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PubMed1998

Analysis of domoic acid in shellfish by thin-layer chromatography.

A thin-layer chromatography (TLC) method has been developed for the semi-quantitative analysis of domoic acid (DA) in shellfish tissues. Tissues were extracted in a single-step homogenization of tissue with 50 % aqueous methanol and then taken through a selective strong anion exchange cleanup. Cleaned extracts were applied directly to silica gel TLC plates and developed with a butanol-acetic acid-water mixture (3:1:1, Rf = 0.45 for DA). As little as 10 microg DA per gram of tissue could be detected after chromatography using a hand-held short-wave UV lamp to detect fluorescence quenching. Confirmation was provided by spraying the plate with ninhydrin, which reacts with the secondary amine of DA to give a distinctive yellow colored product. The extraction, cleanup and TLC procedures are fast and simple, and do not require the use of expensive equipment. This method should prove useful for the routine screening of shellfish tissues in those laboratories not equipped with an LC system. It should also be useful as a chemical confirmation method for DA in samples tested positive by assay methods such as immunoassay.

Animals↗
PubMed2001

Site distribution in resin beads as determined by confocal Raman spectroscopy.

Scanning confocal Raman spectroscopy was used to study the distribution of reactive sites within a resin bead used for solid-phase synthesis. The distribution of NH2 groups in aminomethylated polystyrene resin (APS) was determined by doping with varying amounts of 4-cyanobenzoic acid. The extent of loading was determined by both elemental analysis and ninhydrin assays. The spatial distribution of the coupled 4-cyanobenzamide within the bead was determined to an in-plane resolution of 1 microm and depth resolution of about 4 microm, using the strong Raman CN stretching vibrational transition at 2230 cm(-1). Dry and swollen beads were studied and the distribution was found to be essentially uniform throughout the bead in all cases.

Journal Article↗
PubMed1980

The origin of nuclear bodies: a study of the undifferentiated epithelial cells of the equine small intestine.

During an electron and light microscopic study of the equine intestinal epithelium, it was observed that some secretory granules of the undifferentiated crypt epithelium were incorporated into the nucleus during mitosis. A study was made of the chemical nature of the granules, using standard histochemical techniques: PAS-Alcian blue, Deamination-PAS, and Ninhydrin-Schiff reactions. The granules contained a neutral protein-polysaccharide complex with many terminal amino groups, possibly an antibody (IgA). The intranuclear granules underwent coalescence and degeneration during differentiation. The end-product was identical with the nuclear bodies seen in other somatic cells and described in the literature. These nuclear bodies were seen in absorptive cells, goblet cells, and Paneth cells; but were not observed in any entero-endocrine cells. This study shows that the nucleus is capable of isolating and degrading unwanted material, foreign and internally generated, and, further, it gives an explanation for the origin of nuclear bodies, structures that have long been an enigma in cell biology.

Animals↗
PubMed2006

Synthesis and characterization of the biodegradable polycaprolactone-graft-chitosan amphiphilic copolymers.

A novel synthetic approach to biodegradable amphiphilic copolymers based on poly (epsilon-caprolactone) (PCL) and chitosan was presented, and the prepared copolymers were used to prepare nanoparticles successfully. The PCL-graft-chitosan copolymers were synthesized by coupling the hydroxyl end-groups on preformed PCL chains and the amino groups present on 6-O-triphenylmethyl chitosan and by removing the protective 6-O-triphenylmethyl groups in acidic aqueous solution. The PCL content in the copolymers can be controlled in the range of 10-90 wt %. The graft copolymers were thoroughly characterized by 1H NMR, 13C NMR, FT-IR and DSC. The nanoparticles made from the graft copolymers were investigated by 1H NMR, DLS, AFM and SEM measurements. It was found that the copolymers could form spherical or elliptic nanoparticles in water. The amount of available primary amines on the surface of the prepared nanoparticles was evaluated by ninhydrin assay, and it can be controlled by the grafting degree of PCL.

Biocompatible Materials↗
PubMed2002

Surface engineering of poly(D,L-lactic acid) by entrapment of chitosan-based derivatives for the promotion of chondrogenesis.

