Spectrophotometric investigation of complex formation of an oxime PAM-4Cl with palladium(II) and its analytical application

The colour reaction of 4-hydroxyiminomethyl-1-methylpyridinium chloride (PAM-4Cl) and palladium(II) chloride has been investigated. The optimum reaction conditions, spectral characteristics, conditional stability constant and composition of the yellow water-soluble complex have been established. A new spectrophotometric method is proposed for the microdetermination of PAM-4Cl.     

Chloride transport in the renal proximal tubule

The renal proximal tubule is responsible for most of the renal sodium, chloride, and bicarbonate reabsorption. Micropuncture studies and electrophysiological techniques have furnished the bulk of our knowledge about the physiology of this tubular segment. As a consequence of the leakiness of this epithelium, paracellular ionic transport--in particular that of Cl(-)--is of considerable importance in this first part of the nephron. It was long accepted that proximal Cl(-) reabsorption proceeds solely paracellularly, but it is now known that transcellular Cl(-) transport also exists. Cl(-) channels and Cl(-)-coupled transporters are involved in transcellular Cl(-) transport. In the apical membrane, Cl(-)/anion (formate, oxalate and bicarbonate) exchangers represent the first step in transcellular Cl(-) reabsorption. A basolateral Cl(-)/HCO(3)(-) exchanger, involved in HCO(3)(-) reclamation, participates in the rise of intracellular Cl(-) activity above its equilibrium value, and thus also contributes to the creation of an outwardly directed electrochemical Cl(-) gradient across the cell membranes. This driving force favours Cl(-) diffusion from the cell to the lumen and to the interstitium. In the basolateral membrane, the main mechanism for transcellular Cl(-) reabsorption is a Cl(-) conductance, but a Na(+)-driven Cl(-)/HCO(3)(-) exchanger may also participate in Cl(-) reabsorption.     

Regulatory volume decrease in northern fur seal (<Emphasis Type="Italic">Callorhinus ursinus</Emphasis>) red blood cells

The cation transport and regulatory volume decrease (RVD) were investigated in the red blood cells (RBCs) of northern fur seals (Callorhinus ursinus). Extracellular Ca-dependent Na efflux was increased to threefold by hypotonicity. K–Cl cotransport activity was not detected by hypotonic medium, but measured only by nitrite or N-ethylmaleimide stimulation. RBCs were restored to their original volume after being swollen in hypoosmotic medium with Ca, though this recovery was inhibited by the addition of quinidine. Based on these results, Na/Ca exchange transporter played the major role in the regulatory volume decrease in the RBCs of northern fur seals. H. Fujise has passed away.     

Hydrophobic interactions in Plasmodium falciparum invasion into human erythrocytes

Human glycophorins block in vitro invasion of Plasmodium falciparum merozoites into human erythrocytes. A segment of glycophorin A which appears to be involved in the inhibition, is at, or adjacent to, the membrane-spanning domain of the molecule. To study the role of hydrophobic interactions in the inhibition, a series of proteins were derivatized with lipophilic side groups, and tested for inhibitory activity. Glycophorin A became five times more inhibitory after derivatization with nitrobenzylfurazan groups. Bovine serum albumin was derivatized to different degrees with nitrobenzylfurazan, dinitrobenzyl, trinitrobenzyl, dansyl, disulfonic stilbene, and fluorescein groups. The presence of hydrophobic side groups on the protein rendered it highly inhibitory to invasion, whereas the presence of hydrophilic substitutes such as disulfonic stilbenes did not. Other soluble proteins such as human serum albumin, transferrin, ovalbumin, fetuin and casein derivatized with dinitrobenzyl groups, were also found to block invasion. Inhibition was not a result of toxic effects of the protein derivatives on parasite metabolism or development. A minimum of ten hydrophobic side groups per bovine serum albumin was required in order to elicit appreciable inhibition. The invasion blocking activity was highly correlated with the rate and affinity of binding of the derivatized macromolecules to heptyl-Sepharose. The latter provided a quantitative measure for the capacity of amphiphiles to undergo hydrophobic interactions with insoluble matrices. The results of the present study indicate that hydrophobic interactions may be an essential component in the invasion of P. falciparum merozoites into human erythrocytes.     

Effects of vasopressin on water and NaCl transport across the in vitro perfused medullary thick ascending limb of Henle's loop of mouse,rat, and rabbit kidneys

Direct tubular effects of arginine vasopressin (AVP) on water and NaCl transport across the medullary thick ascending limb of Henle (MAL) were examined by the in vitro perfusion of isolated nephron fragments of mice, rats, and rabbits. Osmotic water permeability of the MAL of mice and rats was low and remained unchanged with 2 mU/ml AVP added to the bath. A dose-dependent increase in transepithelial electrical potential difference (PD) with AVP was observed in the mouse MAL when the ambient medium was isotonic. A similar result was also obtained when 2×10^–4 mol/l dibutyryl adenosine 3,5-cyclic-monophosphate was added to the bath. In this preparation, AVP also caused an increase in the unidirectional Cl efflux from 323±45 to 398±61 pmoles·mm^–1 ·min^–1 (n=6,P<0.05). In contrast, under similar condition, we could not demonstrate any effect of AVP on PD, Cl efflux, or net Na flux in the rat MAL and on PD and Cl efflux in the rabbit MAL. Both PD and Cl efflux in the rat MAL were unaffected by AVP when the perfusate was made hypotonic. However, when the ambient medium was made hypertonic by adding NaCl and urea, a significant increase in PD was observed. In addition, we confirmed that AVP stimulated adenylate cyclase activity in the MAL as well as in the collecting tubule of mice and rats. We conclude that AVP stimulates Cl transport across the MAL of mice and rats by activating adenylate cyclase-cyclic AMP system. However, this effect of AVP may quantitatively vary among species.     

