Exploring the feasibility of general health promotion in UK dental primary care: ENGAGE in Scotland.

Introduction Despite UK dental guidance recommending opportunistic health promotion, it's rare for GDPs to discuss more than oral hygiene with their patients. The ENGAGE intervention incorporates UK guidance and evidence-based behaviour change techniques to motivate patients to make lifestyle changes (reduce smoking, alcohol consumption and/or improve diet). It was designed to take less than five minutes and be delivered during a routine dental check-up, and includes a take-home patient handout signposting to free NHS lifestyle counselling helpline services.Aims To determine the feasibility (patient and GDP acceptance) of implementing ENGAGE in Scottish dental primary care. The overall aim is to examine feasibility UK-wide before testing its effectiveness for influencing patient outcomes in a multi-centre UK trial.Methods Study 1: patient survey: N = 1000 adults from all health boards in Scotland were randomly selected from an NHS data base of medical patients and emailed the study invitation and link to an online questionnaire. Study 2: GDP workshop, audit, survey: N = 50 GDPs across Scotland were invited to participate in the training workshop (limited to the first 20 applicants), implement the intervention with their next 20 adult patients in for a check-up, audit their experience, then complete an online questionnaire.Results Study 1: 200 people completed the survey (52% male; 37% were 55 years or younger; 90% had visited their dentist in the previous 12 months). Less than (<) 15% were asked about their smoking, alcohol intake and/or diet when they last visited their dentist for a check-up; <10% would be embarrassed/offended if their dentist or dental hygienist asked them lifestyle questions during a dental check-up; more than (>) 70% would be reassured by the professionalism of their dentist or dental hygienist if they were asked; <4% would be embarrassed/offended if given a leaflet with NHS helpline information by their dentist. Study 2: N = 18 GDPs from nine out of 14 NHS regional health boards in Scotland delivered the ENGAGE intervention to 335 patients (averaging 18 patients each). N = 17/18 participants agreed that this intervention could be delivered during a check-up, was an improvement on what they currently did and thought that it may make a difference to what their patients thought, felt, and/or did about reducing health risk.Conclusion The ENGAGE intervention is feasible to implement in Scottish dental primary care. Comments from patient and GDP participants will inform its development and further feasibility studies set in other UK regions.

Egg and time limitation mediate an egg protection strategy.

The number of mature eggs remaining in the ovaries and the time left for oviposition determine the reproductive decisions of the hyperdiverse guild of insects that require discrete and potentially limiting resources for oviposition (such as seeds, fruits or other insects). A female may run out of eggs before all available oviposition sites are used (egg limitation), or die before using all of her eggs (time limitation). Females are predicted to change clutch size depending on whether eggs or time is the limiting resource. We extend this framework and ask whether the same constraints influence a strategy in which females modify eggs into protective shields. In response to egg parasitism cues, female seed beetles (Mimosestes amicus) lay eggs in vertical groups of 2-4, modifying the top 1-3 eggs into shields in order to protect the bottom egg from attack by parasitoids. We made contrasting predictions of how egg and time limitation would influence egg size and the incidence and level of egg protection. By varying access to seed pods, we manipulated the number of remaining eggs a female had at the time she received a parasitism cue. Although egg size was not affected, our results confirm that egg-limited females protected fewer eggs and time-limited females protected more eggs. Female body size explained the number of eggs in a stack rather than host deprivation or the timing of parasitoid exposure. Our results clearly show that host availability relative to female age influences the incidence of egg protection in M. amicus. Furthermore, our study represents a novel use of life history theory to explain patterns in an unusual but compelling defensive behaviour.

Reported versus confirmed wheeze and lung function in early life.

