has been verified by LC−mass spectrometry (MS). The presence of these compounds was not readily evident in previous analyses using gas chromatography−MS with cold-on-column injection an...

Hydropericardium syndrome (HPS) in India: a preliminary study on the causative agent and control of the disease by inactivated autogenous vaccine.

Hydropericardium syndrome (HPS) in broiler birds of 3 to 6 weeks of age was recorded for the first time in the Haldwani area of Nainital district (UP) in India in November, 1994. The overall mortality in 6 poultry farms was 61.62 per cent. The disease was experimentally transmitted by bacteria free infected liver homogenate extract passed through membrane filters of 0.22 and 0.1 mu APD. The aetiological agent was inactivated by heat treatment at 56 degrees C for one hour and 80 degrees C for 10 min. A precipitin band was demonstrated in agar gel immunodiffusion and counter immunoelectrophoresis using infected liver homogenate extract as antigen and homologous antisera raised in the laboratory. The disease was effectively controlled by formalinised and heat inactivated autogenous vaccine prepared from the infected livers of birds which died of natural infection.

Structural characterization of the N-glycans from Echinococcus granulosus hydatid cyst membrane and protoscoleces

Infection by the tapeworm Echinococcus granulosus in the intermediate host results in the development of a hydatid cyst which contains the protoscoleces within a fluid-filled cavity enclosed by the bilayered cyst membrane. N-glycans were enzymatically released from crude extracts of homogenates of hydatid cyst membranes and protoscoleces and their structures were defined by high sensitivity fast atom bombardment mass spectrometry in conjunction with sequential exoglycosidase digestions.The major N-glycans from the cyst membrane were found to be non-charged structures having complex-type antennae and core fucosylation. The antennae are either truncated at the first N-acetylglucosamine or are extended with β-galactose to form N-acetyllactosamine (lacNAc). A significant proportion of the lacNAc backbones are capped by α-galactose. The resulting Gal α-Gal β-terminal structures may account for the earlier observation that antibodies against the blood group P1 epitope recognise components of hydatid cyst extracts. The complex-type N-glycans identified in the protoscoleces extracts were the same as the neutral structures found in the cyst membrane but a small proportion of high mannose structures and truncated di- and trimannosyl core structures were also identified. Sialylated N-glycans were identified as minor constituents of the cyst membrane preparation but were not observed in protoscoleces extracts. Whether the sialylated glycans are host derived or endogenously synthesized by the parasite remains to be established. This is the first reported structural analysis of N-glycans from cestodes and provides new insights into protein glycosylation in helminths.

Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR

A sample preparation method based on a two-phase aqueous polymer system, composed of 8% (w/w) polyethylene glycol 4000 and 11% (w/w) dextran 40, was employed to remove PCR-inhibitory substances from human faeces prior to PCR. The majority of the PCR-inhibitory substances, including bile salts, were shown to be distributed in the polyethylene glycol-rich top phase, whereas target bacteria were detected by PCR in the dextran-rich bottom phase. The efficiency of the aqueous two-phase system in removing PCR-inhibitory substances from faeces was evaluated with a standardised PCR assay containing different concentrations of pure Helicobacter pylori DNA. The detection level for a pure water solution of DNA was in the range 10 −12 to 10 −13 g DNA per reaction tube. Untreated faecal homogenate totally inhibited PCR even after 1000 times dilution in water. A positive PCR result was obtained when 10 −6 g H. pylori DNA was present in a 50 μl reaction mixture containing 10 μl homogenised faeces, which had been briefly centrifuged, boiled and diluted 100 times in water. After extraction of an undiluted heat-treated homogenate in the aqueous two-phase system, the detection level was lowered to the range 10 −10 to 10 −11 g DNA. When the aqueous two-phase system was evaluated on five faecal samples the detection level was decreased by three to five orders of magnitude. Finally, the aqueous two-phase system was applied to faecal samples inoculated with H. pylori cells. The sensitivity of the PCR assay after extraction was found to be 40 times above the detection level of H. pylori in a pure water solution. The target bacteria were detected directly by PCR in the dextran-rich bottom phase. Furthermore, most of the H. pylori cells were shown to be present in the dextran-rich bottom phase.