Oxybutyric Acid Research Articles

Shedding Light on Drug-Induced Liver Injury: Activation of T Cells From Drug Naive Human Donors With Tolvaptan and a Hydroxybutyric Acid Metabolite

Exposure to tolvaptan is associated with a significant risk of liver injury in a small fraction of patients with autosomal dominant polycystic kidney disease. The observed delayed onset of liver injury of between 3 and 18 months after commencing tolvaptan treatment, along with rapid recurrence of symptoms following re-challenge is indicative of an adaptive immune attack. This study set out to assess the intrinsic immunogenicity of tolvaptan and pathways of drug-specific T-cell activation using in vitro cell culture platforms. Tolvaptan (n = 7), as well as oxybutyric (DM-4103, n = 1) and hydroxybutyric acid (DM-4107, n = 18) metabolite-specific T-cell clones were generated from tolvaptan naive healthy donor peripheral blood mononuclear cells. Tolvaptan and DM-4103 T-cell clones could also be activated with DM-4107, whereas T-cell clones originally primed with DM-4107 were highly specific to this compound. A signature cytokine profile (IFN-γ, IL-13, granzyme B, and perforin) for almost all T-cell clones was identified. Mechanistically, compound-specific T-cell clone activation was dependent on the presence of soluble drug and could occur within 4 h of drug exposure, ruling out a classical hapten mechanism. However, antigen processing dependence drug presentation was indicated in many T-cell clones. Collectively these data show that tolvaptan-associated liver injury may be attributable to an adaptive immune attack upon the liver, with tolvaptan- and metabolite-specific T cells identified as candidate effector cells in such etiology.

Release of taurine and glutamate contributes to cell volume regulation in human retinal Müller cells: differences in modulation by calcium.

Neuronal activity in the retina generates osmotic gradients that lead to Müller cell swelling, followed by a regulatory volume decrease (RVD) response, partially due to the isoosmotic efflux of KCl and water. However, our previous studies in a human Müller cell line (MIO-M1) demonstrated that an important fraction of RVD may also involve the efflux of organic solutes. We also showed that RVD depends on the swelling-induced Ca2+ release from intracellular stores. Here we investigate the contribution of taurine (Tau) and glutamate (Glu), the most relevant amino acids in Müller cells, to RVD through the volume-regulated anion channel (VRAC), as well as their Ca2+ dependency in MIO-M1 cells. Swelling-induced [3H]Tau/[3H]Glu release was assessed by radiotracer assays and cell volume by fluorescence videomicroscopy. Results showed that cells exhibited an osmosensitive efflux of [3H]Tau and [3H]Glu (Tau > Glu) blunted by VRAC inhibitors 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)-oxybutyric acid and carbenoxolone reducing RVD. Only [3H]Tau efflux was mainly dependent on Ca2+ release from intracellular stores. RVD was unaffected in a Ca2+-free medium, probably due to Ca2+-independent Tau and Glu release, but was reduced by chelating intracellular Ca2+. The inhibition of phosphatidylinositol-3-kinase reduced [3H]Glu efflux but also the Ca2+-insensitive [3H]Tau fraction and decreased RVD, providing evidence of the relevance of this Ca2+-independent pathway. We propose that VRAC-mediated Tau and Glu release has a relevant role in RVD in Müller cells. The observed disparities in Ca2+ influence on amino acid release suggest the presence of VRAC isoforms that may differ in substrate selectivity and regulatory mechanisms, with important implications for retinal physiology. NEW & NOTEWORTHY The mechanisms for cell volume regulation in retinal Müller cells are still unknown. We show that swelling-induced taurine and glutamate release mediated by the volume-regulated anion channel (VRAC) largely contributes the to the regulatory volume decrease response in a human Müller cell line. Interestingly, the hypotonic-induced efflux of these amino acids exhibits disparities in Ca2+-dependent and -independent regulatory mechanisms, which strongly suggests that Müller cells may express different VRAC heteromers formed by the recently discovered leucine-rich repeat containing 8 (LRRC8) proteins.

