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PubMed1981

Serotyping of Haemophilus influenzae a comparison of co-agglutination with countercurrent--immunoelectrophoresis and slide--agglutination.

30 out of 55 strains of H. influenzae could be assigned to one of the six serotypes A--F by slide agglutination, countercurrent-immunoelectrophoresis or co-agglutination. While slide-agglutination appeared less reliable due to non-specific agglutinations countercurrent-immunoelectrophoresis was of high specificity. No cross reactions were encountered in this test and results were unambiguously to evaluate. Co-agglutination is attractive because of its rapidity and simplicity without any need of special equipment. It was found to be of increased sensitivity in detecting type B capsular antigen when compared with countercurrent-immunoelectrophoresis, the problem of cross-reactions remains to be solved however. The use of co-agglutination for serotyping H. influenzae strains in combination with countercurrent-immunoelectrophoresis can be recommended.

Agglutination Tests↗
PubMed1981

[Formation of the ductal and vascular components of the rabbit kidney countercurrent system].

Series of graphic and plastic reconstructed models, injections of vessels with successive clearing, injections with successive reconstruction and other methods have been performed and applied to study certain peculiarities of anlage and formation, as well as topographic-anatomical interconnections in the canalicular and vascular components in the rabbit counterflow system during prenatal ontogenesis. It has been stated that the conoid fasciculus of the tubular formations (collecting tube, descending and ascending parts of the nephron loop, vessels) is the structural unit in the osmoregulating nephric apparatus; the collecting tube occupies the central position, and loops of three nephrons belonging to the same generations and having connections with the tube are situated, in a definite order, around it.

Animals↗
PubMed1981

N2 atom of guanine and N6 atom of adenine residues as sites for covalent binding of metabolically activated 1'-hydroxysafrole to mouse liver DNA in vivo.

Administration of 1'-[2'-3'-3H]hydroxysafrole to adult female mice resulted in the formation of DNA-, ribosomal RNA-, and protein-bound adducts in the liver that reached maximum levels within 24 hr. The levels of all three macromolecule-bound adducts decreased rapidly between 1 and 3 days after injection, at which time the amounts of the DNA-bound adducts essentially plateaued at approximately 15% of the maximum level. The amounts of the protein and ribosomal RNA adducts were very low by 20 days. Comparison by high-performance liquid chromatography of the deoxyribonucleoside adducts obtained from the hepatic DNA with those formed by reaction of deoxyguanosine and deoxyadenosine with 1'-acetoxysafrole, 1'-hydroxysafrole-2',3'-oxide, and 1'-oxosafrole indicated that the four in vivo adducts studied were derived from an ester of 1'-hydroxysafrole. Three of the four in vivo adducts comigrated with adducts formed by reaction of 1'-acetoxysafrole with deoxyguanosine; the fourth adduct comigrated with the major product of the reaction of this ester with deoxyadenosine. Adduct formation in vivo at low levels by the other two electrophilic metabolites was not excluded. The three adducts obtained by reaction of 1'-acetoxysafrole with deoxyguanosine appeared to be substituted on the 2-amino group of the guanine residue on the basis of their partitions between aqueous buffer solutions and 1-butanol:ethyl ether as a function of pH and their retention of 3H from [8-3H]deoxyguanosine. The corresponding three adducts derived from the hepatic DNA of mice given 1'-[2',3'-3H]hydroxysafrole had pH partition patterns not significantly different from the three adducts formed in vitro. Adduct II was further characterized from its nuclear magnetic resonance spectrum as N2-(trans-isosafrol-3'-yl)deoxyguanosine. Adduct IV, derived from the reaction of 1'-acetoxysafrole with deoxyadenosine 5'-phosphate, was characterized in the same manner as N6-(trans-isosafrol-3'-yl)deoxyadenosine.

Animals↗
PubMed1980

[Structural organization of the tubular component of the renal countercurrent system].

The time course of the developing tubules of the dog kidney in the prenatal period of ontogenesis was studied by the methods of microscopy and three-dimensional reconstruction. It was established that the structural functional unit of the osmoregulatory system is a cone-shaped fascicle whose central axis is a collecting tubule. Henle's loops of three juxtamedullary nephrons and accompanying blood vessels are grouped concentrically along the axis. The tubules are grouped in such a manner that the most central position is occupied by the ascending portions of the loop while the descending ones lie somewhat outwards.

