The Effect of Tunicamycin on the Glucose Uptake,Growth, and Cellular Adhesion in the Protozoan Parasite Crithidia fasciculata

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.     

Primary cultures of cardiac muscle cells as models for investigation of protein glycosylation

Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells.Exposure of the cells up to 6 g/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (B_max) to 46.0 ± 17.2% of the untreated control. Similar effects were observed after enzymatic deglycosylation using N-glycosidase F (PNGase F). The results suggest that a posttranslational modification of parts of the cardiac L-type Ca⁺⁺ channel by N-glycosylation is an important determinant for the binding of Ca⁺⁺ antagonists of the DHP-type to the ₁ subunit which itself is not glycosylated. The results suggest a participation of N glycosylation in the assembling of the subunits to the functional channel and/or its turnover. However, a possible effect of tunicamycin on the expression of the Ca channel as an alternative mechanism cannot be excluded.     

Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different^IIα-globin chains were separated from two different^Iα-chains by HPLC. Two α-chains (^Iα¹ and^IIα³), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (^Iα² and^IIα⁴), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.     

The complete primary structure of the marine carnivora,galapagoes fur seal (Arctocephalus galapagoensis,Otariidae) hemoglobins

The complete primary structure of the two hemoglobin components of the fur seal (Arctocephalus galapagoensis) is presented. The two components (HbI and HbII) occur in nearly equal amounts and have identical β-chains; whereas the two α-chains (αI/αII) differ by six exchanges Ile/Val, Met/Thr, Ser/Ala, Pro/His, Lys/Gly, and Thr/Ala at positions 10, 34, 35, 50, 78, and 131, respectively. The components were isolated by DEAE-Sephacel chromatography and were separated into the globin chains by RP-HPLC on a column of Nucleocil-C4. The sequences have been determined by Edman degradation in liquid- and gas-phase sequencer, using the native chains and tryptic peptides. The sequences compared with those of other Carnivora species and an adult human globin chains. An identical β-chain is found in fur seal and walrus, whereas larger differences were found between αI and αII compared to β-chains.     

Complex-type carbohydrates of apolipoprotein-B of human plasma low-density lipoproteins

Two fractions of glycopeptides containingN-glycosidic asparagine-linked glycans were isolated by concanavalin-A-Sepharose affinity chromatography from Pronase digests of apolipoprotein-B of human low-density lipoproteins. Methylation analysis indicated that the non-binding fraction contains about 0.5 mol complex-type tri- or tetra-antennary oligosaccharides per mol lipoprotein. Structures containing a bisectingN-acetylglucosamine were also detected in this fraction. The weakly binding fraction from the chromatography contains the majority of the complex oligosaccharides of apolipoprotein-B, i.e. 5–6 mol of bi-antennary chains of the transferrin-type per mol of lipoprotein. In addition to sialylated structures, branches containing a terminalN-acetylglucosamine residue were detected on the complex-type glycans of apolipoprotein-B.     

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca²⁺ concentrations in the mutant strain Aspergillus awamori 66A containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity of sporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 μM) stimulated spore germination: the number of germinating spores was 2–3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 μM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca²⁺ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 μM) were shown to cause an instantaneous increase in Ca²⁺ concentrations compared to the control and lower amphotericin concentration (2.5 μM). Ca²⁺ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.     

Carnivora: The primary structure of hemoglobin from adult coati(Nasua nasua rufa,Procyonidae)

The complete primary structure of the hemoglobin from the adult coati (Nasua nasua rufa) is presented. The erythrocytes contain one hemoglobin component and two globin chains. The isolation of globin chains was achieved by reversed-phase HPLC on a column of Nucleosil-C4. The primary structure of globin chains and tryptic peptides was determined in liquid- and gas-phase sequenators. The sequence of the α and β-chains of coati compared with those of other Carnivora species. Results are discussed with respect to structural variations and the phylogenetic relationship.     

Nutrient couplings between on-site sewage disposal systems,groundwaters, and nearshore surface waters of the Florida Keys

We performed a one-year study to determine the effects of on-site sewage disposal systems (OSDS, septic tanks) on the nutrient relations of limestone groundwaters and nearshore surface waters of the Florida Keys. Monitor wells were installed on canal residences with OSDS and a control site in the Key Deer National Wildlife Refuge on Big Pine Key. Groundwater and surface water samples were collected monthly during 1987 and analyzed for concentrations of dissolved inorganic nitrogen (DIN = NO_f3/sup- + NO_f2/sup- + NH_4/su+), soluble reactive phosphate (SRP), temperature and salinity. Significant nutrient enrichment (up to 5000-fold) occurred in groundwaters contiguous to OSDS; DIN was enriched an average of 400-fold and SRP some 70-fold compared to control groundwaters. Ammonium was the dominant nitrogenous species and its concentration ranged from a low of 0.77 μM in control wells to 2.75 mM in OSDS-enriched groundwaters. Concentrations of nitrate plus nitrite were also highly enriched and ranged from 0.05 μM in control wells to 2.89 mM in enriched groundwaters. Relative to DIN, concentrations of SRP were low and ranged from 30 nM in control wells to 107 μM in enriched groundwaters. N : P ratios of enriched groundwaters were consistently > 100 and increased with increasing distance from the OSDS, suggesting significant, but incomplete, adsorption of SRP by subsurface flow through carbonate substrata. Nutrient concentrations of groundwaters also varied seasonally and were approximately two-fold higher during the winter (DIN = 1035 μM; SRP = 10.3 μM) compared to summer (DIN = 470 μM; SRP = 4.0 μM). In contrast, surface water nutrient concentrations were two-fold higher during the summer (DIN = 5.0 μM; SRP = 0.50 μM) compared to winter (DIN = 2.5 μM; SRP = 0.15 μM). Direct measurement of subsurface groundwater flow rate indicated that tides and increased groundwater recharge enhanced flow some two-fold and six-fold, respectively. Accordingly, the observed seasonal coupling of OSDS-derived nutrients from groundwaters to surface waters is maximum during summer because of seasonally maximum tides and increased hydraulic head during the summer wet season. The yearly average benthic flux of anthropogenic DIN into contiguous canal surface waters is 55 mmol m^-2 day^-1, a value some five-fold greater than the highest rate of benthic N-fixation measured in carbonate-rich tropical marine waters.     

Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-β-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic β-globin which can be folded in the presence of cyanohemin and native α-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.     

Production of extracellular lysine by a strain ofBacillus coagulans

A naturally deficient thiamine and methionine requiring strain ofBacillus coagulans (Ms15) accumulates lysine in medium only when exogenous pyridoxine (optimal concentration, 0.1 μg/ml) and threonine (optimal concentration, 100 μg/ml) are supplied. Threonine exerts an inhibitory effect at higher concentrations but pyridoxine does not.     

Studies on choline acetyltransferase isolated from human brain

The purified choline acetyltransferase from human striatal tissue was found to have aK _m value of 8 μM for acetyl-coenzyme A and 250 μM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimatedS-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCl, NaCl, (NH₄)₂SO₄, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 mM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole.     

Glycosylation of the N-terminal potential N- glycosylation sites in the human α1,3- fucosyltransferase V and -VI (hFucTV and -VI)

Human 1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and hFucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced by glutamine. Western blotting analyses demonstrated that both sites in hFucTV were glycosylated, whereas in hFucTVI only one of the sites (Asn91) was glycosylated. All the single mutants and the hFucTVI N46Q/N91Q double mutant exhibited enzyme activities that did not differ considerably from the wt activities. However, the enzyme activity of the hFucTV N60Q/N105Q double mutant was reduced to approximately 40% of the wt activity. In addition, castanospermine treatment diminished the enzyme activity and hence trimming of the N-linked glycans are required for expression of full enzyme activity of both hFucTV and -VI. The present study demonstrates that both of the N-terminal potential N-glycosylation sites in hFucTV and one of the sites in hFucTVI are glycosylated. Individually, their glycosylation does not contribute considerably to expression of enzyme activity. However, elimination of both sites in hFucTV reduces the enzyme activity.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

In vitro plantlet regeneration through asexual embryogenesis in cotyledonary segments of Corylus avellana L.

Cotyledonary nodes of corylus avellana L. excised from young seedlings cultured in K(h) medium for 5 weeks were used to induce embryoid formation. Embryogenesis was achieved in 60% of the explants over two 20-day culture steps in the presence of IBA (5 μM) plus BAP (0.5 μM) and BAP (5 μM) plus IBA (0.5 μM) respectively. Subsequent proliferation was successfully maintained during 5 subcultures on K(h) basal medium and in an unlimited number of subcultures in the presence of BAP (0.5 μM). Plant regeneration (50–55%) was easily achieved by planting into a basal K(h) medium after 10–20 days.     

NH2-terminal sequence of the α and β chains of human DC-1 antigen isolated from the JY cell line. Homology with murine I-A molecules

The DC-1 antigen has been isolated from the JY cell line (DR4, w6). The amino terminal sequences of its α and β chains are both reported and are homologous to the murine I-A antigens. The JY and previously reported LB DC-1 α chain sequences appear to be variants of the.DC α chains reported by other authors. The JY DC-1 β chain sequence appears to be identical with that deduced from a β chain cDNA clone and thus identifies this clone. The JY and LB DC-1 β-chains are clearly different since the latter has a blocked amino terminus.     

Enzyme preparations fromAspergillus flavus for structural studies of the peach gum polysaccharide

Extra- and intracellular glycanohydrolases were isolated fromAspergillus flavus and partially characterized. Both preparations exhibited β-galactosidase activity. Gel chromatography of the extracellular enzyme preparation on Sephadex revealed one protein fraction containing β-galactosidase activity and a second one exhibiting mainly β-xylosidase activity. Electrophoresis in starch gel and disc electrophoresis in polyacrylamide gel showed that the preparation obtained from the cultivation broth contained five protein fractions, whereas two protein fractions could be detected in the intracellular preparation. Hydrolysis of a partially degraded polysaccharide of peach gum by the above preparations yieldedd-galactose as the main product and traces ofd-mannose,l-arabinose,d-xylose and a number of oligosaccharides.     

Antagonistic effects of Satureja hortensis essential oil against AFB,on human lymphocytes in vitro

Satureja hortensis L. (Lamiaceae) has been used as a folk remedy to treat various such as cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases. In this study, the antagonistic effects of essential oil of S. hortensis (SHE) were studied against aflatoxin B₁ (AFB₁) in human lymphocytes in vitro. The analysis of the essential oil was performed by using Gas chromatography-mass spectrometry (GC-MS). Anti-genotoxic effects of the SHEs was evaluated using sister chromatid exchange (SCE), micronuclei (MN) tests against AFB1. Also level of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities used to determine the anti-oxidative effects of the SHEs. This result showed AFB₁ (5 μM) increased the frequencies of SCE, MN and the level of MDA. AFB1 at the same concentration decreased the activities of SOD and GPx. However, different concentrations of SHE with AFB¹ decreased the frequency of SCE and MN and level of MDA and also increased the activities of SOD and GPx significantly. Especially, the 1.0, 1.5, 2.0 μL dose of SHE are more effective than other doses. The results of this experiment have clearly shown that SHE has strong antioxidative and antigenotoxic effects, these biological activities of SHEs can be due to its component.