Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells.

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.

Lipopeptaibols, a novel family of membrane active, antimicrobial peptides.

Lipopeptaibols are members of a novel group of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of the turn/helix forming alpha-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. The amino acid sequences range from 6 to 10 residues and the fatty acyl moieties from 8 to 15 carbon atoms. The peptide portion of lipopeptaibols can be shorter than those of the nonlipidated peptaibols that range from 10 to 19 amino acid residues. The longest peptides fold into a mixed 3(10)/alpha helix, whereas the shortest peptides tend to adopt a beta-turn/sheet structure. Using solution methodologies, a series of analogues of trichogin GA IV was synthesized which allowed determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity and exploitation of a number of spectroscopic techniques aimed at determining its preferred conformation under a variety of conditions and investigating in detail its mode of interaction with, and its effect on, the phospholipid membranes.

Detection of beta-ureidopropionase deficiency with HPLC-electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level.

The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to beta-alanine and beta-aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl-beta-amino aciduria, due to a deficiency of beta-ureidopropionase, was easily diagnosed at the metabolite level using HPLC-tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of beta-ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of beta-ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and dihydropyrimidinase was present. Thus, HPLC-tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of beta-ureidopropionase.

Substrate autoregulation of glucose transport: hexose 6-phosphate mediates the cellular distribution of glucose transporters.

Exposure of rat skeletal muscle and skeletal muscle cell lines to high glucose levels results in a time- and dose-dependent reduction of the rate of hexose uptake, paralleled by a reduction in the plasma membrane density of glucose transporters. The mechanism of this process was investigated in cultured L8 myocytes. Low concentrations (0.5-2.0 mmol/l) of deoxyglucose mimicked the downregulatory action of 20 mmol/l glucose both regarding the time-course and magnitude of the effect, but in an irreversible manner. A dose-dependent relationship between intracellular accumulation of deoxyglucose 6-phosphate and the magnitude of the downregulatory response was observed. Depletion of intracellular deoxyglucose 6-phosphate restored the rate of hexose transport to the control level. The reduction of hexose transport activity by deoxyglucose occurred independently of ATP depletion which by itself produced the opposite effect. The effects of deoxyglucose and high glucose on hexose transport were associated with reduced transport maximal velocity and GLUT1 transporter abundance in the plasma membranes of myocytes, as assessed by cell surface biotinylation. The reduction of myocyte GLUT1 mRNA content, observed after exposure to high glucose, did not accompany the transport down regulatory action of deoxyglucose. We suggest that hexose 6-phosphate is the mediator of the downregulatory signal for subcellular redistribution of GLUT1 in L8 myocytes. The signal responsible for reducing the GLUT1 mRNA level may be related to glucose metabolites downstream of the hexokinase reaction.

Aib-containing analogues of the insect kinin neuropeptide family demonstrate resistance to an insect angiotensin-converting enzyme and potent diuretic activity.

Analogues of the insect kinin family in which the Xaa2 residue of the C-terminal pentapeptide core sequence Phe-Xaa1-Xaa2-Trp-Gly-NH2 (Xaa1 = Asn, His, Phe, Ser, or Tyr; Xaa2 = Ala, Ser, or Pro) is replaced with sterically hindered aminoisobutyric acid (Aib) prove to be resistant to hydrolysis by housefly (Musca domestica) angiotensin-converting enzyme (ACE), an endopeptidase capable of hydrolysis and inactivation of the naturally occurring insect kinin peptides. The Aib residue is compatible with formation of turn in the active core region that is important for the biological activity of the insect kinins. One of the Aib-containing analogues, pGlu-Lys-Phe-Phe-Aib-Trp-Gly-NH2, is five- and eightfold more active than the most active endogenous insect kinins in cockroach (Leucophaea maderae) hindgut myotropic and cricket (Acheta domesticus) Malpighian tubule fluid secretion assays, respectively. As the analogue is blocked at both the amino- and the carboxyl-terminus and resistant to an endopeptidase present in insects, it is better adapted than the endogenous peptides to survive for long periods in the hemolymph. Enzyme-resistant insect kinin analogues can provide useful tools to insect researchers studying the neuroendocrine control of water and ion balance and the physiological consequences of challenging insect with diuretic factors that demonstrate enhanced resistance to peptidase attack. If these analogues, whether in isolation or in combination with other factors, can disrupt the water and/or ion balance they hold potential utility for the control of pest insect populations in the future.