AIMS: To investigate the relation between parentally reported wheeze (unconfirmed), physician confirmed wheeze, and subsequent lung function. METHODS: Children at risk of allergic disease (one parent atopic) were recruited antenatally and followed prospectively from birth. During the first three years of life parents were asked to contact the study team if their child was wheezy. The presence of wheeze was confirmed or not by the primary care or study physician. Respiratory questionnaire and specific airway resistance measurement (sR(aw), body plethysmograph) were completed at age 3 years. RESULTS: A total of 454 children were followed from birth to 3 years of age. One hundred and eighty six (40.9%) of the parents reported their child wheezing in the first three years of life, and in 130 (28.6%) the wheeze was confirmed. A total of 428 children attended the three year clinic review, of whom 274 (64%) successfully carried out lung function tests. There was no significant difference in sR(aw) (kPa.s; geometric mean, 95% CI) between children who had never wheezed (n = 152; 1.03, 1.00 to 1.06) and those with a parentally reported but unconfirmed wheeze (n = 36; 1.02, 0.96 to 1.07, p = 1.00). sR(aw) was significantly higher in children with a physician confirmed wheeze (n = 86; 1.17, 1.11 to 1.22, p < 0.001) compared to those with no history of wheeze or with unconfirmed wheeze. CONCLUSIONS: Children with physician confirmed wheeze have significantly poorer lung function compared to those with parentally reported but unconfirmed and those who have never wheezed. A proportion of parents may have little understanding of what medical professionals mean by the term "wheeze".

Interactions of bioactive glasses with osteoblasts in vitro: effects of 45S5 Bioglass, and 58S and 77S bioactive glasses on metabolism, intracellular ion concentrations and cell viability.

In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass caused extra- and intracellular alkalinization, a rise in [Ca2+]i and [K+]i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H+] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca2+]i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.

An update on concurrent malaria and typhoid fever in Cameroon.

Malaria and typhoid fever are endemic diseases in Cameroon, with overlapping signs and symptoms. While the high prevalence of malaria is an established fact, it is only within the past 5 years that an unusually high number of illnesses have been diagnosed as malaria co-existing with typhoid fever. The Widal test is widely used as the sole laboratory test for the diagnosis of typhoid fever. To investigate the extent of the malaria and typhoid fever association, we used blood and stool cultures as additional diagnostic tests for typhoid fever. We report that, of 200 patients presenting with fever, 17.0% had concurrent malaria and typhoid fever (Salmonella typhi) based on bacteriological proven diagnosis as compared with 47.9% based on the Widal test. A higher proportion of patients (32.5%) had malaria coexisting with S. typhimurium when compared to S. paratyphi (2%) and S. typhi (P < 0.05). We conclude that the number of fever cases diagnosed as malaria co-existing with typhoid fever is overestimated.

Viral induction, transmission and apoptosis among cells infected by a Human Intracisternal A-type retrovirus.

Sjogren's Syndrome, a systemic autoimmune disease, is characterized by lymphocytic infiltration of the salivary or lacrimal glands, producing xerostomia or xerophthalmia. Although definitive proof of viral etiology has not been established, a cell line containing viral particles termed Human Intracisternal A-type Particles (HIAP) resulted from co-culture with patient lip biopsies. We stimulated these chronically infected cells with phorbol myristate acetate (PMA) in an effort to enhance production of viral particles for further characterization. We report that the virus present in the HIAP cell line can be induced to become lytic when subjected to PMA and that there is a difference in the effects of PMA on H9 and HIAP cell groups, with apparent protection from apoptosis due to PMA being exerted by viral presence. Delayed apoptosis may prolong exposure of the foreign/self complex, thus enhancing an autoimmune response. Polyacrylamide gel electrophoresis (PAGE) revealed the presence of new peptides in pellets of supernatants of PMA-stimulated HIAP cells, with prominent bands at 55 and 43 kDa, and several fainter ones. HIAP infection was transferred by cell-free filtered supernatants from stimulated cells to H9 cells, which became identical to parent HIAP cells by PAGE and fluorescence activated cell sorter.

Reactivity of sera from systemic lupus erythematosus and Sjögren's syndrome patients with peptides derived from human immunodeficiency virus p24 capsid antigen.

