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Physicochemical characterization of nanoparticles in highly diluted preparations and exploratory plasma proteomic correlates in an N-of-1 study.

The physicochemical properties of highly diluted homeopathic preparations remain insufficiently characterized. This study investigated particulate features of Kali carbonicum (K2CO3) at 50-millesimal potencies (LM4-LM7, ∼1:50,000 dilutions per step) and explored plasma proteomic changes in a placebo-controlled N-of-1 trial. Scanning electron microscopy showed larger particle size in Kali carbonicum (67.3 nm) than in the lactose control (47.5 nm) at LM4 in a descriptive comparison. Dynamic light scattering showed no significant differences in size, polydispersity, or zeta potential among Kali carbonicum, lactose control, and solvent blank, accounting for vial-level clustering. Atomic force microscopy showed more compact dendritic assemblies in Kali than in lactose controls, suggesting trituration influences self-organization. Raman spectroscopy of LM7 detected carbonate-associated bands absent in controls. Plasma proteomics identified six FDR-significant proteins during Kali exposure, including increased S100A9, with exploratory enrichment for inflammation, cytoskeletal, and motility terms. These findings are exploratory and do not imply causality.

Proteomics

Modulation by cell trypsinization of Sendai virus expression in African green monkey kidney cells: first infection and establishment of a carrier state.

Two African green monkey kidney (AGMK) cell lines, 37RC (interferon-producing) and Vero (non-interferon-producing), were infected by egg-grown Sendai virus passaged in eggs at high and low m.o.i. The appearance of haem-adsorption, and cytopathic effect (c.p.e.) as well as the presence of haemagglutinating virions in the supernates were much more pronounced with a virus seed obtained with 10(-3) diluted passages than with a seed obtained with undiluted inoculum. They were also independent of interferon production (data obtained in 37RC and Vero cells were almost superimposable). In studies carried out with the virus seed prepared at high dilution the establishment of infection was maximal when monolayers were infected as soon as 5 h after trypsinization and cell seeding, regardless of cell density. Virus in supernates obtained from cultures infected 5, 20 or 50 h after seeding exhibited a greatly reduced infectivity for monkey cells, but not apparently for chick embryos. Trypsin treatment of the virus supernates restored their infectivity for AGMK cells more efficiently for virus released from cells infected 5 h after seeding than for virus released from cells infected later after seeding. In keeping with these observations, virus in supernates from cultures infected 5, 20 or 50 h after seeding contained increasing amounts of auto-interfering virions. The decreased infectivity obtained in cell supernates was not accounted for by major differences in virus RNA synthesis. Similarly, the optimum infection established in cells seeded only for 5 h was not due to increased virus adsorption. Several lines of cells surviving first infection with egg-grown Sendai virus have been obtained and kept in cultures for 3 to 18 months. Virus release and c.p.e. apparently become reduced in the cells surviving the first infection until the newly repopulated monolayers must undergo trypsinization. Weekly protease treatments of the cells reactivate all parameters of virus infection which again will tend to disappear slowly and then reappear following each trypsinization ('intermittent' carrier state). The establishment and the 'intermittent' reactivation of these lines were not prevented by the inclusion in the medium of anti-Sendai virus serum. Temperature-sensitive ts functions do not seem to play an important role in this virus-host relationship.

Adsorption

The allosteric regulation of hexokinase C from amphibian liver.

A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.

Adenosine Triphosphate

Clonal transformation of human leukocytes by Epstein-Barr virus in soft agar.

The B95-8 strain of Epstein-Barr virus (EBV) induced colony formation of human umbilical cord-blood leukocytes in soft agar medium. One-hit response relationship between the number of colonies and the virus dose was observed with high dilutions of the virus preparation. However, there was a presumed cell-killing effect with low dilutions of virus. The colonies were similarly induced, but with a lower efficiency, in adult peripheral blood leukocyte cultures infected with the virus. The colony-forming activity of EBV was neutralized by anti-EBV-positive but not by negative human sera. The cells in colonies were capable of growing continuously and carried EBV-associated nuclear antigen. Thus, it was evident that the colony formation was caused by clonal transformation by EBV.

Agar

Homologous radioimmunoassay for canine prolactin.

