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PubMed1982

Physicochemical and biological comparison of polyene macrolide antibiotics fungichromin, lagosin and cogomycin.

The three polyene macrolide antibiotics, fungichromin, lagosin, and cogomycin, previously described as having some stereochemical differences at one or more centers, are shown by countercurrent distribution, high-performance liquid chromatography, carbon-13 nuclear magnetic resonance spectroscopy, circular dichroism, and biological studies to be identical in all respects, including stereochemical aspects. The differences observed earlier in their properties have now been ascribed to varying amounts of impurities, which are separable by high-performance liquid chromatography. All three antibiotics contain one major and several minor components.

Antibiotics, Antineoplastic↗

PubMed1982

Characterization of the antifungal and antiprotozoal antibiotic partricin and structural studies on partricins A and B.

Partricin, a heptaene macrolide antibiotic, has been separated into three polyene components, partricins A, B and C, and one non-polyene component by countercurrent distribution. Treatment of partricin with base gave p-(methylamino)acetophenone and p-aminoacetophenone from partricins A and B, respectively, identifying both as members of the aromatic subgroup of the heptaene antibiotics. Both partricins A and B yield mycosamine on mild acid hydrolysis. NMR and mass spectral studies on products of ozonolysis or hydrogenolysis of acetyl derivatives provided evidence for the partial structures 1 approximately 9.

Antifungal Agents↗

PubMedDate not supplied

Assessment of the acrosomal status of ram spermatozoa by RCA lectin-binding and partition in an aqueous two-phase system.

The acrosome reaction is an important marker for sperm function. Because different laboratory techniques may be used to detect this exocytotic process, the objective of this study was to investigate the use of fluoresceinated lectins to assess the acrosomal status of nonpermeabilized ram spermatozoa. In addition, we used centrifugal countercurrent distribution (CCCD) in an aqueous 2-phase system to assess the sperm surface modifications associated with the acrosome reaction by observing changes in their partition behavior. We analyzed the binding of 5-fluorescein isothiocyanate (FITC)-conjugated lectins to ram sperm to select a lectin that bound preferentially to the acrosomal region, which would allow differentiation of acrosome-intact from acrosome-damaged ram spermatozoa. Ricinus communis agglutinin (RCA) bound intensely to the anterior and weakly to the equatorial acrosomal regions. Acrosomal labeling changed when spermatozoa were induced to acrosome-react with calcium ionophore A23187. RCA acrosomal labeling significantly increased (P < .0001) after incubation (84% versus 28% in control samples). To determine if RCA lectin labeling could be used to assess the acrosomal status of fresh ram spermatozoa in suspension, we compared the percentage of acrosome-reacted sperm detected by the carboxyfluorescein diacetate/propidium iodide (CFDA/PI) double-fluorescent staining with the percentage detected by FITC-RCA labeling. The incidence of acrosome-reacted spermatozoa detected by CFDA/PI was not significantly different (P = .704; 13 comparisons in 6 different experiments) from the incidence of spermatozoa detected by FITC-RCA staining. The evaluation of the spontaneous acrosome reaction by RCA labeling (5.83%) was not significantly different (P = .644) from that assessed by CFDA/PI (6.88%). The percentage of induced acrosome reactions detected by CFDA/PI staining (56%) significantly correlated (P < .0001; r = 0.876) with that detected by RCA labeling (56.67%). We simultaneously carried out a comparative CCCD in an aqueous 2-phase system to analyze sperm surface changes associated with the acrosome reaction. Results revealed that sperm surface hydrophobicity decreased in samples that had been incubated with ionophore compared with the untreated-control samples. Likewise, RCA binding after CCCD showed that all acrosome-reacted cells were stained, whereas only 42% of cells were lectin-labeled in the untreated semen sample. This change in lectin reactivity of acrosome-reacted spermatozoa signals the presence of some deep membrane or intracellular residues that would affect partitioning. Therefore, the FITC-RCA-labeling procedure can be used to accurately assess the acrosomal status of ram spermatozoa in suspension.

Acrosome Reaction↗

PubMed1975

Isolation and characterization of a new pancreatic polypeptide hormone.

