Abnormal chloride and potassium conductances in cultured embryonic tongue muscle from trisomy 16 mouse.

Trisomy 16 (Ts16) mouse is considered an animal model of Down syndrome (human trisomy 21). Whole-cell patch-clamp was used to evaluate potassium and chloride currents of cultured tongue muscle cells from fetal Ts16 and diploid mice. No difference was found in membrane capacitance between the two groups. K(+) and Cl(-) currents were pharmacologically isolated. K(+) conductance was reduced by 31% in Ts16 cells (373 pS/pF) compared with diploid cells (539 pS/pF). Cl(-) conductance was 51% larger in Ts16 cells (103 pS/pF) compared with diploid cells (68 pS/pF). However kinetics for K(+) and Cl(-) currents did not differ between the cell types. An increase in Cl(-) conductance and a decrease in K(+) conductance in Ts16 muscle cells, if present in muscle of Down syndrome subjects, might account for the observed hypotonia in these subjects.

Characterization by ionization mass spectrometry of lactosyl beta-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site.

The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.

Quantification of acetylcholine in cell culture systems by semi-micro high-performance liquid chromatography and electrospray ionization mass spectrometry.

Electrospray ionization (ESI) was used to adapt the thermospray ionization liquid chromatographic/mass spectrometric (LC/MS) method of Liberato et al. to the determination of acetylcholine and choline released from cells in culture. A clonal cell line derived from a rat pheochromocytoma (PC12) was used and cultures depolarized in the presence of cholinesterase inhibitor to maximize the recovery of acetylcholine from the culture medium and protect against its hydrolysis. Concentration of acetylcholine and choline released into the incubation medium was accomplished with Sep-Pak C16 cartridges. The preformed analyte cations were resolved by reversed-phase high-performance liquid chromatography and analyzed without derivatization by electrospray ionization mass spectrometry with the use of acetyl[2H9]choline as internal standard. LC/MS allows for the direct determination of acetylcholine and choline, which is not possible with other methods.

New opportunities from the isolation and utilization of whey proteins.

Management of dairy whey has often involved implementation of the most economical disposal methods, including discharge into waterways and onto fields or simple processing into low value commodity powders. These methods have been, and continue to be, restricted by environmental regulations and the cyclical variations in price associated with commodity products. In any modern regimen for whey management, the focus must therefore be on maximizing the value of available whey solids through greater and more varied utilization of the whey components. The whey protein constituents offer tremendous opportunities. Although whey represents a rich source of proteins with diverse food properties for nutritional, biological, and functional applications, commercial exploitation of these proteins has not been widespread because of a restricted applications base, a lack of viable industrial technologies for protein fractionation, and inconsistency in product quality. These shortcomings are being addressed through the development of novel and commercially relevant whey processing technologies, the preparation of new whey protein fractions, and the exploitation of the properties of these fractions in food and in nontraditional applications. Examples include the following developments: 1) whey proteins as physiologically functional food ingredients, 2) alpha-lactalbumin and beta-lactoglobulin as nutritional and specialized physically functional food ingredients, and 3) minor protein components as specialized food ingredients and an important biotechnological reagents. Specific examples include the isolation and utilization of lactoferrin and the replacement of fetal bovine serum in tissue cell culture applications with a growth factor extract isolated from whey.

Electrophysiological and metabolic effects of a convulsant barbiturate on dissociated mouse primary sensory neurons.

1. The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) depolarizes dorsal root ganglion (DRG) neurons. We have applied microfluorimetric and whole-cell patch clamp techniques to investigate the mechanisms underlying this response in freshly dissociated mouse DRG cells. 2. Application of CHEB (2-200 microM) raised cytosolic calcium concentration ([Ca2+]i) rapidly and reversibly in 55% of eighty-three neurons tested. This population did not correlate with other classifications of sensory neurons based on either cell size or the expression of membrane currents. 3. The response was dependent on external calcium and was reduced by 81 +/- 22% by Ruthenium Red. A rise in [Ca2+]i was still seen with the membrane potential clamped at -70 mV, excluding membrane depolarization and activation of voltage-dependent Ca2+ channels as the principal mechanism for the response. 4. The rise in [Ca2+]i was associated with an increase in membrane conductance and a current, ICHEB, which was inward at -70 mV. Both the rise in [Ca2+]i and the current showed 'run-down' under whole-cell recording conditions. When K+ conductances were blocked, the reversal potential of ICHEB was close to 0 mV. This was independent of the Cl- reversal potential, suggesting that ICHEB is carried as a non-specific cation current. 5. In contrast to the change in [Ca2+]i, ICHEB was not dependent on external Ca2+ and the current was still seen when [Ca2+]i as strongly buffered by the pipette filling solution. These data suggest that CHEB opens a non-selective cation channel permeant to Ca2+, raising [Ca2+]i and further depolarizing the cell membrane potential. The exact nature of this conductance remains unknown. These actions could readily account for the convulsant actions of the drug, depolarizing neurons and increasing transmitter release. 6. It was also noted that CHEB increases autofluorescence derived from mitochondrial NAD(P)H. Further examination of this phenomenon using the dye rhodamine 123 to follow changes in mitochondrial potential (psi m) suggested that CHEB is a potent inhibitor of mitochondrial respiration, probably acting at complex I. These effects appeared to be quite distinct from the action of CHEB at the level of the plasma membrane.

