Immunization with r-Lactococcus lactis expressing outer membrane protein A of Shigella dysenteriae type-1: evaluation of oral and intranasal route of administration.

AIMS: To evaluate the comparative immunogenic potential of food grade Lactococcus lactis expressing outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. METHODS AND RESULTS: OmpA of SD-1 was cloned and expressed first in Escherichia coli and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and immunoblot analysis. Using immobilized metal affinity chromatography, OmpA was purified from recombinant E. coliBL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and faecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. CONCLUSION: Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared with the nasal route. SIGNIFICANCE AND IMPACT OF THE STUDY: Using food grade recombinant L. lactis has implications in the development of a prophylactic against multidrug-resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella.

Investigating the Role of Plasma Glucose Concentration as a Phenotypic Marker for CYP2C9 Genetic Variants, in the Diabetic Population of Gujarat.

The present study was aimed to investigate the role of plasma glucose concentration as a phenotypic marker and to study the frequency distribution of CYP2C9 genetic variants in Gujarat state diabetic population. One hundred and nine unrelated diabetes mellitus patients treated with sulfonylureas were genotyped for CYP2C9*2 and CYP2C9*3 alleles. Their pre- and posttreatment postprandial blood glucose levels were recorded and mean glucose drop per milligram of drug values were calculated and further used as an index for phenotypic correlation. The frequencies of CYP2C9*1, CYP2C9*2 and CYP2C9*3 alleles in the Gujarat state diabetic population were 0.84, 0.07 and 0.09, respectively. The distribution of CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes were 0.73, 0.08, 0.13, 0.0, 0.06 and 0.0, respectively. Patients with CYP2C9*1/*2 genotype did not show any significant difference in the mean glucose drop per milligram of drug values when compared with wild-type patients in glipizide-treatment group. Patients with CYP2C9*1/*3 genotype showed greater mean glucose drop per milligram of drug values than patients with CYP2C9*1/*1 wild-type genotype for both glipizide and glimepiride while patients with CYP2C9*2/*3 genotype showed greater drop than patients with CYP2C9*1/*1 genotype only in the glipizide-treatment group. The presence of CYP2C9*3 allele significantly affected plasma glucose drop per milligram of drug values in patients taking glipizide and glimepiride, while effects of CYP2C9*2 allele were insignificant. Further studies are needed to confirm the effects of CYP2C9*2 allele on plasma glucose drop per milligram of drug values. However, plasma glucose concentration is a complex physiological marker that cannot be used to establish perfect genotype-phenotype correlation. Hence studies exploring robust phenotypic markers must be initiated.

Synthesis, Antiinflammatory and HIV-1 Integrase Inhibitory Activities of 1,2-Bis[5-thiazolyl]ethane-1,2-dione Derivatives.

Based on principles of pharmacophore delineation and drug designing, compounds containing diketofunctionallity namely 1,2-bis[5-thiazolyl]ethane-1,2-diones were designed and synthesized as antiinflammatory agents. The compounds were evaluated in carrageenan-induced rat-paw edema method. G-3, G-6, G-17, G-20, G-23, G-22, L-708 and 906 showed good antiinflammatory activity. In addition as diketo functionality containing compounds are reported to have HIV-1 integrase inhibitory property, and these compounds contains diketo functionality, so these compounds were screened in assay for HIV-1 integrase inhibition. Few compounds showed weak HIV-1 integrase Inhibitory activity.

Involvement of endometrial membrane sulphydryl groups in blastocyst implantation: sulphydryl groups as a potential target for contraceptive research.

The role of membrane sulphydryl groups in blastocyst implantation was studied by masking the membrane sulphydryl groups in the endometrium of Swiss albino mice, Mus musculus, using 10(-5) M cobalt chloride and 0.05 mM as well as 0.005 mM n-ethylmaleimide. Here we show that the blocking of sulphydryl groups with cobalt resulted in a decrease in superoxide radical surge and an increase in superoxide dismutase levels at the time of implantation. We hypothesize that it may be due to either a decrease in membrane fluidity or the unavailability of sulphydryl groups of endometrial membrane, thus preventing blastocyst implantation. These sulphydryl groups can be targeted for future contraceptive research.

HPTLC method for the estimation of alkaloids of Cinchona officinalis stem bark and its marketed formulations.

