An analytical strategy for the identification of carbamates, toxic alkaloids, phenobarbital and warfarin in stomach contents from suspected poisoned animals by thin-layer chromatography/ultraviolet detection.

In this study, an analytical strategy to identify brucine, strychnine, methomyl, carbofuran (alkaline compounds), phenobarbital, and warfarin (acid compounds) using thin-layer chromatography (TLC) screening with ultraviolet (UV) detection at 254 nm in stomach content is shown. The optimum mobile phase was found to be a chloroform: ethyl acetate: diethylamine (0.5:8.5:1) mixture for alkaline substances while a mixture of chloroform: acetone (9:1) has given better results for acidic substances. As for extraction, an equal proportion between distillated water and crude material (1:1) is required. For alkaline compounds, a filtration system was created in order to avoid any interferences from the biological matrix while for acidic compounds only centrifugation (4000 rpm/10 minutes) was required to obtain an appropriate sample. After the respective pretreatments, a one-step liquid-liquid extraction (LLE) has been employed for alkaline substances using a 3 mL of chloroform: ethyl ether (2:1) mixture for 2 min while acidic analytes used 3 mL of chloroform only during 5 min. For both methodologies described, the respective organic layers were dried down and re-suspended with 50 µL of methanol for further TLC plate application. The methodologies have been developed, successfully validated and applied to gastric contents from real case samples of suspected animal poisoning. Positive results from TLC/UV screening were confronted with HPLC-UV and confirmed by GC-MS.

Galloyl derivatives from Caesalpinia coriaria exhibit in vitro ovicidal activity against cattle gastrointestinal parasitic nematodes.

Gastrointestinal nematodes (GIN) are responsible for enormous economic losses worldwide. The use of anthelmintic drugs reduces the parasitic burden in ruminants. However, the excessive use of these drugs triggers anthelmintic resistance in these parasites, which leads to a worrisome inefficacy of most of the commercially available antiparasitic drugs. Caesalpinia coriaria is an arboreal legume possessing medical properties, although the antiparasitic potential of this plant against animal parasitic nematodes has not yet been studied. The aim of this study was to assess the in vitro ovicidal activity of a hydro-alcoholic extract (HA-E) from C. coriaria fruits against GIN and to identify the compounds responsible for this activity through an egg hatch inhibition (EHI) assay. GIN eggs obtained from cattle faeces were used in bio-guided assays. The HA-E was subjected to a liquid-liquid extraction using water and ethyl acetate to obtain two fractions, an organic fraction (EtOAc-F, 27% yield) and an aqueous (Aq-F, 73% yield) fraction. The chromatographic fractionation of the EtOAc-F (2 gr) was performed on a glass column packed with silica gel and eluted with dichloromethane/methanol with 10% ascending polarity. The bioactive compounds were analysed using high-performance liquid chromatography (HPLC) with UV detection, nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS). The HA-E extract and the EtOAc-F showed ovicidal activity at a LC50 of 0.92 and 0.16 mg/mL, respectively. A concentration-dependant effect was observed in both treatments. Chromatographic fractionation of the EtOAc-F, allowed for the isolation and characterisation of three important compounds: methyl gallate (1), gallic acid (2) and an unidentified compound (UC). The bioactive molecules (2 and UC) displayed an ovicidal activity close to 100% at 1 mg/mL concentration. The results of this work show that gallic acid (2) isolated from C. coriaria fruits is responsible for its ovicidal activity. The use of Caesalpinia coriaria could be explored in future studies as an environmentally-friendly alternative for the control of GIN in ruminants.

Determination of lipid profiles in serum of obese ponies before and after weight reduction by using multi-one-dimensional thin-layer chromatography.

Obesity is a key component of equine metabolic syndrome, which is highly associated with laminitis. Feed restriction and/or exercise are known to alleviate the detrimental effects of insulin resistance in obese ponies. However, little is known about changes in the serum lipid patterns due to weight reduction and its association with disease outcomes. Therefore, the lipid patterns in the serum of 14 mature ponies before and after a 14-week body weight reduction program (BWRP) were investigated by multi-one-dimensional thin-layer chromatography (MOD-TLC). Additionally, sensitivity to insulin (SI), body condition scores (BCS) and cresty neck scores (CNS) were measured. A BWRP resulted in a significant loss of body weight (P<0.001), which was associated with beneficial decreases in BCS and CNS (both, P<0.001). Serum lipid compositions revealed significantly increased free fatty acid (FFA), sphingomyelin (SM; both P<0.001), total cholesterol (C) and cholesterol ester (CE) (both P<0.01) and triacylglycerol (TG; P<0.05) densities. Improvement of SI after the BWRP was associated with increases in neutral lipids (C, CE and TG, all P<0.01), FFA and the phospholipid SM (both, P<0.001). The results show that a BWRP in obese ponies was effective and associated with changes in the concentrations of neutral lipids and the phospholipid SM, indicating that SM may play a role in insulin signaling pathways and thus in the pathogenesis of insulin resistance and the progression of metabolic syndrome in obese ponies.