Animals↗
PubMed1995

Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells.

High-dose therapy with autologous marrow or peripheral blood stem cell (PBSC) rescue has been extensively applied in the treatment of multiple myeloma (MM) patients during the past 10 years resulting in improved event-free and overall survival when compared with standard chemotherapy. However, relapses are common and cure is unlikely in the majority of patients. Because both bone marrow and PBSCs are contaminated with myeloma cells it is conceivable that relapse after autotransplantation originates at least in part from autografted tumor cells. In this study, mobilized PBSCs were examined for the presence of myeloma cells based on immunophenotyping and sensitive polymerase chain reaction (PCR)-based techniques. In addition, CD34+ Lin- Thy+ stem cells were purified from mobilized PBSC harvests of 10 MM patients by sequentially using counterflow elutriation centrifugation, treatment with phenylalanine methylester, and flow sorting, using 5-parameter gating (propidium iodide, forward scatter, side scatter, CD34+ v Lin- and CD34+ v Thy+). Virtually all mobilized unsorted PBSC preparations contained myeloma cells in sufficient quantities (range, < 0.01 to > 10%) potentially causing a disease relapse. Stem cell purification led to an overall enrichment by about 50-fold in all 10 patients; approximately 90% of the final cell population expressed CD34+ Lin- Thy+ with no evidence of myeloma cell contamination based on flow cytometric analysis of CD38bright cells (< 0.1%). Quantitative PCR amplification of patient-specific complementarity determining region III (CDRIII) DNA sequences showed depletion of clonal B cells by 2.7 to 7.3 logs, with the highest log reduction noted in the samples initially containing the most tumor cells. Our results show that purification of CD34+ Lin- Thy+ cells depletes myeloma cells to undetectable levels from up to 10% present in unsorted PBSCs, thus offering a tool to investigate whether MM relapse after autotransplantation can be reduced markedly.

ADP-ribosyl Cyclase↗
PubMed1993

[Immunophenotyping study of bone marrow fractions obtained by elutriation in allogeneic bone marrow transplantation].

PURPOSE: Bone marrow transplantation (BMT) is an effective treatment for acute and chronic leukaemias. Lymphocyte depletion of donor bone marrow for preventing GVHD has been associated with a higher incidence of relapse after allogeneic BMT. This association suggests an antileukaemic effect of donor lymphocytes. In vitro studies show that cytotoxic T lymphocytes (CD3+ CD56+) and NK cells (CD3-CD56+) have an antileukaemic effect. To know which specific subpopulation of lymphocytes are depleted by counterflow centrifugation or elutriation, we analysed B, T, NK cells and hematopoietic precursors in the marrow fractions after this procedure. PATIENTS AND METHODS: Eight patients (6 CML, 1 ALL, 1 B-CLL) received an allogeneic BMT with lymphocyte depletion of the bone marrow graft using elutriation. After a Percoll gradient, donor marrow mononuclear cells (MNC) were separated with this method in five fractions (F1 to F5). RESULTS: Lymphocyte depletion of donor marrow was in average of 1.7 log. This depletion was also selective, the last fraction containing higher number of cytotoxic T lymphocytes and NK cells than the other fractions. Recovery of CD34+ cells in the four fractions concerning to post-Percoll marrow was 84%, most of them being in the last fraction. CONCLUSIONS: The use of elutriation for lymphocyte depletion is a good method for graft manipulation with the feasibility to adjust a lymphocyte/Kg. dose. Elutriation may be effective in reducing the incidence and severity of graft versus host disease and preserving the antileukaemic effect.

Adult↗
PubMed1977

[Study of the dynamics of hepataene and pentaene formation by a culture of Streptoverticillium mucoheptinicum strain 44B/1 in the process of biosynthesis].