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.

Determination of alpha-dialkylamino acids and their enantiomers in geological samples by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde.

Derivatization with o-phthaldialdehyde (OPA) and the chiral thiol N-acetyl-L-cysteine (NAC) is a convenient and sensitive technique for the HPLC detection and resolution of protein amino acid enantiomers. The kinetics of the reaction of OPA-NAC with alpha-dialkylamino acids was investigated. The fluorescence yield of alpha-dialkylamino acids was only about 10% of that of protein amino acids when the derivatization was carried out at room temperature for 1-2 min, which is the procedure generally used for protein amino acid analyses. The fluorescence yield of alpha-dialkylamino acids can be enhanced by up to ten-fold when the derivatization reaction time is increased to 15 min at room temperature. The OPA-NAC technique was optimized for the detection and enantiomeric resolution of alpha-dialkylamino acids in geological samples which contain a large excess of protein amino acids. The estimated detection limit for alpha-dialkylamino acids is 1-2 pmol, comparable to that for protein amino acids.

Morphological alterations accompanying the effect of peptaibiotics, alpha-aminoisobutyric acid-rich secondary metabolites of filamentous fungi, on Culex pipiens larvae.

The effect of different representatives of the group of peptaibiotics, alpha-amino-isobutyric acid rich secondary metabolites of filamentous fungi, on Culex pipiens larvae was studied. Light and transmission electron microscopy techniques were used to localize the intracellular damage and to determine the target organells for the mode of action of peptaibols in mosquito larvae. Though different in insecticidal activity, all tested compounds induced the same type of tissue damage, which was characterized by heavy challenge of mitochondria followed by partial swelling, crystaeolysis and destruction of mitochondrial walls. It is concluded that the mode of action of peptaibols in mosquito larvae is mediated through the damage of mitochondria. The structure-mosquitocidal effect of these compounds, their potential mode of action and role in the natural fungal entomopathogenic process are briefly discussed.

Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio.

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

The absence of gamma-glutamyltransferase activity in transport-dependent methotrexate-resistant hepatoma cells.

A cell line derived from H35 hepatoma cells resistant to methotrexate (MTX) as a result of a defective transport system for MTX has been examined to determine how closely the variant resembles the parent cells with regard to other biochemical properties. The capacity of extracts of resistant cells to catalyze the poly-gamma-glutamylation of MTX was approximately twice as great as that of wild-type cell extracts. Evidence of similarity between wild-type and H35 R0.3 cells was derived from the equitoxic activity to both cell lines of nonclassical antifolates and other miscellaneous antineoplastics which act by a variety of mechanisms. Two phenotypic markers of hepatic cell function, alpha-aminoisobutyric acid (AIB) transport and tyrosine aminotransferase (TAT) activity inducibility, were present in both cell types, demonstrating the maintenance of these phenotypic properties in the H35 R0.3 cells. gamma-Glutamyltransferase (GGT, EC 2.3.2.2) activity differed in that it was present in wild-type cells and barely detectable in H35 R0.3 cells. The GGT activity reappeared in the H35 cells when they regained MTX sensitivity after incubation for 14-20 weeks in MTX-free media. Although defective MTX transport appeared to be correlated with the disappearance of GGT activity in an H35 variant cell line, no functional relationship between them is apparent at this time. It is possible that a lack of GGT activity may be evidence of a more differentiated phenotype in the transport-resistant cell line.

Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques.