We have previously demonstrated that about one-third of patients with either Sjögren's syndrome (SS) or systemic lupus erythematosus (SLE) react to human immunodeficiency virus (HIV) p24 core protein antigen without any evidence of exposure to, or infection with, HIV itself. Herein, we further characterize the specificity of this reaction using enzyme-linked immunosorbent assay to peptides representing fragments of p24. Characteristic epitope-specific profiles were seen for SS and SLE patients. SS patients had significantly increased responses to peptides F (p24 amino acids 69 to 86) and H (amino acids 101 to 111) and diminished reactivity to peptides A (amino acids 1 to 16) and P (amino acids 214 to 228). SLE patients had increased reactivity to peptides E (amino acids 61 to 76), H, and P. Utilization of peptide P hyporeactivity as the criterion to select for SS patients results in a screen that is moderately sensitive (64%) and specific (79.3%). Adding hyperreactivity to one other peptide (F or H) as an additional criterion yields an expected decrease in sensitivity (to 41%) while increasing specificity (to 93.1%). All sera-reactive peptides from regions of known structure of HIV p24 were located in the apex of the p24 molecule. Thus, the specificity of the peptide reactivities described here indicates a specific pattern of a nonrandom cross-reactivity between HIV type 1 p24 and autoimmune sera which may be partially syndrome specific. The future focus of our work will be to optimize assays of the peptide as diagnostic tools.

Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells.

Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na+]i and [Ca2+]i rose while [K+]i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na+]i and [K+]i observed during energy depletion were gradual and progressive whereas those in [Ca2+]i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na+]i were usually smaller than reductions in [K+]i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.

Limitation of glycolysis by hexokinase in rat brain synaptosomes during intense ion pumping.

Incubation of rat brain synaptosomes under conditions of either increased energy utilization (addition of Na+ channel opener, veratridine, or ionophores, monensin and nigericin) or inhibition of oxidative phosphorylation (addition of rotenone), or a combination thereof, decreased [ATP], increased [ADP] and stimulated glycolysis. The rates of lactate generation were linear over a 15-min interval in the presence of rotenone alone but decreased in the other two conditions. During the first 5 min, the amount of lactate formed with veratridine, monensin or nigericin was as high or higher than with rotenone, but it was lower in the last 10 min. With a combination of one of the stimulators of ion movements and rotenone the rate of glycolysis was always markedly lower than with each compound added singly. The stimulated rates of lactate formation correlated positively with the synaptosomal content of [ATP]. After 15 min, [ATP] was 0.9-1.0 nmol/mg with rotenone, 0.5-0.9 nmol/mg with veratridine (or ionophores), and <0.3 nmol/mg with a combination of the two. Under the conditions used, calcium did not affect glycolytic activity directly. The Lineweaver-Burk plot of the rate of lactate formation against [ATP] yielded a straight line with a Km for ATP of about 0.1 mM, which is very similar to the Km for this nucleotide of brain hexokinase bound to mitochondria. In C6 cells glycolytic rate measured with a combination of an ionophore and rotenone was higher than with each of these compounds added singly while [ATP] never declined below about 9 nmol/mg prot. It is concluded that in synaptosomes, the high rate of energy utilization required for intense ion movement decreases [ATP] to a level that limits hexokinase activity kinetically. This may contribute to a reduction in the rate of glycolysis and hence energy production in brain hypoxia and ischemia.

The effect of pH on glycolysis and phosphofructokinase activity in cultured cells and synaptosomes.

The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK-21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [P(i)] ([ATP]i, [ADP]i, and [P(i)]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [P(i)], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] and lower [glucose-6-phosphate] and [fructose-6-phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [P(i)] rose under all pHe conditions. Simultaneously [glucose-6-phosphate] and [fructose-6-phosphate] decreased whereas [fructose-1,6-bisphosphate] and [glyceraldehyde-3-phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of antibody levels to the 96 tR recombinant protein of Plasmodium falciparum in children over a six-month period.

Several Plasmodium falciparum asexual stage antigens have been reported as possible candidate antigens for vaccines against malaria. One such is the 96-kDa antigen. We used the 96 tR recombinant protein to determine the levels of antibodies to this protein over a 6-mo period in children 4 mo to 15 yr old, who make up the population at risk in Cameroon. The mean enzyme-linked immunosorbent assay (ELISA) on 83 children at the start of the study in June was 0.270, with 13% of these children having ELISA values considered to be positive (> or = 0.36). In the follow-up study in early December, the mean ELISA was 0.320 with 20% of the children having positive values. In the period between June and December, 85% of the children who returned for follow-up had had 1 or more attacks of malaria. The mean ELISA values of this latter group had increased from 0.235 in June to 0.318 in December, with 6% of the children in June having positive values compared with 18% in December. These findings reinforce our evidence from a previous study that reactivity to the 96-kDa antigen is related to adequate exposure or repeated infection.