We report a sensitive method for the estimation of quinine (Qn), cinchonine (Cn), and cinchonidine (Cnd) and a new method based on fluorescence enhancement and detection and quantification of quinidine (Qnd) from Cinchona stem bark and its formulations, using HPTLC. Standard solutions of Qn, Qnd, Cn, and Cnd were applied on precoated HPTLC plates and developed with chloroform/diethylamine (9.6:1.4 v/v). The plates were scanned and quantified at 226 nm for Qn, Cn, Cnd and for Qnd at 366 nm in fluorescence and reflectance mode ([symbol: see text] K400 filter). The method was validated for precision, accuracy and repeatability. Further, the stem bark of Cinchona officinalis and some herbal and homeopathic formulations were evaluated for their individual alkaloid content applying the developed method.

Sensitive high-performance thin-layer chromatographic method for the estimation of diospyrin, a tumour inhibitory agent from the stem bark of Diospyros montana Roxb.

Diospyrin, a tumour inhibitory agent from the stem bark of Diospyros montana was isolated and characterised. A sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the estimation of diospyrin. The method was validated for precision (intra- and inter-day), repeatability and accuracy. The method was found to be precise, with the RSDs for intra-day in the range of 0.72-1.85% and RSDs for inter-day in the range of 1.06-2.95%, for different concentrations. Instrumental precision and repeatability of the method were found to be 0.086 and 0.937 (% CV), respectively. Accuracy of the method was checked by performing the recovery study at two levels and average percentage recovery was found to be 97.87%. The developed HPTLC method was adopted for the estimation of diospyrin content of the stem bark of D. montana from different regions, which varied from 0.35 to 0.47% (w/w) in the samples.

Localization of cyclic-AMP receptors with acidosomes in Dictyostelium discoideum.

Earlier studies have shown that in Dictyostelium discoideum, a buoyant membrane fraction contained approximately 90% of the vacuolar proton pump (V-H(+)-ATPase) activity, leading to its designation acidosomes. It was proposed that acidosomes may be involved in endocytosis, specially in the acidification of endosomes. In this study we further investigated the putative function(s) of acidosomes. The findings suggest that acidosomes contain abundant receptors for cyclic AMP (CAR1) and that it may be the site for recycling of internalized receptors. Acidosomes also contain an abundance of Rab4 (Bush et al. 1994), a marker for early endosomes. By these criteria, we suggest that the acidosomes are analogous to early or recycling endosome present in mammalian cells. These findings suggest that the structure earlier defined biochemically, morphologically and immunologically as acidosomes may represent early and/or recycling endosomes in this protist.

Electromagnetic purification of endocytic vacuoles and acidosomes from Dictyostelium.

Earlier studies have shown that, in Dictyostelium discoideum, approximately 90% of the vacuolar proton pump (V-H(+)-ATPase) activity is present in a buoyant membrane fraction called "acidosomes." In the presence of Mg2+, acidosomes and endocytic vacuoles copurified on equilibrium sucrose gradients, suggesting their reversible association. The association depended on Mg2+ and cytosolic proteins (H. Padh et al., 1991, J. Biol. Chem. 266, 5514-5520, 12123-12126). To further characterize the putative association of acidosomes and endocytic vacuoles, cells were fed dextran-coated superparamagnetic iron colloid plus FITC-dextran to load and label their endocytic vacuoles. The endocytic vesicles were then purified approximately 20-fold at > 60% yield by their retention on a column of fine steel wire in an electromagnetic field in the absence of Mg2+. The fraction retained on a magnet column contained only about 5% of total cellular V-H(+)-ATPase and traces of other organelle markers. In the presence of 1.5 mM Mg2+, however, the retention of V-H(+)-ATPase as well as FITC-dextran was approximately 60% with only traces of contaminant markers. When such preparations were washed with buffer lacking Mg2+ while still in the magnetic field, the endocytic marker (FITC-dextran) remained on the column while V-H(+)-ATPase was eluted selectively. The elute was shown by negative-stain electron microscopy to contain purified acidosomes (saccular membranes studded with V-H(+)-ATPase). The parent material, recovered from the column in the presence of Mg2+, was rich in endocytic vacuoles bearing colloidal iron. In an electron microscope, the endocytic vacuoles were often seen associated with pump-studded acidosomes. The results independently support and extend earlier observation that acidosomes and endocytic vacuoles physically associate in a Mg(2+)-dependent manner. In addition, the procedure provides a rapid method of purifying acidosomes.