Differential expression of membrane currents in dissociated mouse primary sensory neurons.

The whole cell configuration of the patch clamp technique has been applied to identify the membrane currents expressed by populations of dissociated mouse primary sensory neurons. Three discrete populations of cells were distinguished on the basis of cell size and the array of currents expressed. Group 1 cells (capacitance 10-30 pF) expressed a Na+ current resistant to tetrodotoxin (1 microM) and a prominent, low threshold, inactivating, K+ current sensitive to 4-aminopyridine (IA). A population (53%) of these small cells responded to capsaicin (10 microM) with an inward current, suggesting a functional correlate with nociceptive "C"-cells. The cells of Group 2 (capacitance 55-85 pF) were characterized by the expression of a Na+ current sensitive to tetrodotoxin and a prominent inward current activated by hyperpolarization (IH). They also showed a variant of the A-type K+ current, which was a low threshold, but sustained K+ current, sensitive to dendrotoxin (30 nM). Group 3 cells, of intermediate size (capacitance 30-55 pF) were similar to Group 2 cells, in that they expressed a tetrodotoxin-sensitive Na+ current and (through reduced in amplitude), IH. The most notable feature of Group 3 cells was the expression of a transient, low threshold Ca2+ current. The differential expression of these conductances was reflected in the behaviour of cells under current clamp control. Each group of cells could thus be distinguished by the selective expression of specific ionic conductances which correlated clearly with cell size, suggesting a correlation with well recognised functional differentiation of sensory neurons. The selective expression of specific subsets of membrane channels may provide valuable markers in studying the developmental regulation of phenotype in this population of cells.

Heat-related changes to the hydrophobicity of cheese whey correlate with levels of native beta-lactoglobulin and alpha-lactalbumin.

Correlations were identified between levels of the native whey proteins, beta-lactoglobulin and alpha-lactalbumin and the surface and total hydrophobicities of cheese whey in response to different heat treatments. Heat-induced changes in the native beta-lactoglobulin content and surface hydrophobicity of whey exhibited the most significant linear relationship while correlations between total hydrophobicity and the native proteins were less significant because of an atypical rise in the n-heptane-binding capacity of whey after high-temperature treatment. The content of native beta-lactoglobulin in whey was more sensitive to heating than the content of native alpha-lactalbumin, while heat-related changes in the total hydrophobicity of whey were generally greater than similar changes in surface hydrophobicity.

New nonpeptide angiotensin II receptor antagonists. 2. Synthesis, biological properties, and structure-activity relationships of 2-alkyl-4-(biphenylylmethoxy)quinoline derivatives.

A novel series of nonpeptidic angiotensin II (AII) receptor antagonists is reported, derived from linkage of the biphenylcarboxylic acid or biphenylyltetrazole moiety found in previously described antagonists via a methyleneoxy chain to the 4-position of a 2-alkyl quinoline. When evaluated in an in vitro binding assay using a guinea pig adrenal membrane preparation, compounds in this series generally gave IC50 values in the range 0.01-1 microM. Structure-activity studies showed the quinoline nitrogen atom and a short alkyl chain at the quinoline 2-position to be essential for receptor binding. On intravenous administration in a normotensive rat model, the more potent compounds inhibited the AII-induced pressor response with ED50 values in the range 0.1-2.0 mg/kg. One of the compounds, 2-ethyl-4-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methoxy]quinoline (5g), demonstrated good oral activity in two rat models. At doses in the range 1-10 mg/kg in AII-infused, normotensive rats, the compound exhibited a dose-related inhibition of the pressor response with a good duration of action at the higher doses. In a renal hypertensive rat model, compound 5g showed a rapid and sustained lowering of blood pressure at a dose of 5 mg/kg. On the basis of its profile, this compound, designated ICI D8731, has been selected for clinical evaluation.

Structure/activity studies related to 2-(3,4-dichlorophenyl)-N-methyl-N-[2-(1-pyrrolidinyl)-1-substituted- ethyl]acetamides: a novel series of potent and selective kappa-opioid agonists.