In the process of growth Streptoverticillium mycoheptinicum, strain 44B/I produced simultaneously a two-component pentaenic antibiotic (mycopenten) and a heptaenic antibiotic (mycoheptin) by the 1st day of cultivation. The ratio of the components remained constant during the whole fermentation process. When grown on the medium with sodium thiosulfate (under conditions of the changed medium potential) the culture produced the heptaen and a mixture of the components with the background absorption of the UV spectrum at 290--360 nm. The ratio of the heptaen and the mixture was also constant during the whole fermentation process.

Anti-Bacterial Agents↗
PubMed1996

[Droplet countercurrent chromatography to isolate ecdysteroids from the herb Silene tatarica (L>)].

Our research project was to isolate ecdysteroids from Silene tatarica (L.) Pers., which plant belongs to Caryophyllaceae family. This plant as a species of the Silence genus has been considered as a good potential source of ecdysteroids, including new, hitherto never identified natural product(s). The raw material of isolation was the herba of Silene tatarica (L.) Pers. that was cultivated in the fields of the Ecological and Botanical Research Institute of the Hungarian Academy of Sciences (Vácrátót, Hungary) and the plants were collected in June of 1994. The isolation procedure started with methanol extraction of the ground and milled herba. To improve the recovery, the extraction was done with boiling methanol under reflux for 20 minutes. The crude extract was purified using simple techniques, such as fractioned precipitation (with acetone), solvent-solvent partition (benzene-aqueous methanol). Further purification includes droplet counter-current chromatography (chloroform-methanol-water, 65:20:20, v/v/v) and straight-phase HPLC (silica stationary phase, dichloromethane-150 propanol-water. 125:25:2 v/v/v, mobile phase). Structural identification was done by using mass spectrometry and NMR spectroscopy. Three of the isolated ecdysteroids were identified as 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone and 20-hydroxyecdysone-22-benzoate these compounds have never been detected in Silene tatarica (L.) Pers. Structural elucidation of the fourt isolated ecdysteroid resulted in a new natural product with the chemical structure of 2-deoxy-20-hydroxyecdysone 22-benzoate.

Countercurrent Distribution↗
PubMed2003

Effect of blood flow on thermal equilibration and venous rewarming.

In this study we have explored the feasibility of using an isolated rat limb as an animal model for studying countercurrent arterial thermal equilibration and venous rewarming in muscle tissue. Unlike in vivo experiments in which animal models have been used for studying thermoregulation or temperature response in tissue under various physiological conditions, isolated organ or tissue provides for better control and more accurate measurement of the blood perfusion rate. It has been shown that the induced perfusion rate in the rat limb can vary from 3 ml/(min x 100 g) at normal physiological conditions to 25 ml/(min x 100 g) during hyperemic conditions. Temperature distributions along the countercurrent arteries and veins have been measured using fine thermocouple wires. We observed a 25%-78% thermal equilibration along the femoral artery and its branches in intermediate size vessels between 700 and 300 microm diameter. This equilibration depends strongly on the local perfusion rate. In comparison, local perfusion rate plays a minor role in determining the overall venous rewarming in the rat hind limb. Approximately 70%-80% of the heat leaving the artery is recaptured by the countercurrent vein. This agrees well with our previous theoretical and experimental results, which show a dramatic shift in thermal equilibration between the supply artery and vein tissue cylinder and the secondary vessel tissue cylinder as the flow rate changes.

Adaptation, Biological↗
PubMed1986

Subcellular distribution of rat pituitary homogenates by poly(ethylene glycol)-dextran countercurrent partitioning.

Rat pituitary homogenates were subjected to two phase countercurrent partition in a poly(ethylene glycol)-dextran mixture using a simple apparatus with enhanced gravity to facilitate the phase separations. Assay of the fractions for organelle marker enzymes and prolactin after 17 transfers showed similar distributions for endoplasmic reticulum, lysosomes, prolactin granules and plasma membrane at the lowest dextran concentrations. Increasing the dextran concentrations had a differential effect on the various organelles. Excellent resolution of endoplasmic reticulum from the other organelles was obtained and marked organelle heterogeneity was demonstrated. Two-phase countercurrent partition thus offers an alternative approach to the subcellular fractionation of pituitary homogenates and should prove useful in separating endoplasmic reticulum from plasma membrane and other cell components.

Acetylglucosaminidase↗
PubMed2006

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method.

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.

Chromatography, High Pressure Liquid↗