Chitosan and chitosan-amino acid derivatives were explored to engineer poly(D,L-lactic acid) (PDL-LA) as an extracellular matrix-like surface to promote cell adhesion and growth. Four kinds of chitosan-amino acid derivatives were prepared to mimic the carbohydrate moieties of cell matrix glycoprotein. The chitosan-amino acid derivatives were characterized by using Fourier transform infrared and ultraviolet spectra. The amino acid content on chitosan-amino acid derivatives was determined by using a ninhydrin-ultraviolet method. A new strategy, entrapment, was therefore used to modify the PDL-LA membrane with chitosan and chitosan-amino acid derivatives. The results of X-ray photoelectron spectroscopy, attenuated total reflectance-Fourier transform infrared, and contact angle confirmed that a stable thin film of chitosan and its derivatives can be entrapped on the surface of the PDL-LA membrane. From the results of chondrocyte cytocompatibility, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and cell morphology, the chitosan-amino acid derivative modified PDL-LA membranes were shown to promote chondrogenesis. The novel surface treatment method combines the good mechanical property of PDL-LA with the good cytocompatibility of chitosan derivatives, which may have potential for tissue engineering.

Amino Acids↗
PubMed1979

Grafting of proteins onto polymer surfaces with the use of oxidized starch.

A study has been carried out on the coupling of proteins onto crosslinked poly(vinyl alcohol) hydrogel membranes and ethylene-vinyl alcohol copolymer(EVA) films previously grafted with oxidized starches having many pendant aldehyde groups. The coupling reaction of proteins is based on the Schiff's base formation between the amino groups of proteins and the aldehyde groups of the oxidized starches which have been grafted onto the substrate membrane or film through acetalization of the aldehyde of starch with hydroxyl groups of the substrate polymers. The grafting of oxidized starches onto the EVA films seems to be restricted to the film surface, since no detectable change is observed in the weight and the attenuated total reflection infrared spectrum of the grafted films. The amount of grafted protein, determined by the ninhydrin method, reveals that, at least, plasma proteins such as serum albumin and fibrinogen are grafted to the film surface in a monomolecular layer without undergoing a marked denaturation. The alpha-amylase grafted onto the EVA film showed a distinct enzymatic activity in hydrolysis of amylose and starch, but the activity was very low compared with that of the ungrafted, soluble alpha-amylase.

Adsorption↗
PubMed2003

Paraffin spheres as porogen to fabricate poly(L-lactic acid) scaffolds with improved cytocompatibility for cartilage tissue engineering.

Three-dimensional poly(L-lactic acid) (PLLA) scaffolds with high porosity and pore size ranging from 150 to 700 microm were conveniently prepared with paraffin spheres used as porogen. PLLA/1,4-dioxane solution containing a given amount of paraffin spheres was frozen at -25 degrees C to obtain a solidified mixture, followed with freeze drying and subsequent leaching with hexane to remove the 1,4-dioxane and paraffin spheres, respectively. The fabricated PLLA scaffolds were highly porous with evenly distributed and interconnected pores. The microstructures of the PLLA scaffolds as a function of paraffin-sphere size, paraffin-sphere dosage, and PLLA concentration were characterized by confocal laser scanning microscopy (CLSM) and scanning-electronic microscopy (SEM). To improve the cytocompatibility of the bioinert material, a hybrid PLLA scaffold containing Type I collagen was prepared by pressing the collagen solution into the scaffold under reduced pressure. The amounts of the collagen introduced in the scaffolds were detected by ninhydrin method. The distribution of the collagen in the scaffolds was studied with CLSM. Finally, in vitro cell culture was performed by injecting a chondrocyte suspension into the scaffolds. The results showed that the chondrocytes were more evenly distributed and more spread out in the collagen-modified PLLA scaffolds than in the unmodified ones.

Animals↗
PubMed2006

Poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymers as surface additives for promoting chondrocyte attachment and growth.

The poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymer was explored to engineer poly(D,L-lactic acid) (PLA) material to promote chondrocyte attachment and growth. The poly(D,L-lactic acid)-block-poly(ethylene glycol) copolymer (PLE) was synthesized by a coupling reaction between PLA and poly(ethylene glycol) (PEG) (M(n) 1000, 2000, and 4000 respectively), with the use of 4,4'-methylenediphenyl diisocyanate (MDI). Then the PLE was activated by methyl sulfonyl chloride and the amino acids or arginine-glycine-aspartic acid tripeptide (RGD) was attached, which was verified by the ninhydrin-UV method. The modified PLA films were simply prepared by blending PLA with PLE derivatives. ATR-FTIR, XPS, contact angle, and AFM results clearly showed that the PEG chain stably enriched on the surface of PLE-modified PLA films. The chondrocyte cytocompatibility test showed the modified PLA films could significantly improve chondrocyte attachment and proliferation.

Cell Adhesion↗
PubMed1977

Interrelationships between water and cellular metabolism in Artemia cysts. VII. Free amino acids.

The concentration of total ninhydrin-positive material (NPM) soluble in 5% trichloroacetic acid was measured in cysts of the brine shrimp. Artemia salina, as a function of hydration level. No net change in NPM was observed until the cysts had achieved a water content of about 0.65 g H2O/g of initially dry cysts. Above this hydration threshold the NPM content increased markedly. Examination of the free amino acid composition of cysts incubated at selected hydration levels revealed that almost all of the amino acids underwent net change above the hydration threshold. However, just below this threshold, the free amino acid composition was essentially the same as in fully dried cysts. The activity generating net changes in the concentration of free amino acids above the hydration threshold was shown to be metabolic in nature and restricted to the cellular component of the cyst.

Amino Acids↗
PubMed1978

The isolation of a low molecular weight serum factor which enhances the viability of SV3T3 cells.

A serum factor which enhances the growth/viability of SV40 transformed 3T3 mouse fibroblasts, but not untransformed 3T3 cells in tissue culture has been partially purified from calf serum. The purification, which involves acidification to pH 2, chromatography on Sephadex G-100 at pH 2 followed by either Sephadex G-25 or BioGel P-2, results in material exhibiting two ninhydrin positive spots on thin layer chromatography and five to six dansylatable bands on SDS gels. This substance, termed Peak III or Serum Factor III, has been found in every serum examined thus far (including calf, rat, mouse, goat, lamb, rabbit, pig, horse, and chicken) and appears to be a low molecular weight, heat-insensitive molecule. Attempts to characterize it by chemical analyses and specific enzymatic inactivation have been either negative or inconclusive. Its biological mode of action is presently obscure. While it does not effect DNA synthesis, it does greatly increase the viability of SV3T3 cultures growing in low serum (0.15-0.30% calf serum) medium and, when added to "stationary phase" cells, restores healthy proliferation. This and evidence reported by others suggest that Peak III may affect SV3T3 cells in either overcoming a potentially lethal block in G1 or in passage through G2/M.

Blood↗
PubMed2005

Amino acid signaling through the mammalian target of rapamycin (mTOR) pathway: Role of glutamine and of cell shrinkage.

Mammalian target of rapamycin (mTOR) mediates a signaling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth rate, participating to a versatile checkpoint that inspects the energy status of the cell. The pathway is activated by branched-chain amino acids (BCAA), leucine being the most effective, whereas amino acid dearth and ATP shortage lead to its deactivation. Glutamine- or amino acid-deprivation and hyperosmotic stress induce a fast cell shrinkage (with marked decrease of the intracellular water volume) associated to mTOR-dependent S6K1 dephosphorylation. Using cultured Jurkat cells, we have measured the changes of cell content and intracellular concentration of ATP, of relevant amino acids (BCAA) and of ninhydrin-positive substances (NPS, as measure of NH(2)-bearing organic osmolytes) under conditions that deactivate (leucine-deprivation, glutamine-deprivation, amino acid withdrawal, sorbitol-induced hyperosmotic stress) or reactivate a previously deactivated, mTOR-S6K1 pathway. We have also assessed the mitochondrial function by measurements of mitochondrial transmembrane potential in cells subjected to hypertonic stress. Our results indicate that diverse control signals converge on the mTOR-S6K1 signaling pathway. In the presence of adequate energy resources, the pathway senses the amino acid availability as inward transport of effective amino acids (as BCAA and especially leucine), but its activation occurs only in the presence of an extracellular amino acid complement, with glutamine as obligatory component, and does not tolerate decrements of cell water volume incapable of maintaining adequate intracellular physicochemical conditions.