A method is described for isolation, from chicken pancreas, of an avian pancreatic polypeptide which may be a new hormone. This method involves acid-alcohol extraction, gel filtration, DEAE-cellulose chromatography, and droplet countercurrent distribution. The peptide contains 36 amino acids, has a molecular weight of 4240 and the isoelectric point if pH 6 to 7. The average amount of avian pancreatic polypeptide extractable from chicken pancreas was 4 mg/100 g of pancreas. The amino acid sequence of the peptide is Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.

Amino Acid Sequence↗

PubMed1975

The carboxylic acid groups of bovine luteinizing hormone. The effects of their modification on receptor site binding and subunit-subunit interaction.

The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies.

Amino Acid Sequence↗

PubMed1990

Isolation and structural elucidation of biotransformation products from acarbose.

Following oral administration the a-glucosidase inhibitor acarbose (O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl) -2-cyclohexen-1-yl]amino]-a-D-glucopyranosyl-(1----4)-O-a-D-glu copyranosyl-(1----4)-D-glucopyranose, Bay g 5421) is degraded by digestive enzymes and/or intestinal microorganism. The effect of anaerobic intestinal bacteria can be studied in an in vitro model which involves the incubation of acarbose with human or animal intestinal flora. Acarbose and nine biotransformation products can be isolated from the incubation mixture. These products were identified by nuclear magnetic resonance and mass spectrometry as so-called component 2 (loss of the terminal glucose), component 1 (loss of both glucose rings), hexose homologues of acarbose and component 2, methyl homologues of acarbose, butyric acid ester of component 2, basic disaccharide (loss of the cyclitol ring of component 2), delta-aminovaleric acid and gamma-aminobutyric acid. Following oral administration of [14C]-acarbose to healthy volunteers, 35% of the radioactivity was excreted in the form of at least 13 metabolites in the urine. Three of the metabolites were isolated by Craig countercurrent distribution and ion-pair HPLC and characterized by virtue of their nuclear magnetic resonance and mass spectra as derivatives of 4-methylpyrogallol. Two were shown to be monomethylether-monosulphates while the third was a monosulphate-monoglucuronide. The synthesis of ten reference substances and the comparison of HPLC and UV data clearly indicated that the majority of the non-isolated metabolites were also 4-methylpyrogallol derivatives. The peculiarities of the nuclear magnetic resonance and mass spectra of this type of compound are discussed.

Acarbose↗

PubMed1985

Enrichment of a fraction toxic to guinea-pigs from Pachystigma pygmaeum (Schltr.) Robyns.

Pachystigma pygmaeum is one of several species of rubiaceous plants which cause delayed heart failure among ruminants after their ingestion at relatively high doses. Using guinea-pigs for toxicity determinations, we were able to separate and enrich a toxic fraction from a fermentation extract of the plant material by countercurrent distribution. It contained virtually no potassium salts, passed through a 500 dalton selective membrane, exhibited lability under acid conditions and was toxic at 1 g/kg per os, with a delayed response of 3-4 days.

Animals↗

PubMed1979

Partial synthesis of harringtonine.

The partial synthesis of harringtonine from cephaltotaxine has been described. A key intermediate, 5, 5-dimethyl-2-hydroxytetrahydrofuran-2-carboxylic acid (V), prepared from 4-methyl-1, 4-valerolactone, was dehydrated smoothly to give 5, 5-dihydrofuran-2-carboxylic acid (VI), Through its sodium salt, VI was converted into the corresponding acyl chloride VIII, which reacted with cephalotaxine in the presence of pyridine to give ester IX. After being treated with hydrichloric-acetic acid, ester IX unerwent Reformatsky reaction to give a mixture (XIV) of harringtonine and its diastereoisomer (epiharringtonine) as the final product which was purified either by countercurrent distribution or column chromatography on neutral alumina. The amounts of the two epimers in the mixture shown by TLC were roughly equal.

Alkaloids↗

PubMed1978

[Hemoglobins, XXI: sequence analysis of porcine hemoglobin (author's transl)].