An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum.

Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.

A post-lysosomal compartment in Dictyostelium discoideum.

Fluorescein isothiocyanate (FITC)-dextran and pyranine were fed to the social amoeba, Dictyostelium discoideum. These membrane-impermeable, pH-sensitive fluorophores initially entered a approximately equal to neutral endocytic compartment. They encountered maximal acidity (pH approximately equal to 5) about 15 min after ingestion, in what appeared to be digestive vacuoles (lysosomes). The environment of the probes returned to near neutrality by 30 min. At that time, the probes accumulated in a decreasing number of vacuoles of increasing size; ultimately, there were only a small number of vacuoles per cell with diameters of up to 3 microns. The late vacuoles sedimented more rapidly than did proton pumps, acid hydrolases, and recently ingested cargo. Unlike the vacuoles harvested immediately after the cells were fed FITC-dextran, the late vacuoles were not acidified by MgATP in vitro. Egestion of ingested FITC-dextran commenced after a lag of approximately equal to 45 min. A similar lag was observed for the resurfacing of two endocytosed bilayer-intercalated fluorophores. These results suggest that, in Dictyostelium, undigested endocytic cargo accumulates in and is returned to the cell surface through a distinctive compartment of large and nearly neutral post-lysosomal vacuoles. It will be important to determine the degree to which internalized plasma membrane components follow this post-lysosomal pathway.

Acidosomes from Dictyostelium. Initial biochemical characterization.

The acidosome, a newly described organelle in Dictyostelium discoideum, is rich in vacuolar proton pumps (V-H(+)-ATPases) and is responsible for the acidification of endocytic vacuoles. Purified acidosomes were not significantly contaminated by lysosomes, endosomes, or plasma membranes but contained a small fraction of contractile vacuole markers. The specific activity of the proton pump in these acidosomes reached 30 mumol/min/mg protein, the highest yet reported for any V-H(+)-ATPase. The V-H(+)-ATPase was the predominant protein in acidosomes. Based on gel electrophoresis and densitometry, its 8 polypeptides had the following apparent molecular mass (in kDa) and stoichiometry: 90(1), 68(3), 53(3), 42(1), 37(3), 25(3), 17(6), and 15(1). These values suggested a Mr congruent to 8 x 10(5), consistent with the hydrodynamic properties and electron microscopic image of the purified pump. The 90- and 17-kDa polypeptides were integral, while the others were peripheral; only the 90-kDa subunit was biosynthetically labeled by [3H]glucosamine and 35SO4. The specific content of phosphatidylcholine and phosphatidylserine in the acidosomes was the highest of any subcellular fraction tested, while sterols and sphingolipids were the lowest. Acidosomes had congruent to 10% of the lipid biosynthetically labeled with [3H]glucosamine. This organelle contributed 5% of cellular protein and 15% of the phospholipid in stationary cultures. We conclude that the acidosome in Dictyostelium is a biochemically discrete organelle, produced by the endoplasmic reticulum/Golgi apparatus but distinct from other endomembranes as well as from the plasma membrane.

Reconstitution of the association of endocytic vacuoles and acidosomes from Dictyostelium.

In the amoeba, Dictyostelium discoideum, endocytic vacuoles are acidified by proton pumps which reside not in their membranes but in an associated organelle which we call the acidosome. These two organelles can be dissociated in vitro, and we now describe conditions for their functional reassociation. Fluorescein 5-isothiocyanate-dextran was fed to amoebae to report on the pH of their endocytic vacuoles. Following homogenization, the endocytic vacuoles were dissociated from acidosomes by removing Mg2+ and cytosol and purged of their native acidity by transient exposure to nigericin. The endocytic vacuoles could then be reacidified by ATP if first preincubated under these optimized conditions: 30 degrees C for 30 min in the presence of acidosomes, a 4-fold excess of cytosol, and 5 mM Mg2+ at pH 7.4. Reacidification was observed with early but not late endocytic compartments. Mn2+ and Ca2+ were poor substitutes for Mg2+; albumin did not substitute for cytosol. Neither Ca2+, ATP, nor adenosine 5'-O-(3-thiotriphosphate) affected reconstitution appreciably; guanosine 5'-O-(3-thiotriphosphate) inhibited reacidification by 50% when present during preincubation at 0.1 mM. Warming the cytosol to 50 degrees C or exposing it to protease abolished its activity but N-ethylmaleimide did not. Molecular sieving indicated that the cytosolic factor was a macromolecule. We conclude that the specific functional association of acidosomes and endocytic vacuoles can be reconstituted in vitro with soluble proteins plus Mg2+.