The effect of a herbal paste and oil extract on the lipid content of canine hair fibres.

BACKGROUND: Application of herbal paste and oil to a dog's coat and body before rinsing (often combining with shampooing) is a cosmetic therapy available in Japan. It is highly appreciated by users, who claim that the treatment makes the coat shinier, improves volume and eliminates tangles. However, there has been no scientific evaluation of such treatments. HYPOTHESIS/OBJECTIVES: Improvement of hair condition is derived from oils such as sebum and conditioning oils because chemicals are not used. Therefore, we examined nonpolar lipids (the primary lipids in dog hair) and the botanical oils used in this therapy. ANIMALS: Hair samples were obtained from six beagle dogs. METHODS: Groups were based on different combinations of the following processes: rinsing, shampooing, herbal therapy and herbal therapy with oil extract. Analysis of lipids was performed by high performance thin layer chromatography. RESULTS: The processes of shampooing and herbal therapy were associated with an equivalent reduction in cholesterol ester and triglyceride (TG). However, hair treated by herbal therapy combined with oil extract had an almost three-fold higher TG content, even after shampooing. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrated that the herbal therapy was able to coat hair samples with TG that was not removed with rinsing. Further investigation is required to evaluate the possible benefits of the application of botanical products containing lipids, such as TG, on hair coat quality in dogs.

Development of a regulatory testing procedure to study the metabolism of pesticides in farmed fish.

BACKGROUND: Diets used in commercial fish farming use significant proportions of crop-derived commodities, and it is important to understand the potential for transfer of any pesticide residues on the crop into edible tissues in fish. It is a current requirement in the EU that fish metabolism studies must be performed when a pesticide is used in crops where commodities or processed fractions are fed to farmed fish. Fish metabolism studies in both rainbow trout and common carp have been carried out, following the new working document on the nature of pesticide residues in fish using (14) C-labelled pesticide. RESULTS: The ingestion of experimental diets by rainbow trout and common carp resulted in the uptake and metabolism of the test item, as shown by liquid scintillation counting combined with radio-thin-layer chromatography. The metabolite profiles for trout and carp were qualitatively similar regarding the main residue. However, species-specific differences were found regarding the remaining residue with rainbow trout showing additional metabolites in comparison to carp. CONCLUSIONS: Metabolism studies for regulatory purposes can be carried out with both fish species under laboratory conditions. The experimental design reported is suitable for quantifying the transfer of residues to edible tissues and enables characterisation of the chemical nature of residues.

Modulación farmacológica de la cicatriz glial para la reparación de lesiones del SNC / Pharmacological modulation of the glial scar for CNS injury repair

Objetivo: Estudiar la actividad de la neurostatina obtenida enzimáticamente y purificada mediante un nuevo método, en cultivos de células implicadas en la formación de la cicatriz glial. Material y métodos: La neurostatina se obtuvo mediante una reacción enzimática a partir del gangliósido GD1b comercial y se purificó con un método nuevo simplificado. La actividad de la neurostatina se probó en ensayos de proliferación con MTT en pericitos y microglía de rata. Resultados: La actividad de la neurostatina purificada fue similar a la obtenida mediante purificación a partir de cerebro de mamífero. La neurostatina inhibió la proliferación de los pericitos inducida por el factor de crecimiento PDGF-B y la proliferación de la microglía de rata inducida por la toxina bacteriana LPS a concentraciones nanomolar. Conclusión: La nueva metodología de obtención y purificación de la neurostatina y su actividad justifican su ensayo en modelos de lesión del SNC en animales, para evaluar su posible uso como terapia en pacientes con lesiones del SNC (AU)

Objective: To study the activity of neurostatin obtained enzymatically and purified using a new method, in cultures of cells involved in the formation of the glial scar. Material and methods: Neurostatin was obtained from the commercial ganglioside GD1b, using enzymatic Oacetylation, and was purified by a new simplified method. The activity of neurostatin was tested by an MTT proliferation assay in pericytes and rat microglial cells. Results: The activity of neurostatin obtained and purified by this new method was similar to the neurostatin obtained by the purification from mammalian brain. Neurostatin inhibited the PDGF-B growth factor induced proliferation of pericytes and LPS bacteria toxin induced proliferation of rat microglial cells at nanomolar concentrations. Conclusion: This new methodology to synthesize and purify neurostatin and its activity justify further studies to test its effect in animal models of CNS injuries and to evaluate its possible use as a therapy in patients with CNS injuries (AU)

Ultrasonographic characteristics of lipiduria in clinically normal cats.