Water, sodium, potassium, ATP, amino acids, and sugars are not uniformly distributed in Rana pipiens oocytes. Concentration differences exist between nucleus (germinal vesicle) and ooplasm and between animal and vegetal ooplasmic regions. The mechanisms responsible for these differences were investigated using intracellular reference-phase (iRP) analysis. The iRP is an artificial "organelle" that has the solvent properties of a dilute salt solution and is in diffusional equilibrium with water and solutes present in other cellular compartments. Ooplasm/iRP solute distributions show that ooplasm differs from ordinary aqueous solutions--exhibiting both solute exclusion and solute binding. Yolk platelets are an important cause of this behavior, largely because their proteins are present as hydrate crystals, which are rich in anionic sites and which interact intensely with associated water. Because of yolk's abundance, it obscures the solvent and binding properties of ooplasmic ground substance. The oocyte nucleus is yolk and organelle free and the nuclear envelope is readily permeable. Consequently, nucleus/iRP solute concentration differences reflect the binding and solvent properties of nuclear ground substance. Nucleoplasm binds approximately 19 meq of potassium. Furthermore, the monosaccharides, 3-O-methylglucose, L-glucose, and D-xylose, are selectively excluded, their nucleus/iRP concentration ratios averaging about 0.7; ratios for other solutes studied are unity. We interpret monosaccharide exclusion to mean that nuclear ground substance water is different in its "instantaneous" structure from ordinary saline water. Because of this difference, hydrogen bond interaction between nuclear water and certain sterically restricted solutes, of which ringed monosaccharides are examples, is reduced. Some implications of modified ground substance water and selective solute exclusion are discussed.

Regional measurements of blood-to-tissue transport in experimental RG-2 rat gliomas.

Regional measurements of blood-to-tissue transport were performed in transplanted RG-2 rat gliomas using [alpha- 14C]aminoisobutyric acid (AIB), quantitative autoradiography, and equations to express a unidirectional transfer constant. Thirty-eight intracranial tumors in ten rats were analyzed according to location; 23 intraparenchymal tumors, eight meningeal tumors, six fourth-ventricular tumors, and one third-ventricular tumor were studied. Except for the small third-ventricular tumor, the transfer constant (K) for AIB was similar in all groups and ranged from 0.031 to 0.038 ml/g/min. Within individual tumors, regional variation of K was also small, although some local variation could be correlated with histological features. The K for AIB decreased in brain around tumor and, at a distance of 300 microns from tumor edge, had returned to values similar to those of normal cortex (0.002 ml/g/min). An average extraction fraction (E) of 0.09 was calculated for AIB in the RG-2 tumors. The low E suggests that delivery of water-soluble chemotherapeutic drugs to RG-2 tumors should be limited more by capillary permeability or surface area than by blood flow. RG-2 is an ideal experimental tumor with which to test drug delivery and the methods that attempt to increase drug delivery in brain tumors.

Paracelsin, a peptide antibiotic containing alpha-aminoisobutyric acid, isolated from Trichoderma reesei Simmons. Part A.

A peptide antibiotic has been isolated from Trichoderma reesei QM 9414. Although crystalline and uniform in TLC, this antibiotic could be resolved by HPLC into 3 sequence analogues. The close relationship to alamethicin was proved by chemical and spectroscopic methods, and the formation of ion-conducting pores in lipid bilayers.

Dual-column cation-exchange chromatographic method for beta-aminoisobutyric acid and beta-alanine in biological samples.

A rapid, automated chromatographic method has been developed for the quantitation of the nucleic acid catabolites beta-aminoisobutyric acid and beta-alanine in urine, serum, and other physiological fluids. The analyses were performed on a modified Beckman 121M amino acid analyzer with dual ion-exchange columns and the use of a single sodium citrate buffer (pH 4.38, 0.20 mol/liter). By carefully matching the elution pattern for the two ion-exchange columns and alternating use of these columns, analyses are completed every 40 min. The chromatography, regeneration, and equilibration of the two columns are precisely programmed, thus the detector sees only the elution of beta-aminoisobutyric acid and beta-alanine alternately from each column. Long-term precision and analytical recovery for the two metabolites in urine were 1.9 and 102%, and 3.3 and 101%, respectively. Their normal physiological values were determined in human serum and urine. Their excretion in the urine was also studied as a function of collection time, to validate a more convenient, less costly method of sampling. This study shows that randomly collected samples are acceptable when the concentration of the two metabolites are expressed in terms of creatinine excretion. In addition, the distribution of the free and conjugated forms of the two metabolites in urine and serum was studied. A preparative method was also developed for the quantitative isolation of beta-amino-isobutyric acid from urine samples. The alternating dual-column technique may be applied to any ion-exchange chromatographic method where many analyses must be performed. This method is currently used in our laboratories for measuring these beta-amino acids in urine and serum of patients with various types of cancers.