Endosomes are acidified by association with discrete proton-pumping vacuoles in Dictyostelium.

The endocytic compartment in the amoeba Dictyostelium discoideum was labeled by feeding fluorescein 5-isothiocyanate-dextran. In homogenates containing 2 mM Mg2+, the compartments so labeled copurified with all of the vacuolar H(+)-ATPase activity in a dense peak. The fluorescence properties of the probe showed that these dense vacuoles were inherently acidic. Furthermore, after purging their residual acidity, they could be re-acidified by the addition of ATP. These data suggest that the H(+)-ATPase was structurally and functionally coupled to the endocytic space. The association of the H(+)-ATPase and endocytic compartment was reversed by the removal of either Mg2+ or traces of the cytosol. Endocytic vacuoles prepared in this way were deficient in vacuolar H(+)-ATPase activity and were not acidified upon addition of MgATP. The missing proton pumps were recovered in large buoyant vacuoles that lacked ingested fluorescein 5-isothiocyanate-dextran, acid hydrolases, and residual acidity. These vacuoles were also less susceptible than endosomes to disruption by digitonin, suggesting that their bilayers were low in sterols. These results indicate that the endocytic circuit in Dictyostelium is acidified by a discrete and separable proton-pumping organelle.

Vitamin C: newer insights into its biochemical functions.

Ever since the discovery of vitamin C (ascorbic acid), scientists have been intrigued as to how ascorbic acid deficiency can lead to the diverse symptoms exhibited in scurvy. Only in recent years has it been appreciated that ascorbic acid has important functions in many cellular reactions and processes in addition to its role in collagen synthesis. The few such reactions that are understood at the molecular level make it apparent that ascorbic acid does not directly participate in enzyme-catalyzed conversion of substrate to product. Instead, the vitamin regenerates prosthetic metal ions in these enzymes in their required reduced forms. This is in agreement with other antioxidant functions of vitamin C, e.g., scavenging of free radicals. Ascorbate and other antioxidant nutrients are presumed to play a pivotal role in minimizing the damage from oxidative products, including free radicals. This protective function is twofold: the already-oxidized groups in prosthetic centers of enzymes are reduced and the oxidants and free radicals are removed.

Cellular functions of ascorbic acid.

It has long been suspected that ascorbic acid is involved in many cellular reactions. This is evident from the multitude of seemingly unrelated symptoms seen in scurvy. However, until recently, our understanding of its involvement was confined to its role in the synthesis of collagen. Studies in the past few years have unveiled mechanisms of its actions in collagen formation and many other enzymatic reactions. In addition, numerous physiological responses are reportedly affected by ascorbic acid. From the well-characterized enzymatic reactions involving ascorbic acid, it has become clear that in animal cells the ascorbate does not seem to be directly involved in catalytic cycles. Rather its major function seems to keep prosthetic metal ions in their reduced form. The role of ascorbate as a reductant in these enzymatic reactions complements its other antioxidant functions which have been recently appreciated, including that as a scavenger of free radicals. Therefore, it seems that the major function of ascorbate is to protect tissues from harmful oxidative products and to keep certain enzymes in their required reduced forms. However, it remains unclear how the deficiency of ascorbate leads to the pathological symptoms found in scurvy.

Characterization of a vacuolar proton ATPase in Dictyostelium discoideum.

Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.

Ascorbic acid transport by 3T6 fibroblasts. Regulation by and purification of human serum complement factor.