Echoes are frequently seen in the urinary bladder of cats during abdominal ultrasound. These have been attributed to hematuria, pyuria, crystalluria, and lipid. However, sonographic findings have not been previously correlated with urinalysis. We prospectively evaluated 40 clinically normal cats via ultrasound, serum chemistry, and urinalysis. Thin layer chromatography was performed on the urine to determine the amount (mg) of lipid subfractions including diacylglycerol, triglyceride, phospholipid, free fatty acid, cholesterol, and cholesterol ester. Ninety percent (36/40) of the cats in our population had sonographic echoes suspended in the urinary bladder, with most having a subjective score of mild echoes (n = 20). None of the sonographic echoes were gravity dependent or caused distal acoustic shadowing, reverberation, or twinkle artifact. Of the cats with sonographic echoes in the urine, 66% (24/36) had no significant findings on urinalysis other than the presence of lipid. The total amount of subjective sonographic echoes was not significantly related to the total amount of fat measured on thin layer chromatography or the number of lipid droplets seen on urinalysis. An increased amount of urine diacylglycerol was significantly associated with clumping of echoes (P = 0.02) and the amount of lipid droplets seen on urinalysis (P = 0.04). An association between increased amounts of urine diacylglycerol and the amount of echoes seen on ultrasound approached significance (P = 0.05). Findings from this study support previously published theories that sonographic echoes within the urinary bladder of clinically normal cats may be due to urine lipid.

Hepatoprotective action of celery (Apium graveolens) leaves in acetaminophen-fed freshwater fish (Pangasius sutchi).

Acetaminophen (APAP)-induced liver damage is one of the most common problems among the population. Therefore, the study was aimed to investigate the hepatoprotective effect of celery leaves on APAP-induced toxicity in a freshwater fish, Pangasius sutchi. Fish were divided into four experimental groups of 6 fish each. Group 1 served as control. Group 2 fish were exposed to APAP (500 mg/kg) for 24 h. Groups 3 and 4 fish were exposed to APAP + celery leaf powder (CE) (500 mg/kg) and CE for 24 h, respectively. The severity of liver damage, hepatic lipid, glycogen, ions status and histological alterations was examined. The characterization of CE extract was also performed. APAP-exposed fish showed elevated levels of both circulating and tissue hepatotoxic markers (AST, ALT and ALP), reduced hepatic glycogen and lipid contents (TG and cholesterol), increased tissue lipid peroxidation markers (TBARS, LHP and PCO), altered tissue levels of enzymatic (SOD, CAT, GPx and GST) and non-enzymatic (GSH) antioxidants and cellular thiol levels (T-SH, P-SH and NP-SH), and reduced hepatic ions (Na(+), K(+) and Ca(2+)) and abnormal liver histology. The abnormalities associated with APAP exposure were reversed on treatment with CE. The TLC separation and HPLC quantification of petroleum ether/acetone extract of CE showed the peaks for highly efficient flavonoids such as rutein, quercetin and luteolin. The observed hepatoprotective effect of CE might be due to its rich flavonoids.

Comparison of myelination between large and small pig fetuses during late gestation.

We compared myelination of the cerebellum, brain stem, and spinal cord in the largest and smallest pig fetuses within a litter during late gestation. Gilts were killed on Days 92, 100, and 110 of gestation and these neural tissues were obtained from the largest and smallest fetuses in each litter. Myelin basic protein (MBP) mRNA was quantified in each tissue using real time reverse transcriptase polymerase chain reaction (rtPCR). Myelin was recovered from each tissue and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) was used to measure MBP and lipids, respectively. MBP mRNA increased with advancing gestation in all three tissues examined (P≤0.05) and was less in brain stem of small piglets compared to large piglets (P<0.01). Two coomassie stained protein bands (HMBP and LMBP) were observed by SDS-PAGE. Six prominent lipid bands were obtained by TLC (cholesterol, hydroxy(h)-cerebroside, nonhydroxy(nh)-cerebroside, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin). Significant day by fetal size interactions for cerebellar MBP and lipids indicated that cerebellar myelination in the smallest fetuses was less compared to the largest fetuses on Days 100 and 110 of gestation. Myelin MBP and lipid obtained from brain stem increased with advancing gestation and LMBP and lipids were less in small piglets compared to large piglets. In contrast, myelination in spinal cord increased with day of gestation but was not different between smallest and largest fetuses. These results confirm that myelination of the cerebellum and brain stem, but not spinal cord, is reduced in small fetuses during late gestation.