The hemoglobin of a bavarian domestic pig (Suidae) was isolated. The chains were separated by countercurrent distribution, then cleaved with trypsin. The isolated peptides were sequenced with hydrophilic phenylisothiocyanate I and IV, or with a dimethylaminopropyne program in the sequenator. The sequences of the chains are given. Some methodical aspects of automatic sequencing are discussed and the sequences of human porcine hemoglobin are compared. The role of adult pig haemoglobin as foetal hemoglobin is discussed.

Animals↗

PubMed1981

Affinity partitioning of membranes. Evidence for discrete membrane domains containing cholinergic receptor.

Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high as 20,000 micrometers-2. Intense homogenization of the electroplax releases membrane fragments enriched in nicotinic receptor from basal lamina and other synaptic cleft and presynaptic elements. Ideally, preparations of membrane fragments, highly enriched in nicotinic receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near the levels observed after receptor dispersal in detergents and subsequent affinity chromatography. We report the application of affinity partitioning, combined with multiple extraction techniques, to yield preparations of virtually homogeneous membranes enriched in nicotinic receptor alpha, beta, gamma, and delta subunits as well as the 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves to resolve three populations of receptor-containing membranes. One fraction is refractory to affinity partitioning and may represent aggregates of receptor-rich membranes with fragments derived from nonsynaptic membranes. The second and third fractions contain membrane fragments derived from the subsynaptic membrane and are highly enriched in nicotinic receptor (5.1 to 7.8 nmol of alpha-bungarotoxin binding sites/mg of protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of all three fractions indicates that alpha, beta, and gamma subunits are present in stable stoichiometric ratios, while the 43,000-dalton peripheral protein content varies by 33% between the fractions. However, removal of 90% of the 43,000-dalton component by mild alkali treatment does not result in conversion of one fraction into the other. The combination of affinity partitioning and counter-current distribution techniques utilized in this study should prove useful in the resolution of a variety of subcellular particles that contain specific binding sites.

Animals↗

PubMed1994

[Askostatin--a fungicide from Streptomyces viridovulgaris].

Askostatin is a fungicide isolated from the culture of Streptomyces viridovulgaris. It was separated by the countercurrent distribution procedure into components A and B. By the mass and NMR spectra and the biological findings the components were identified with cycloheximide and isocycloheximide respectively.

Antifungal Agents↗

PubMed1993

[Variation of behavioral traits in natural Drosophila melanogaster populations from Eurasia].

Two behavior traits--phototactic response and locomotor activity--were studied in 13 populations of Drosophila melanogaster with countercurrent distribution technique. These traits were characterized by three parameters: average phototactic (locomotor) activity, normalized variance of activity and proportion of inactive flies. Males had bigger average phototactic activity and average locomotor activity, but less proportion of inactive flies than females. For phototaxis, no correlation was found between all estimated parameters and geographic localization of populations. For locomotor activity, females exhibited clinal variation in proportion of inactive flies.

Animals↗

PubMedDate not supplied

Improvement of cryopreserved ram sperm heterogeneity and viability by addition of seminal plasma.

The effect of seminal plasma, as well as some seminal plasma fractions, on the heterogeneity and viability of frozen-thawed ram spermatozoa was studied. Fresh and frozen sperm samples were simultaneously assessed by a routine semen-quality assay and centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system analysis. All samples used for this study were obtained from Salz rams by an artificial vagina and frozen according to the Fiser method. Sperm samples, consisting of either raw or washed semen, were frozen in the presence of either whole ram seminal plasma (RSP) or > 10 kDa seminal plasma fraction. Two control samples were considered: one diluted in Fiser's extender and an undiluted sample. Cell-membrane integrity (viability), hypoosmotic swelling (HOS) test, and sperm motility were assessed in all cases after collection, before freezing (at 5 degrees C), after thawing, and after removing the diluent before CCCD analysis. Our results show that sperm cell partition behavior was dependent on the sperm treatment and that the freezing-thawing process accounted for a clear loss of sperm heterogeneity and membrane integrity. These losses were less apparent when seminal plasma had been removed from semen samples before freezing. In addition, washed semen frozen in the presence of either > 10 kDa seminal plasma fraction or whole re-added seminal plasma maintained a higher rate of sperm heterogeneity and viability.

Animals↗

PubMed1963