Segmental heterogeneity of swelling-induced Cl transport in rat small intestine

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (I _sc) and potential difference (V ^ms) in the jejunum, whereas the ileum responded with an increase in I _sc and V ^ms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl⁻ and was blocked by the Cl⁻ channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in I _sc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl⁻ secretion induced by hypotonicity was blocked by the K⁺ channel blocker quinine and was reversed into a decrease in I _sc when serosal Ca²⁺ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl⁻ channels and in the ileum by a Ca²⁺-mediated opening of K⁺ channels which enhances apical Cl⁻ efflux. Received: 27 June 1995/Received after revision: 8 December 1995/Accepted: 28 December 1995     

Hypotonicity stimulates translocation of ICln in neonatal rat cardiac myocytes

 Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them. Received: 31 October 1997 / Received after revision and accepted: 23 March 1998     

Microelectrode measurements of the effects of basolateral adenosine in polarized human intestinal epithelial cells in culture

 Activation of the basolateral receptor for adenosine in HT-29cl.19A cells, by 100 μM adenosine, increased the equivalent short-circuit current (ΔI _sc= 24±2 μA/cm²), depolarized the intracellular potential (ΔV _a= 26±2 mV) and decreased the fractional apical membrane resistance (ΔfR _a=–0.48). The changes in all parameters reached their peak values simultaneously. This suggests that the primary action of the adenosine-activated pathway is on only one membrane. Bumetanide inhibited the transepithelial response and repolarized the cell potential. After preincubation with 100 μM forskolin, application of 300 μM adenosine caused a significant further change in V _a, I _sc, the transepithelial potential (V _t) and fR _a. Together with the results from ion-replacement studies, the observations indicate that adenosine activates channels other than the cystic fibrosis transmembrane conductance regulator (CFTR). The rank order of potencies of adenosine and adenosine analogues implies that the receptor is of the A₂ subtype. Preincubation with 4-bromophenacyl bromide (4-BPB) inhibited the effect of an adenosine analogue by 50%, indicating that activation of phospholipase A₂ may be involved in the adenosine-induced response. Received: 5 August 1998 / Received after revision: 12 October 1998 / Accepted: 5 November 1998     

Identification of five pyrrolidinyl substituted cathinones and the collision‐induced dissociation of electrospray‐generated pyrrolidinyl substituted cathinones

This article reports on the analytical properties of five pyrrolidinyl substituted cathinones: α ‐pyrrolidinononaphenone (α ‐PNP, 1 ), 4‐chloro‐α ‐pyrrolidinopropiophenone (4‐Cl‐α ‐PPP, 2 ), 4‐chloro‐α ‐pyrrolidinovalerophenone (4‐Cl‐α ‐PVP, 3 ), 5‐dihydrobenzofuranpyrovalerone (5‐DBFPV, 4 ), and 2‐(pyrrolidin‐1‐yl)‐1‐(5,6,7,8‐tetrahydronaphthalen‐2‐yl)hexan‐1‐one (β ‐THNPH, 5 ). These identifications were based on liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry (LC–QTOF–MS), gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance spectroscopy (NMR). To our knowledge, no analytical data about α ‐PNP, 4‐Cl‐α ‐PPP, 4‐Cl‐α ‐PVP, and β ‐THNPH have appeared until now, making this the first report on these compounds. Moreover, in order to study the collision‐induced dissociation (CID) characteristic fragmentation routes of pyrrolidinyl substituted cathinones, a total number of 13 pyrrolidinyl substituted cathinones were selected and discussed. The major fragmentation pathways under CID mode are produced, leading to the formation of characteristic ions. Product ions of [M‐C₄H₉N]⁺ and C_nH_2nN⁺ indicate the presence of pyrrolidinyl substitution. Characteristic fragments are also produced via the cleavages of the CH–N(CH₂)₄ bond and the CO‐CHN bond. Copyright © 2016 John Wiley & Sons, Ltd.     

Glycosidic intermediates identified in 1H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways

The glycosylation process, through the addition of carbohydrates, is a major post‐translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N‐linked or O‐linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β‐linked N‐acetylglucosamine. In both cases, the key precursors are the uridine diphospho‐N‐acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho‐N‐acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho‐N‐acetylhexosamines visible in high‐resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho‐N‐acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes. Copyright © 2010 John Wiley & Sons, Ltd.