A homologous double antibody radioimmunoassay has been developed for canine prolactin. Purified canine prolactin was iodinated by lactoperoxidase/H2O2 to an average of 101 "+/- 10.1 muCi/microgram. Antiserum was used at a final dilution of 1:80 000 and at this concentration bound approximately 20% of the added tracer in the absence of competing unlabelled prolactin. Partial cross-reaction was observed with ovine and bovine prolactin but there was no cross-reaction with a highly purified canine growth hormone preparation. Dilutions of pregnant and lactating bitch sera were parallel to the purified canine prolactin standard curve. Mean prolactin levels in normal male and anoestrous females were 8.8 +/- 0.8 and 12.6 +/- 2.6 ng/ml, respectively. Thyrotrophin-releasing hormone (TRH) induced a consistent elevation in prolactin levels 15--30 min after intravenous injection.

Animals

Fluorimetric assay of tobacco leaf dehydrogenases with resazurin.

A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.

Dihydrolipoamide Dehydrogenase

Gastrin-histamine as a normal sequence in gastric acid stimulation in the rabbit.

In a suspension of isolated gastric glands the effect of secretagogues on the oxyntic cells can only be detected by direct stimulation. An indirect stimulus like gastrin inducing one type of cell to liberate histamine which then acts on the oxyntic cells will not be detectable because of the very high dilution of the liberated substance. Thus the isolated gland preparation presents a means by which two steps in a sequential stimulation can be separated. There is no evidence that gastrin acts directly on the oxyntic cells but it does liberate histamine in a dose-effect relationship, which would in an intact stomach give histamine concentrations sufficient to effectively stimulate the acid secretion. Thus in the rabbit histamine seems to be a normal physiological mediator for gastrin stimulation.

Animals

Ferredoxin-Sepharose 4B as a tool for the purification of ferredoxin-NADP+ reductase.

Ferredoxin immobilized on Sepharose 4B was prepared by reaction of CNBr-Sepharose 4B with spinach ferredoxin. The ferredoxin-Sepharose 4B conjugated ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, [EC 1.6.7.1]) in dilute buffer solution and released it in high salt concentrations. A novel method of preparation for the reductase was established by a combination of affinity adsorption on the ferredoxin-Sepharose 4B column with usual purification procedures. It was found using the new method, that there are two forms of ferredoxin-NADP+ reductase, FNR I and FNR II, in spinach. Comparative studies of the two components suggest that FNR I may be a dimer of FNR II.

Chromatography, Affinity

Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands.

A highly purified preparation of high-molecular-weight adrenocorticotropic hormone (ACTH) was prepared from ovine pituitary glands by dilute acetic acid extraction, oxycellulose fractionation. Sephadex gel filtration, and affinity chromatography on immobilized alphap(1-39)ACTH antibodies. Two ACTH peptides of molecular weights of 24 000 and 34 000 were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis in this preparation. It appeared that the immobilized antibodies adsorbed two forms equally well and could not distinguish between them under the conditions used. These two ACTH peptides were found to be present in crude extracts of ovine pituitary glands, indicating that they were not artifacts produced by the purification procedure. The high-molecular-weight forms of ACTH were found to be susceptible to degradation by tissue enzymes. They could be easily destroyed during the extraction, if precautions were not taken. Moreover, they were poorly adsorbed by oxycellulose which had been used for the adsorption of ACTH activity from crude preparations by most investigators. These properties probably accounted for the fact that high-molecular-weight forms of ACTH remained undetected until very recently.

Adrenocorticotropic Hormone

Serum reactions. An analysis of commercial antivenoms and the possible role of anticomplementary activity in de-novo reactions to antivenoms and antitoxins.

Infusion of concentrated heterologous serum proteins may precipitate severe reactions in humans. Some of these reactions are not due to a preexisting hypersensitivity to the foreign protein, but rather to the strongly anticomplementary activity (ACA) of the infused protein. The latter mechanism could account for the occasional anaphylactoid reaction seen in a patient who has had no known previous exposure to the foreign protein. Similarly, it could account for the patient who fails to react to a preliminary subcutaneous trial dose, but then collapses when he is infused later with the same preparation. Nine commercial antivenoms from nine countries were examined and most were found to have high levels of anticomplementary activity. Considerable variation was found in both the immunochemical properties and the anticomplementary activity of these antivenoms. Antitoxins of equine origin to the toxins of diphtheria, tetanus and gas gangrene were also found to have high anticomplementary activity. Because of this activity these preparations should always be diluted and infused slowly. Lack of reaction to a test dose will never guarantee an uneventful infusion, and thus all infusions of heterologous immunoglobulin should be undertaken with the greatest caution and vigilance.