Definition of populations at risk for Plasmodium falciparum infection in three endemic areas of Cameroon.

Blood samples were collected from 285 individuals attending hospitals in 1 of 3 different regions of Cameroon. Of these, 89 had Plasmodium falciparum parasitemia. Prevalence in the Douala region was drastically reduced above the age of 19. In the Njinikom and Bamenda regions, on the other hand, an appreciable decline in prevalence was not observed until over the age of 49. Enzyme-linked immunosorbent assay (ELISA) values indicate that in Douala, high antibody titers to P. falciparum were present in all age groups tested. In Njinikom and Bamenda, an age dependence was seen in the response, with sera from individuals above 20 having significantly higher ELISA values compared with those below age 20. Generally, individuals with high antibody titers had low or no parasitemia. Results suggest that future malaria control and treatment measures might target high risk populations such as those defined in this study.

Correlation between serum levels of antibodies to the 96-kD antigen of Plasmodium falciparum and protective immunity in Cameroon: a longitudinal study.

A longitudinal study was conducted in the Yaounde area of Cameroon that involved 211 individuals in June 1990, and 70 individuals for the follow-up study in December 1990. Sera from these subjects were tested against the recombinant 96-thermoresistant antigen of Plasmodium falciparum and the kinetics of antibody production to this protein show that titers tend to increase with age and are also related to antigen exposure. The increase in antibody titers with age correlates positively with the ability of the individual to prevent development of a high parasitemia. Adults who maintained stable high titers generally did not experience clinical attacks during the study period. The data suggest that antibodies against the 96-kD antigen participate in conferring some immunity to falciparum malaria.

Relations between intracellular ions and energy metabolism under acidotic conditions: a study with nigericin in synaptosomes, neurons, and C6 glioma cells.

Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i, [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 microM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+]i and [Na+]i. The increase in [Na+]i in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+]i depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia.

Effect of inhibitors of N-linked oligosaccharide processing on the high-affinity transport of D-aspartate by C6 glioma cells.

The effect of several inhibitors of oligosaccharide-processing on the high-affinity transport of D-aspartate was investigated in C6 glioma cells. Swainsonine, an inhibitor of mannosidase II, had no effect on the uptake of the amino acid. Castanospermine (100 micrograms/ml) and 1-deoxynojirimycin (1 mM), inhibitors of glucosidases, and 1-deoxymannojirimycin (1 mM), an inhibitor of mannosidase I, reduced the rate of transport by 35-45%. All inhibitory compounds decreased the Vmax for transport without affecting the Km which suggests that inhibition of oligosaccharide trimming reduces the number of competent transporters on the surface of the plasma membrane. Returning the cells to a drug-free medium for 24 h, following a 24 h exposure, resulted in complete recovery of uptake. Treatment of cells with neuraminidase from V. cholerae also decreased the Vmax for transport by about 20%. The results suggest that: (i) a partial complex carbohydrate chain on the high-affinity transporter for acidic amino acid transmitters is sufficient for activity and (ii) sialic acid residues may be necessary for normal operation of the transporter.

Relations between intracellular ions and energy metabolism: a study with monensin in synaptosomes, neurons, and C6 glioma cells.