This paper describes the synthesis of a series of N-[2-(1-pyrrolidinyl)ethyl]acetamides 1, variously substituted at the carbon adjacent to the amide nitrogen (C1), and related analogues, together with their biological evaluation as opioid kappa agonists. In the first part of the study, the variants in N-acyl, N-alkyl, and amino functions were explored when the substituent at C1 was 1-methylethyl and the optimum was found to be exemplified by 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(1-methylethyl)-2- (1-pyrrolidinyl)ethyl]acetamide (13). Subsequently, racemic or chiral amino acids were used to introduce other alkyl and aryl substituents at C1 of the ethyl linking moiety. A series of potent compounds, bearing substituted-aryl groups at C1, were discovered, typified by 2-(3,4-dichloro-phenyl)-N-methyl-N-[(1R,S)-1-(3-aminophenyl)-2-(1- pyrrolidinyl)ethyl]acetamide (48), which was 5-fold more active as the racemate than 13 in vitro and exhibited potent naloxone-reversible analgesic effects (ED50 = 0.04 mg/kg sc) in a mouse abdominal constriction model.

Human IgE-binding synthetic peptides of bovine beta-lactoglobulin and alpha-lactalbumin. In vitro cross-reactivity of the allergens.

The allergenicity of cow's milk whey proteins, purified by high performance liquid chromatography (HPLC), was examined by the radio-allergosorbent test (RAST) against the sera of children immediately hypersensitive to milk. beta-lactoglobulin and alpha-lactalbumin bound specific IgE in the sera of 63% and 75% of these patients respectively. These allergens were tested for cross reactivity with each other by RAST inhibition. Both inhibited the binding of IgE, in the sera of allergic patients, to the other protein. Two possible determinant peptides, one from beta-lactoglobulin and one from alpha-lactalbumin, were selected by computer prediction of antigenic sites and synthesized by the fluorenylmethoxycarbonyl (FMOC)-polyamide method. The peptides were adsorbed to nitrocellulose discs and used in further RAST studies with sera from the allergic children. Both peptides bound specific IgE in the RAST assay.

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 1. Synthesis and biological properties of alkyl alcohol and statine derivatives.

A series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity, which incorporate (1S,2S)-2-amino-1,3-dicyclohexyl-1-hydroxypropane, statine (Sta), and (3S,4S)-4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid (ACHPA) transition-state mimetics, have been prepared. Structure-activity relationships for renin inhibitory activity in the series are consistent with the 2-[8-isobutyl-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3-yl]-3-(3 pyridyl)propionic acid moiety 10b acting as a non-peptidic replacement for the P4-P2 (Pro-Phe-His) residues of the natural substrate angiotensinogen. Compounds 12m, 12o and 12q were potent inhibitors of partially purified human renin (IC50 values 1.7, 6.8, and 3.7 nM, respectively), and also effectively lowered blood pressure in anesthetized, sodium depleted marmosets following intravenous administration. On oral administration however, no blood pressure lowering activity could be detected, and absorption studies in bile duct cannulated rats indicate that this may be due primarily to poor oral absorption, rather than rapid biliary excretion. The reason for the observed poor oral activity is not clear, but it seems unlikely that poor aqueous solubility or metabolic instability to gut enzymes are rate-determining, and other factors such as high molecular weight may also be very important.

Pharmacological studies in vivo with ICI 169,369, a chemically novel 5-HT2/5-HT1C receptor antagonist.

ICI 169,369 (2-(2-dimethylaminoethylthio-3-phenylquinoline hydrochloride) has been tested in vivo for its potency and selectivity as an antagonist at 5-HT2 and 5-HT1C receptors. It caused a 50% inhibition of 5-HTP-induced head twitches in mice and fenfluramine-induced hyperthermia in the rat at approximately 1 mg/kg following parenteral administration. Results showed that ICI 169,369 had good oral bioavailability, since in the fenfluramine test the oral and s.c. ID50 values were similar. ICI 169,369 was a selective antagonist of 5-HT-induced bronchoconstriction in the guinea-pig and 5-HT-induced pressor effects in the anaesthetised dog. In a series of other tests in vivo the compound was shown to be devoid of significant activity at alpha 1- and alpha 2-adrenoceptors, dopamine (D2), muscarinic (M1) and histamine (H1) receptors at 30-100 times its ID50 values used in the 5-HT tests. Thus, ICI 169,369 is a selective, orally active 5-HT2/5-HT1C antagonist that should prove useful in the analysis of the role of 5-HT in physiological and pathological states.

The xanthene-9-spiro-4'-piperidine nucleus as a probe for opiate activity.