A sensitive method for qualitative screening of bile salt hydrolase-active lactobacilli based on thin-layer chromatography.

A sensitive protocol based on thin-layer chromatography (TLC) was developed to screen qualitatively bile salt hydrolase (BSH)-active lactobacilli. The sodium salts of glycocholic acid and taurocholic acid were used as substrates, and bacterial BSH activity was confirmed by detecting cholic acid as a product of the bile conjugates using a TLC assay with direct visual observation. Forty-five lactobacilli isolated from human fecal samples were tested for BSH activity by the TLC assay, a conventional plate assay, and a quantitative colorimetric assay. With the TLC and quantitative colorimetric assays, the same 24 BSH-positive strains were detected. No false-positive or false-negative results were detected by the TLC assay. However, only 20 BSH-positive strains were detected with the conventional plate assay. Compared with the conventional plate assay, the TLC assay is more sensitive for the detection of BSH activity of lactobacilli and, thus, more suitable for screening of BSH-active lactobacilli of human origin.

Utilization of oil by-product from the purification process of conjugated linoleic Acid as feeding supplements for the accumulation of conjugated linoleic acid in the egg yolk.

This study was performed to examine an efficient way to use oil by-product which is obtained during the purification process of conjugated linoleic acid (CLA) from safflower oil. The CLA by-product (CBP) was fed to the laying hens to accumulate CLA in the egg yolk. Egg yolk samples from 3 different dietary groups were analyzed: control; CBP, 2%; and CLA-80 (which contained 80% free form of CLA), 2%. Fatty acids from the yolk were analyzed by gas chromatography, and the parameters of egg quality were determined. During the feeding trial, there was little alteration in the egg quality and egg production of the tested groups. The CLA contents in the test group with CBP, which contain CLA as glyceride form, significantly increased in the first week of feeding and had the highest level among the tested groups throughout the feeding trials, whereas the CLA group showed an increase in CLA contents from the third week. Moreover, the contents of total CLA isomers in CLA-80 and CBP oils were decreased by 12.92 and 0.51% after heat treatment in 48 h, and the contents of linoleic acid (LA) isomer in LA-80 (which contained 80% free form of LA) and LA by-product (LBP) oils were decreased by 19.63 and 5.78%, respectively. It was confirmed that a major form of fats in CBP was mainly esterified forms, whereas the fats in CLA-80 and LA-80 were composed of free fatty acids. There was no significant difference in the egg quality and production among the tested groups. It is meaningful that the oil by-product could be utilized as a source for functional foods of animal origin without influencing egg quality and production.

Conjugated alpha-linolenic acid isomers in bovine milk and muscle.

Conjugated linolenic acids (CLnA) are octadecatrienoic fatty acid isomers with at least 2 conjugated double bonds. Various CLnA isomers occur naturally, and some isomers could be formed by ruminants from dietary alpha-linolenic acid. Ruminant biohydrogenation of polyunsaturated fatty acids gives rise to the formation of numerous metabolites having conjugated or nonconjugated structures. The objectives of this study were to identify and characterize CLnA isomers in milk fat and muscle lipid extracts from cattle fed a high-forage diet. The analysis of total fatty acid methyl esters revealed levels of total CLnA of 0.39% in a single milk lipid extract and 0.34% in a single muscle lipid extract. Fatty acid methyl esters were fractionated by argentation thin-layer chromatography. A fraction containing dienoic fatty acids as well as CLnA isomers was isolated and analyzed. The double bond positions of CLnA isomers (cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15 18:3) were confirmed by mass spectrometry of their 4,4-dimethyloxazoline derivatives. Mass spectra of the cis-9, trans-13, cis-15 18:3 isomer was characterized by an intense ion at m/z 236 attributable to the formation of 2 stabilized allylic radical fragments, whereas this intense ion corresponding to the stabilized radical fragments was located at m/z 262 for the cis-9, trans-11, cis-15 18:3 isomer. The gap of 12 amu between m/z 250 and 262 confirmed the occurrence of a double bond in position delta13. Configuration of the double bonds of standards having similar mass spectra and gas-liquid chromatographic retention times was confirmed by 1H nuclear magnetic resonance. We also showed that both CLnA isomers were found in the muscle lipid extract, whereas only the cis-9, trans-11, cis-15 18:3 isomer was identified in the milk lipid extract. This study appears to be the first to identify 2 CLnA isomers in bovine muscle lipid extract.