Treatment of rat brain synaptosomes with 10 microM monensin stimulated activity of the Na/K pump, which enhanced oxygen consumption and lactate production. Glycolytic flux was also increased independently of the pump activation by a fall in [H+]i. Under such conditions, glycolysis provided 26% of ATP for the ouabain-sensitive ATPase, a value substantially greater than the 4% obtained in veratridine-treated preparations (Erecinska and Dagani, 1990). In C6 glioma cells, a glia-derived line endowed with high rates of aerobic lactate synthesis, the cytosolic and mitochondrial ATP generation contributed 50% each for the support of the pump in the presence of 10 microM monensin. The fraction of energy utilized by the pump was greater in synaptosomes than in C6 cells. Enhancement of ion movements was accompanied by changes in the levels of high-energy phosphate compounds. Measurements with ion-sensitive microelectrodes in C6 cells and cultured neurons showed that monensin caused an increase in pHi by 0.4-0.5 unit and a parallel rise in [Na+]i. The increases in [Na+]i were about twofold in both types of cells, but the absolute values attained were much higher in neurons (40-50 mM) than in C6 cells (10-12 mM). Membrane potentials transiently declined by less than 10 mV and returned to their original values after 20 min of treatment. Rises in [Ca2+]i were small in neurons as well as in C6 cells. These changes could be explained by the known mechanism and/or consequences of monensin action. In contrast, in synaptosomes monensin caused an internal alkalinization of 0.1-0.15 pH unit, a large depolarization of the plasma membrane, and massive leakage of potassium into the external medium. The decrease in plasma membrane potential was accompanied by an increase in [Ca/+]i and release of the neurotransmitter amino acids GABA, aspartate, and glutamate. The depolarization and loss of K+ were unaffected by calcium withdrawal, replacement of chloride with gluconate, and addition of 1 mM 4-acetamido-4'-isothiocyanostilebene-2,2'-disulfonic acid (SITS), but was markedly attenuated by elimination of Na+. It is proposed that in synaptosomes monensin and/or the consequences of its action open a nonspecific cation channel that allows Na+ entry and K+ exit, with a consequent decrease in membrane potential.

Antiseptic toxicity to breast carcinoma in tissue culture: an adjuvant to conservation therapy?

Of 50 patients who had scrape cytology of the excision cavity after conservative surgery for breast carcinoma, 10 (20%) had malignant cells remaining in the cavity recognised by cytology. Of these patients, 18 had histological evidence of tumour at the resection margin, giving an accuracy of the cytology of 84%, a sensitivity of 56%, and a specificity of 100%. When assayed for cytotoxicity against a breast tumour cell line (MCF7) or human fibroblasts, chlorhexidine gluconate was the most effective of eight antiseptics or antitumour agents (100% cytotoxicity at a 1/10,000 dilution) in killing breast tumour cells and had 70% toxicity to human fibroblasts at the same dilution. Hydrogen peroxide appeared to be the most useful agent overall with 94% cytotoxicity to breast tumour cells with only a 12% cytotoxicity to human fibroblasts at a dilution of 1/1,000,000. We suggest that free malignant cells left in the cavity after conservative surgery for breast cancer may be a cause of local recurrence. They can be recognised by scrape cytology at operation and the topical use of antiseptics as cytotoxic agents may be beneficial and warrants further investigation.

Inhibition by trypsin of the high-affinity acidic amino acid transport system in C6 glioma cells.

The high-affinity uptake of the acidic amino acid D-aspartate was inhibited in a dose- and time-dependent manner, when C6 cells were exposed to trypsin. The protease decreased the maximal velocity for uptake but not its Km, consistent with a reduction in the number of competent carriers at the plasma membrane. Cellular energy production and [K+]i were unaffected, indicating that the transporter itself was the site of trypsin action. Maximum inhibition of uptake was 50%, which suggests the presence of a heterogeneous population of transporters, only half of which is sensitive to trypsin. These results support our earlier postulate that in glial cells, the high-affinity transporter for acidic amino acids is a transmembrane protein, part of which extends into the external environment.

Effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of acidic amino acid neurotransmitters in C6 glioma cells.

The effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of D-aspartate was investigated in C6 astrocytoma cells. A concentration of tunicamycin (1 microgram/ml) that after 24 h exposure inhibited the rate of transport by 70% and incorporation of [3H]mannose by 82-95% had only a small effect on [14C]leucine incorporation into protein and cell growth (20% reduction). Tunicamycin decreased the Vmax for transport without affecting the Km, which suggests that inhibition of glycosylation reduces the number of competent transporters on the surface of the plasma membrane. The decrease in the velocity of uptake was attenuated when C6 cells were treated with tunicamycin in the presence of protease inhibitors, indicating that the underglycosylated carriers are subject to enhanced proteolytic degradation. Incubation in drug-free medium following treatment with 1 microgram/ml of tunicamycin for 24 h resulted in recovery of D-aspartate transport within 48 h. This recovery was prevented by the presence of cycloheximide, which indicates that synthesis of new transporters is necessary for the restoration of normal rates of D-aspartate uptake. These results support our earlier postulate that the high-affinity carriers for amino acid transmitters are transmembrane glycoproteins.