Animals

Skin reactions to propylene glycol.

Propylene glycol (PG), ethylene glycol (EG), and polyethylene glycol 400 (PEG 400) were tested, as is, in a total of 1,556 cases of eczema using the chamber test method. All the year round, the number of positive reactions to PG was 12.5% to EG 4.9%, and to PEG 400 0.3%. A total of 30% of the positive reactions to PG were allergic in appearance. Also the new fatty alcohol-PG bases of Metosyn ointment and Topilar ointment as well as Metosyn ointment (fluocinonide) itself provoked reactions in a great number of patients with positive reactions to PG, as is. The reactions to PG were considered to be truly allergic in four cases. In them, positive reactions were elicited by testing with high dilutions of PG and by applying the glycol in the patients' armpits as an open test. It is concluded that PG and topical preparations containing it in high concentrations should not be used with occlusion, and that allergic reactions must be watched.

Anti-Inflammatory Agents, Non-Steroidal

Prepatent periods of a tropical strain of Plasmodium vivax after inoculations of tenfold dilutions of sporozoites.

Eleven patients requiring malaria therapy were inoculated intradermally with graded doses of sporozoites of the Chesson strain of Plasmodium vivax. Estimated doses of 10 sporozoites were given to four patients, of 100 to three patients, of 1,000 to two patients, and of 10,000 to two patients. Parasitaemia was detected in all patients 12 to 17 days after inoculation; fever began on the 14th to 19th days. The results of the trials are compared with earlier work on a temperate strain of P. vivax in which patients given small doses of sporozoites exhibited long prepatent periods of 257 days or more. It is concluded that the differences in the two strains can be explained by the assumption that, in varying proportions, all strains of P. vivax produce two types of sporozoites, one eliciting short prepatent periods (Type I), and the other lying dormant or developing slowly to give rise to long prepatent periods (Type II). The latter type greatly predominates in temperate strains, but not in tropical strains; at high dilutions, therefore, pure suspensions of Type II sporozoites of temperate strains can be prepared. It is thought that relapses of P. vivax are in reality a dalyed parasitaemia arising from Type II sporozoites.

Humans

Enhancement of tumor induction in rats with Moloney murine sarcoma virus by a "new" method based on direct injection into fetuses.

Undiluted, fivefold-diluted, and 25-fold-diluted doses of a stock of Moloney murine sarcoma virus were injected directly, in a volume of 0.025 ml, into the backs of fetal Sprague-Dawley rats by laparotomy through the uterine wall at 18 days of gestation. During the first 8 weeks after birth the young responded to the virus with remarkably high but dose-dependent incidences of neoplasms. When a one-fifth dilution of the virus preparation was inoculated at fetal ages 16, 18, and 20 days, the incidences of lesions decreased with advancing fetal age. The tumors developed preferentially at the virus inoculation site and/or in the proximal parts of the extremeties; all were considered to be of mesenchymal derivation, i.e., malignant mesenchymoma, rhabdomyosarcoma, osteosarcoma, fibrosarcoma or fibromyxosarcoma, hemangiosarcoma, plasmacytoma, and a giant cell tumor. This injection procedure provided us with a valuable experimental tool for the rapid screening or testing of potential chemical carcinogens and other biologic studies.

Animals

Development and application of homologous radioimmunoassays for porcine gonadotrophins.

Antisera were raised against highly purified preparations of porcine luteinizing hormone (pLH) and follicle-stimulating hormone (pFSH). Highly specific and sensitive radioimmunoassay systems were developed. The antisera to LH and FSH were used at working dilutions of 1:500,000 and 1:200,000 respectively and the sensitivities of the assays were 0.1 ng LH/ml serum (3 x 10(-12) mol/l) and 0.5 ng FSH/ml serum (1.5 x 10(-11) mol/l). The LH and FSH preparations used as standards were 1.2 and 81 times as potent as NIH-LH-S15 and NIH-FSH-P1 respectively. Both assays were validated and adapted for the measurement of the gonadotrophin content of porcine serum. The concentrations of LH and FSH in blood were measured simultaneously in prepubertal sows throughout a 24 h period, in adult sows during the oestrous cycle and in both prepubertal and adult animals after treatment with LH releasing hormone.