Cromatografia em camada delgada para o diagnóstico da intoxicação por aldicarb ("chumbinho") em cães e gatos / Thin-layer chromatography for aldicarb poisoning diagnosis in dogs and cats

Avaliou-se a cromatografia em camada delgada (CCD) como método de diagnóstico toxicológico para os casos de intoxicação por aldicarb em cães e gatos, utilizando-se 50 amostras de conteúdo gástrico obtidas durante a necropsia e 50 amostras de alimentos utilizados como iscas para intoxicar criminalmente os animais. Todas as amostras resultaram positivas para o aldicarb, mostrando ser a CCD uma técnica qualitativa eficiente, rápida e de baixo custo, com uso potencial na toxicologia veterinária forense

The present study concerns about the identification of aldicarb residues using thin-layer chromatography (TLC) in 50 samples of gastric content obtained from the necropsy of dogs and cats and 50 samples of foods suspected of being used as baits. All samples resulted positive for aldicarb showing that the TLC is an efficient, fast and not expensive qualitative method for the detection of aldicarb, being useful for this purpose in the forensic veterinary toxicology

Quantitative ultrasound, magnetic resonance imaging, and histologic image analysis of hepatic iron accumulation in pigeons (Columbia livia).

Iron overload was induced by iron dextran i.v. in clinically healthy adult pigeons, Columbia livia, (n = 8). Hemosiderosis was induced in all treated birds. Two control pigeons received no iron injections. Pigeons did not show clinical signs of iron overload during the 6-wk study. Ultrasound examination of the liver in the pigeons receiving iron dextran was performed on days 0, 13, 28, and 42. No ultrasound images were collected on the control pigeons. Magnetic resonance imaging was performed on days 0, 13, 28, and 42 on all study pigeons and imaging sequences were collected in three different imaging formats: T1, T2, and gradient-recalled echo (GRE). Surgical liver biopsies were performed on pigeons receiving iron dextran on days 2, 16, and 45 (at necropsy). A single liver sample was collected at necropsy from the control birds. Histologic examination, quantitative image analysis, and tissue iron analysis by thin-layer chromatography were performed on each liver sample and compared to the imaging studies. Although hemosiderosis was confirmed histologically in each experimental pigeon, no significant change in pixel intensity of the ultrasound images was seen at any point in the study. Signal intensity, in all magnetic resonance imaging formats, significantly decreased in a linear fashion as the accumulation of iron increased.

Evaluation of lung maturity by amniotic fluid analysis in equine neonate.

The aim of this study was to gather useful new data for evaluation of lung maturity in the neonatal foal. Because equine neonatal intensive therapy is very expensive, a precocious diagnosis could help to express a prognosis and to offer a respiratory support early after birth, increasing the survival rate and reducing complications. Amniotic fluid was collected at parturition on n=18 mares. Lamellar bodies were isolated in the amniotic fluid and measured with transmission electron microscopy (TEM). Furthermore two tests on amniotic fluid that are commonly used in humane medicine were utilized: lecithin/sphingomyelin ratio (L/S) and lamellar body count (LBC). L/S ratio was determined using thin layer chromatography (TLC) and, for the first time in equine amniotic fluid, with high performance liquid chromatography (HPLC). LBC was performed with an automated blood cell counter. The mean of the L/S ratio obtained in mature foals was 2.5 with TLC and 2.7 with HPLC. The mean LBC in the same group was 48x10(3)/microL. The Spearman's Rank correlation test found a significant correlation between TLC and Apgar score (R=0.66, p<0.01), between TLC and cord pH (R=0.65, p<0.05), between HPLC and Apgar score (R=0.63, p<0.01) and between cord pH and Apgar score (R=0.82, p<0.01). The Student's t-test did not found a significant difference between L/S ratio performed with TLC and with HPLC. These methods may be useful for evaluation of lung maturity in the equine species, but further studies on a large number of mature and premature foals are necessary to establish equine pulmonary maturity standards.