A series of novel 1'-methylxanthene-9-spiro-4'-piperidines has been prepared in the search for opiate analgesics with improved pharmacological properties. It has been found that introduction of a hydroxyl group into the 4-position of the xanthenespiropiperidine nucleus produces a potent mu-opiate agonist. The structure-activity relationship of the series has been explored by use of isosteric replacements of the phenolic hydroxyl group. Moreover, the effect of altering the conformation of the piperidine ring has been studied. It was interesting to note that, in compounds lacking the phenolic hydroxyl group, opiate activity could be produced by introduction of the (phenylamino)ethyl group instead of methyl at the 1'-position.

In vitro studies with ICI 169,369, a chemically novel 5-HT antagonist.

ICI 169,369 is a chemically novel 5-HT antagonist that has higher affinity for the 5-HT2 binding sites in rat cortex than it has for 5-HT1 sites (Ki 1.79 x 10(-8) and 1.58 x 10(-6) M, respectively). In isolated tissue preparations ICI 169,369 was shown to be a competitive antagonist of 5-HT on the rabbit aorta, pig coronary artery and rat caudal artery. In the latter preparation it had a similar pA2 value to ketanserin (pA2 8.18 +/- 0.5 and 8.42 +/- 0.06, respectively). Unlike ketanserin, which was inactive, ICI 169,369 was a non-surmountable antagonist at the rat stomach fundus 5-HT 'D' receptor, recently reclassified as 5-HTIC. It was inactive (greater than 10(-6) M) at the 5-HT3 receptors found in the isolated perfused rabbit heart and the myenteric plexus of the guinea-pig ileum. At receptors other than those for 5-HT (alpha 1, alpha 2, beta 1, beta 2, H1, H2 and muscarinic), ICI 169,369 was inactive at concentrations of either 10(-6) or 10(-5) M. Thus the profile of ICI 169,369 should make it useful in the analysis of the role of 5-HT in physiological and pathological states.

Studies on a partially purified bovine milk lysozyme.

Bovine milk lysozyme has been partially purified by a method developed in this laboratory. We have shown, by preliminary sequential analysis, and by gel filtration on HPLC, that the product is a mixture of two components. One of these, the enzymically active one, differs in its N-terminal sequence from that of "lysozyme 2", a bovine stomach mucosal enzyme, by 7 residues within the first 39 residues. However, some of its properties differ markedly from those of lysozyme 2. The other component, comprising 70% by weight of the total mixture, bears no sequential resemblance to any protein known to us. Our two component system appears to be the same as the preparation of Chandan et al. (Biochim. Biophys. Acta 110, 289 (1965], which they concluded was an homogeneous preparation of lysozyme.

Synthesis and 5-hydroxytryptamine antagonist activity of 2-[[2-(dimethylamino)ethyl]thio]-3-phenylquinoline and its analogues.

A series of 2-[(2-aminoethyl)thio]quinolines substituted at the 3-position with alkyl, aryl, or heteroaryl groups has been prepared in the search for novel and selective 5-HT2 antagonists. The affinity of the compounds for 5-HT1 receptor sites was measured by their ability to displace [3H]-5-HT from rat brain synaptosomes whereas the affinity for 5-HT2 receptor sites was measured by their ability to displace [3H]spiperone from synaptosomes prepared from rat brain cortex. The 5-HT2 antagonist properties of the compounds were measured in vivo by their antagonism of 5-hydroxytryptophan-induced head twitches in the mouse and by their antagonism of hyperthermia induced by fenfluramine (N-ethyl-alpha-methyl-m-(trifluoromethyl)phenethylamine hydrochloride) in the rat. The structure-activity relationships in this series are discussed and the properties of 2-[[2-(dimethylamino)ethyl]thio]-3-phenylquinoline hydrochloride (70) are highlighted.

Heat stability in concentrated and non-concentrated milks--the effect of urea and beta-lactoglobulin levels and the influence of preheating.

Heat stability as a function of pH has been studied in concentrated and non-concentrated milk systems. The influence of preheating has also been examined. The concentration of beta-lactoglobulin has been shown to affect markedly the heat stability behaviour in both systems but with different characteristics. Increasing the level of urea resulted in increased heat stability in non-concentrated milks, but corresponding concentrated milks showed a small decrease in maximum heat stability. It has not been possible to extrapolate heat stability data from non-concentrated to concentrated milks.

Amino acid analysis by gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters. Complete resolution using a support-coated open-tubular capillary column.

Amino acids have been separated by gas-liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenaschuk have been introduced. Acylation by heating at 150 degrees was shown to be destructive; 110 degrees has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of beta-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxy-peptidase digestion.