Adenosine Triphosphate↗
PubMed2001

Early carbonic anhydrase induction in the gills of the blue crab, Callinectes sapidus, during low salinity acclimation is independent of ornithine decarboxylase activity.

Carbonic anhydrase (CA) induction in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to lowered environmental salinity. Simultaneous measurements of ornithine decarboxylase (ODC) activity were made in gills and nonbranchial tissues to determine whether ODC activity and the resultant synthesis of polyamines played a role in the initiation and regulation of CA induction. CA induction in the seventh gill pair (G7) was proportional to the decrease in ambient salinity, but activity in the third gill pair (G3) remained unchanged. Induction began by 24 hr after low salinity transfer, much earlier than previously reported, and peaked after 4 days. The magnitude of salinity change affected the magnitude of CA induction only, not the time course. A general cell volume regulatory response, as measured by the appearance of total ninhydrin-positive substances (TNPS) in the hemolymph, was initiated within 4 hr of low salinity transfer and was complete by 24 hr post-transfer. General cell swelling may be the initial signal in the pathway of CA induction. ODC activity in the gills of acclimated animals was not influenced by salinity. For crabs transferred from 35 to 25 ppt, ODC activity did not change significantly over the time course of acclimation. There was an early but transient increase in ODC activity in all tissues for crabs acclimated to 28 ppt and transferred to 15 ppt. Induction of ODC activity does not appear to be a precursor for CA induction; therefore, it does not appear that polyamines are substantially involved in the up-regulation of transport enzyme activity in low salinity. ODC, and resultant polyamine synthesis, may, however, have a role in cell volume regulation.

Adaptation, Physiological↗
PubMed1976

Respiratory behaviour of sea-urchin spermatozoa. II. Sperm-activating substance obtained from jelly coat of sea-urchin eggs.

A sperm-activating substance (SAS) was obtained from the jelly coat of sea-urchin ova and its chemical properties were investigated in three sea-urchin species. The SAS was partially purified from the jelly coat of Pseudocentrotus eggs through several steps of purification by procedures consisting of charcoal adsorption, ion-exchange chromatography on DEAE-Sephadex A-25 column, and gel-filtration on Sephadex G-15 columns. The partially purified SAS was found to contain a ninhydrin-positive material and is inactivated by pronase digestion. The molecular weight of SAS was estimated as about 630 by gel-filtration through Sephadex G-25 and the isoelectric-point of SAS is located at about pH 5.3 by isoelectrofocusing method. The SAS is non-volatile, alcohol-soluble, and labile in a diluted alkaline or acid solution. The origin of SAS is discussed.

Animals↗
PubMed1993

Changes of amino acid and monoamine levels after neonatal 6-hydroxydopamine denervation in rat basal ganglia, substantia nigra, and Raphe nuclei.

The effects of a neonatal dopaminergic deafferentation with the neurotoxin 6-hydroxydopamine (6-OHDA) on endogenous tissue levels of catecholamines, indoleamines, and amino acids were investigated in discrete rat brain regions. After producing the lesion at postnatal day 3 by intraventricular injections of 6-OHDA, with a desipramine pretreatment to protect noradrenaline neurons, the animals were kept for 3 months. Their brains were dissected to obtain samples of neostriatum, Globus pallidus, Substantia nigra, and Raphe nuclei, which were then analyzed by high-performance liquid chromatography, coupled either to electrochemical detection for aromatic monoamines, or to post-column ninhydrin derivatization with spectrophotometry for amino acids. The neonatal 6-OHDA treatment depleted dopamine (DA) levels in neostriatum, Globus pallidus, and Substantia nigra, but in Raphe nuclei DA was increased. The main metabolites of DA were also decreased in neostriatum, Globus pallidus, and Substantia nigra but remained unchanged in Raphe nuclei. Serotonin (5-HT) and its metabolite 5-hydroxy-indole-3-acetic acid increased in neostriatum and Raphe nuclei; in Substantia nigra there was a slight increase in 5-HT only. The 6-OHDA lesion caused heterogeneous alterations in amino acid contents, which varied according to the region. In the neostriatum there were increases of gamma-aminobutyric acid (GABA), aspartic acid, and glycine. In the Globus pallidus taurine, GABA, glutamic acid, glutamine, aspartic acid, serine, and alanine were elevated. In the Substantia nigra only increases in taurine, GABA, glutamic acid, and glutamine could be documented. This study shows important changes in amino acid levels and in some of their ratios, occurring in different anatomical subdivisions of the basal ganglia and related brainstem nuclei following a neonatal treatment with 6-OHDA. The results thus demonstrate major biochemical modifications in amino acids in the aftermath of a DA denervation and/or a 5-HT hyperinnervation during an early developmental period.