It was earlier reported (Padh, H., and Aleo, J. J. (1987) Proc. Soc. Exp. Biol. Med. 185, 153-157) that the activation of serum complement by endotoxin or immunocomplexes inhibited ascorbate transport in 3T6 fibroblasts. We show here that the inhibitor of 3T6 fibroblasts. We show here that the inhibitor of ascorbate transport increased the Km for ascorbate without affecting the Vmax, indicating that the inhibitor reduces the affinity of the ascorbate transporter for ascorbate without affecting the process of translocation. Inhibition by serum and endotoxin was reversible, and the generated inhibitor was no longer heat-labile (at 56 degrees C for 30 min) suggesting that the inhibitor of ascorbate transport is likely to be a small protein molecule. Utilization of complement components suggested that C3 was consumed during formation of the inhibitor of ascorbate transport while C5 and factor B were not consumed. These data along with other results indicate that the inhibitor is generated at C3 step of complement activation. The inhibitor was purified from inulin activated human serum and it had an apparent molecular mass of around 9000 daltons. The inhibitory effect of the purified factor was abolished by antiserum to C3a suggesting that the 9000-dalton factor could be related to this fragment of complement protein. These data raise the possibility that tissue supply of ascorbate may be compromised during infection or autoimmune processes when serum complement is activated.

Prelysosomal acidic vacuoles in Dictyostelium discoideum.

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.

Characterization of the ascorbic acid transport by 3T6 fibroblasts.

Ascorbic acid transport by 3T6 mouse skin fibroblasts has been characterized using radiometric technique with L-[1-14C]ascorbic acid under the conditions in which oxidation of ascorbic acid was prevented by addition of 1 mM thiourea. The ascorbate transport is temperature-dependent with the energy of activation E and Q10 of 13.3 kcal/mol and 2.0, respectively. The transport requires energy and exhibits Michaelis-Menten kinetics with an apparent Km of 112 microM and Vmax of 158 pmol/min per mg protein, when the extracellular Na+ concentration is 150 mM. The ascorbate transport requires presence of extracellular Na+ and can be inhibited by ouabain treatment. At 40 and 200 microM ascorbate concentrations, respectively, 1.4 and 1.0 moles of Na+ bound the transporter molecule per each mole of ascorbate transported. Increased Na+ binding to the transporter at lower ascorbate concentration may signify multiple Na+-binding sites or ascorbate concentration dependent conformational changes in the transporter molecule. Increasing Na+ concentration decreases Km without affecting Vmax, suggesting that Na+ increases affinity of ascorbate for the transporter molecule without affecting translocation process. An increase in ascorbate concentration reduces the number of Na+ bound to the transporter from 1.4 to 1.0. The ascorbate transport is stimulated by Ca2+ and other divalent cations. The mechanism of stimulation by Ca2+ is not clear. Calcium increases both the Km and Vmax. The data presented support the hypothesis that the ascorbate transport by 3T6 fibroblasts is an energy and temperature-dependent active process driven by the Na+ electrochemical gradient. A potent inhibitor of ascorbate transport is also demonstrated in human serum.

Activation of serum complement leads to inhibition of ascorbic acid transport.

Ascorbic acid is transported into 3T6 fibroblasts by a carrier-mediated, energy-dependent saturable active process with a Km of 112 microM and Vmax of 158 pmole/min/mg protein. The transport is dependent on extracellular Na+ concentration which reduces the Km. It was recently observed in this laboratory that bovine serum contained a heat-labile factor which, after interaction with bacterial endotoxin (lipopolysaccharides), inhibited ascorbic acid transport (J.J. Alleo and H. Padh, Proc Soc Exp Biol Med 179:128-131, 1985). We report here that the inhibition of ascorbic acid transport by endotoxin is mediated by the activation of serum complement. This was done by examining the activation of complement by other activators like zymosan and immunocomplexes (e.g., albumin and antibodies to albumin). Ascorbate transport was inhibited by the mixture of unheated serum and the activators. No inhibition was observed with serum devoid of C3 (component 3 of the complement). When C3-deficient serum was reconstituted by the addition of purified C3, the endotoxin-induced inhibition of ascorbate transport was restored. The implication of these findings is that in spite of a normal intake and blood level of the vitamin, tissues may not be getting adequate vitamin C during disease states when the complement in serum is activated. In other words, what may be considered an adequate intake of vitamin C under health conditions may not be adequate under disease conditions.