Animals

Human monocyte cytotoxicity to tumor cells. I. Antibody-dependent cytotoxicity.

Recent investigations examining mononuclear cell antibody-dependent cell-mediated cytotoxicity against tumor cell lines suggest that K lymphocytes and not monocytes are active in this cytotoxic reaction. We have found, however, that in an allogeneic assay system, human monocyte monolayers as well as lymphocytes mediate substantial lysis of 51Cr-labeled antibody-coated CEM lymphoblast tumor cells. This cytotoxicity is temperature-dependent and rapid, with most 51Cr release occurring in the first 4 hr of co-incubation. Interaction between target cell-bound antibody and the monocyte Fc receptor is necessary as demonstrated by the marked fall in antibody-dependent cell-mediated cytotoxicity (ADCC) produced by staphylococcal protein A, high concentrations of nonspecific immunoglobulin, and dilution of the target cell antiserum. Morphologic and functional characteristics of the monocyte-monolayer preparations establish their relative purity (greater than 95%) and indicate that monocytes and not contaminating lymphocytes are responsible for tumor cell lysis. Furthermore, preincubation of monocyte and lymphocyte preparations with latex particles or low concentrations of immunoglobulin distinguished monocyte from lymphocyte ADCC. Thus, normal human monocytes have the capacity to carry out antibody-dependent cytotoxicity against nucleated malignant target cells.

Antibody-Dependent Cell Cytotoxicity

Sample homogenization procedure for determination of lead in canned foods.

To improve the homogeneity of canned foods before Pb determination, composited samples or single can contents (150--500 g) are treated with an equal weight of dilute nitric acid, and then thoroughly blended in a high efficiency, probe-type Polytron homogenizer; subsamples are then either dry-ashed or wet-ashed in preparation for analysis. Within-can variability is reduced from the 20--50% coefficient of variation range obtained with conventional blending procedures to a coefficient of variation of less than 5% by using the proceudre described. Precision data for Pb, using both the probe-type and conventional blade-type homogenizers, are compared. The procedure described for Pb determination was used to analyze a number of canned products. Data indicate that the Polytron plus dilute acid procedure improves overall Pb homogeneity for subtle as well as severe initial Pb heterogeneity.

Food Analysis

Preparation of stable intermediate-purity factor VIII concentrate with a note on high-purity factor VIII.

Cryoprecipitate prepared by a rapid thawing technique was pooled in batches of 600--720 donor units and washed with ice-cold Tris-citrate-NaCl solution. After dissolving at 37 degrees C, it was adsorbed with Al(OH)3 and kaolin, and cleared by centrifugation. The supernatant, diluted with 5% dextrose was passed repeatedly through a bed of Celite, filtered through a 293 mm X 0.3 micrometer membrane disc and lyophilized. Typical composition was 15 U . ml-1 factor VII, and 40 mg . ml-1 protein with a yield of 300 U/l of starting plasma. The crude factor VIII concentrate was also a suitable material for preparation of high-purity factor VIII by controlled pore glass chromatography.

Blood Donors

Stabilization of interferons.

It is now obvious that there are a number of factors which can influence the stability of interferons. Since human fibroblast interferon is especially unstable both to heat and mechanical stress, the use of stabilizing conditions such as low pH or thioctic acid should facilitate purification and concentration of this interferon. Leukocyte interferon, on the other hand, seems to be more stable even when highly purified; however, losses in activity can occur in very dilute solutions containing low concentrations of protein. Inactivation during storage of interferons may be avoided by using freeze-dried preparations. In order to generate large quantities of purified, stable interferons for clinical use, there is a need to establish an armamentarium of different nontoxic techniques and approaches that will enable investigators to stabilize human interferons at each step in preparation, processing, concentration, purification, shipment and storage.

Fibroblasts