Amino Acids↗
PubMed1976

Structure and toxicity of alkaloids and amino acids of Sophora secundiflora.

Seeds from Sophora secundiflora were extracted into three major fractions: lipids, alkaloids, and amino acids. Most of the lipid material was composed of steroid esters. These esters were hydrolyzed, and the fatty acid compositions were determined. The major alkaloid component was cytisine, with N-methylcytisine, anagyrine, and the fatty acid compositions were determined. The major alkaloid component was cytisine, with N-methylcytisine, anagyrine, and termopsine in lower concentration. The major free ninhydrin-positive compound was gamma-glutamyltyrosine. In addition to several common amino acids, pipecolic acid and 4-hydroxypipecolic acid were identified. Both the amino acid fraction and the alkaloid fractions caused minor pharmacological disorder when injected into rats. However, when both fractions were simultaneously administered, they were lethal.

Alkaloids↗
PubMed1976

Beta-hydroxynorleucine: separation of its isomers and biological studies.

Separation of the four isomers of beta-hydroxynorleucine was accomplished by partition column chromatography and asymmetric enzymatic hydrolysis of the N-chloroacetyl derivatives. From these, the corresponding N-chloroacetyl derivatives were made. The purity and configuration of each isomer of the free acid and N-chloroacetylated derivative were ascertained by: (a) paper chromatography in five solvent systems, (b) elemental analysis, (c) Van Slyke nitrous acid determination of alpha-carbonyl carbon, and (d) Van Slyke ninhydrin determination of alpha-carbonyl carbon, and (e) optical rotation. Comparison of the rate of enzymatic hydrolysis by hog renal acylase I of the N-chloroacetyl derivative of the L-isomers of each diastereomer showed that the acyl B isomer is a better substrate than the acyl A isomer, where A denotes the faster moving diastereomer and B denotes the slower moving diastereomer in a defined chromatographic solvent system. Microbiological assay using Lactobacillus casei in a system selected for screening for possible antitumor activity indicated that while none of the isomers as free amino acids had any growth inhibitory action, the N-acylated isomers showed modest but significant activity. The N-chloroacetyl derivative of the D-enantiomorph of diastereomer B exhibited the greatest growth inhibitory activity, showing about twice the activity of the other three isomers.

Aminocaproates↗
PubMed1991

Simultaneous determination of water-soluble vitamins by over-pressure layer chromatography and photodensitometric detection.

An over-pressure layer chromatographic procedure with photodensitometric detection for the simultaneous determination of water-soluble vitamins in multivitamin pharmaceutical preparations was developed and evaluated. The method uses high-performance TLC (HPTLC) plates with silica gel as the thin-layer, and an n-butanol:pyridine:water mixture (50:35:15, v/v/v) as mobile phase at a rate of 0.25 mL/min for baseline separation. The quantitation was carried out without derivatization (vitamin B1, vitamin B2, vitamin B6, folic acid, nicotinamide, vitamin C) or after spraying ninhydrin reagent (calcium pantothenate) or 4-dimethylaminocinnamaldehyde (vitamin B12, biotin). This was applied to the analysis of multivitamin solutions. Satisfactory relative standard deviations and good recovery were obtained for all the vitamins examined. It was concluded that this method is fast, accurate, specific, and suitable for routine quality control use.

Absorptiometry, Photon↗