Effects of the administration of lactobacilli, maltodextrins and fructooligosaccharides upon the adhesion of E. coli O8:K88 to the intestinal mucosa and organic acid levels in the gut contents of piglets.

The influence of the administration of Lactobacillus plantarum, maltodextrin Maldex 150 and Raftifeed IPX fructooligosaccharides on the inhibition of adhesion of E. coli O8:K88 to the mucosa of the jejunum, ileum and colon as well as on the organic acid levels was investigated in 33 conventional piglets. The counts of E. coli K88 adhering to the jejunal mucosa were significantly decreased (p < 0.05) in Lact. plantarum + Maldex 150 and Lact. plantarum + Maldex 150 + Raftifeed IPX groups. The counts of E. coli K88 adhering to the colonic mucosa of Lact. plantarum + Maldex 150 + Raftifeed IPX and Lact. plantarum + Raftifeed IPX groups were significantly lower (p < 0.05) than in Lact. plantarum and Lact. plantarum + Maldex 150 animals. The acetic acid levels in the ileum and colon of the Lact. plantarum + Maldex 150 + Raftifeed IPX group and Lact. plantarum + Raftifeed IPX group were significantly higher (p < 0.05) than in the Lact. plantarum and Lact. plantarum + Maldex 150 group. The combination of Lact. plantarum, maltodextrin Maldex 150 and Raftifeed IPX proved to be the most effective one to inhibit the counts of E. coli O8:K88 adhering to the intestinal mucosa of the jejunum and colon of conventional piglets.

Erythrocyte and plasma fatty acid patterns in dogs with atopic dermatitis and healthy dogs in the same household.

Recent studies have indicated that dogs with canine atopic dermatitis (CAD) may have a disorder of fatty acid metabolism: possibly low or absent activity of delta6-desaturase or delta5-desaturase, or both. To clarify this possibility, we examined the erythrocyte and plasma fatty acid patterns of 8 dogs with CAD and their 8 healthy housemates. Atopic dermatitis was diagnosed according to the criteria proposed by Willemse; other causes of dermatitis were excluded clinically and by appropriate tests. Erythrocyte ghosts were prepared from blood samples. Membrane lipids were extracted and separated by thin-layer chromatography. From plasma and lipid fractions, fatty acid content was determined by gas chromatography. In erythrocytes, but not in plasma, we observed significant differences in the fatty acid pattern that suggested a reduction in the n6 fatty acid products of the delta6- and delta5-desaturases in dogs with atopic dermatitis when compared with healthy housemates.

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea).

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.

Analysis of neutral glycosphingolipids from Trypanosoma brucei.

Neutral glycosphingolipids (GSLs) were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC), TLC/secondary ion mass spectrometry (TLC/SIMS), and liposome immune lysis assay (LILA). Three species of neutral GSLs, designated as N-1, -2, and -3 were separated on TLC. N-1 GSL migrated very close to glucosylceramide (GlcCer) and N-2 GSL showed the same mobility as lactosylceramide (LacCer). On the other hand, the mobility of N-3 GSL on the TLC plate was slower than globotetraosylceramide (Gb4). In order to characterize the molecular species of neutral GSLs from T. brucei, N-1, -2 and -3 GSLs were analyzed by TLC/SIMS. The TLC/SIMS analysis of N-1 of the parasites revealed a series of (M-H)- ions from m/z 698 to 825 representing the molecular mass range of ceramide monohexoside (CMH) (GlcCer or galactosylceramide). On the other hand, the TLC/SIMS spectra of N-2 GSL revealed a series of (M-H)- ions from m/z 944-987 indicating the molecular mass range of LacCer. In the TLC/SIMS analysis of N-3 GSL, however, the characteristic molecular ions that can elucidate the structure of N-3 GSL were not obtained. In order to confirm the results obtained from TLC/SIMS, N-1, -2, and -3, GSLs were tested by LILA with specific antibodies against GlcCer, LacCer, and Gb4, respectively. N-1 GSL had reactivity to anti-GlcCer antibody and N-2 GSL reacted with the antibody against LacCer. However, N-3 GSL was not recognized by anti-Gb4 antibody. Using anti-GlcCer and anti-LacCer antibodies, furthermore, we studied the expression of GlcCer and LacCer in T. brucei parasites. Both GlcCer and LacCer were detected on the cell surface of T. brucei.