Blockade of the swelling-induced chloride current attenuates the mouse neonatal hypoxic-ischemic brain injury in vivo.

Activation of swelling-induced Cl- current (ICl,swell) during neonatal hypoxia-ischemia (HI) may induce brain damage. Hypoxic-ischemic brain injury causes chronic neurological morbidity in neonates as well as acute mortality. In this study, we investigated the role of ICl,swell in hypoxic-ischemic brain injury using a selective blocker, 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB). In primary cultured cortical neurons perfusion of a 30% hypotonic solution activated ICl,swell, which was completely blocked by the application of DCPIB (10 μmol/L). The role of ICl,swell in neonatal hypoxic-ischemic brain injury in vivo was evaluated in a modified neonatal hypoxic-ischemic brain injury model. Before receiving the ischemic insult, the mouse pups were injected with DCPIB (10 mg/kg, ip). We found that pretreatment with DCPIB significantly reduced the brain damage assessed using TTC staining, Nissl staining and whole brain imaging, and improved the sensorimotor and vestibular recovery outcomes evaluated in neurobehavioural tests (i.e. geotaxis reflex, and cliff avoidance reflex). These results show that DCPIB has neuroprotective effects on neonatal hypoxic-ischemic brain injury, and that the ICl,swell may serve as a therapeutic target for treatment of hypoxic-ischemic encephalopathy.

Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation

Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4′-diisothiocya-natostilbene-2,2′- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl− channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl− channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM.

Inhibition of gastric H+,K+-ATPase by 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), an inhibitor of volume-regulated anion channel

4-(2-Butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB) has been used as an inhibitor of volume-regulated anion channel (VRAC), which is expressed in almost all cells (IC50 is around 4µM). Here, we found that DCPIB significantly inhibited the activities of gastric proton pump (H+,K+-ATPase) in isolated gastric tubulovesicles and the membrane sample of the H+,K+-ATPase-expressing cells, and their IC50 values were around 9µM. In the tubulovesicles, no significant expression of leucine rich repeat containing 8 family member A (LRRC8A), an essential component of VRAC, was observed. The inhibitory effect of DCPIB was also found in the membrane sample obtained from the cells in which LRRC8A had been knocked down. On the other hand, DCPIB had no significant effect on the activity of Na+,K+-ATPase or Ca2+-ATPase. In the H+,K+-ATPase-expressing cells, DCPIB inhibited the 86Rb+ transport activity of H+,K+-ATPase but not that of Na+,K+-ATPase. DCPIB had no effect on the activity of Cl− channels other than VRAC in the cells. These results suggest that DCPIB directly inhibits H+,K+-ATPase activity. DCPIB may be a beneficial tool for studying the H+,K+-ATPase function in vitro.

Volume-sensitive outwardly rectifying Cl(-) channels contribute to butyrate-triggered apoptosis of murine colonic epithelial MCE301 cells.

Butyrate is a fatty acid with an important role in the maintenance of colonic homeostasis. It induces apoptosis in colonic epithelial cells and contributes to the pathogenesis of ulcerative colitis. However, just how how butyrate triggers apoptosis is poorly understood. In our model system of mouse colonic epithelial MCE301 cells, whole-cell patch-clamp recordings revealed the presence of swelling-activated outwardly rectifying chloride ion (Cl(-)) currents. These currents exhibited time-dependent inactivation upon strong depolarization, a low field strength anion selectivity (I(-) > Br(-) > Cl(-) > F(-)), and a sensitivity to 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), a specific blocker of volume-sensitive outwardly rectifying (VSOR) Cl(-) channels. The results of flow cytometric analysis and caspase 3/7 assays demonstrated that exposure of MCE301 cells to sodium butyrate for 16 h triggered apoptotic cell shrinkage, phosphatidylserine exposure, and caspase 3/7 activation. Importantly, the VSOR Cl(-) channel blocker was able to inhibit sodium butyrate-induced apoptotic processes. These results suggest that activation of the VSOR Cl(-) channel is essential for sodium butyrate-triggered apoptosis in MCE301 cells.

Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt.

Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl− currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl− channel by 4,4'-diisothiocya-natostilbene-2,2'-disulfonic acid (DIDS), a non-selective Cl− channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl− channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl− channel blockers against ER stress-associated cardiac anomalies.

Modulation of Staurosporine-Activated Volume-Sensitive Outwardly Rectifying Cl- Channel by PI3K/Akt in Cardiomyocytes

Chloride (Cl⁻) channels participate in the regulation of cardiac function in response to stress although the underlying regulatory mechanism remains poorly understood. This study was designed to examine the impact of the pro-apoptotic stimulus staurosporine (STS) on the volume-sensitive outwardly rectifying Cl⁻ current (I(Cl,Vol)) in cardiomyocytes and possible regulatory mechanism involved with a focus on phosphatidylinositol-3 kinase (PI3K)/Akt. Primary cultured rat neonatal cardiomyocytes were subjected to hypotonic and isotonic environment in the presence or absence of STS prior to whole-cell voltage-clamp evaluation of Cl⁻ current. Whole-cell recordings revealed that STS activated an outwardly rectifying Cl⁻ current with phenotypic properties reminiscent of I(Cl,Vol). These currents were outwardly rectifying with a time-dependent inactivation at positive potentials and were sensitive to 4,4'-diisothiocya-natostilbene- 2,2'- disulfonicacid (DIDS), a non-selective Cl⁻ channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl)oxybutyric acid (DCPIB), a selective VSOR Cl⁻ channel blocker. DIDS and DCPIB inhibited I(Cl,Vol) by 92.6% ± 7.3% and 78.4% ± 5.5%, respectively. Our data further revealed that the PI3K inhibitor LY294002 facilitated the current with the peak amplitude of 19.54 ± 2.70 pA/pF. To the contrary, insulin partially inhibited the current amplitude with the peak current amplitude of 15.4 ± 2.13 pA/pF. Taken together, our data depicted staurosporine is capable of activating I(Cl,Vol) channel in cardiomyocytes via possibly a PI3K/Akt-dependent mechanism.

Bradykinin‐induced astrocyte–neuron signalling: glutamate release is mediated by ROS‐activated volume‐sensitive outwardly rectifying anion channels

Glial cells release gliotransmitters which signal to adjacent neurons and glial cells. Previous studies showed that in response to stimulation with bradykinin, glutamate is released from rat astrocytes and causes NMDA receptor-mediated elevation of intracellular Ca(2+) in adjacent neurons. Here, we investigate how bradykinin-induced glutamate release from mouse astrocytes signals to neighbouring neurons in co-cultures. Astrocyte-to-neuron signalling and bradykinin-induced glutamate release from mouse astrocytes were both inhibited by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. Glutamate release was also sensitive to 4-(2-Butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB), a specific blocker of the volume-sensitive outwardly rectifying anion channel (VSOR). Astrocytes, but not neurons, responded to bradykinin with activation of whole-cell Cl- currents. Although astrocytes stimulated with bradykinin did not undergo cell swelling, the bradykinin-activated current exhibited properties typical of VSOR: outward rectification, inhibition by osmotic shrinkage, sensitivity to DIDS, phloretin and DCPIB, dependence on intracellular ATP, and permeability to glutamate. Bradykinin increased intracellular reactive oxygen species (ROS) in mouse astrocytes. Pretreatment of mouse astrocytes with either a ROS scavenger or an NAD(P)H oxidase inhibitor blocked bradykinin-induced activation of VSOR, glutamate release and astrocyte-to-neuron signalling. By contrast, pretreatment with BAPTA-AM or tetanus neurotoxin A failed to suppress bradykinin-induced glutamate release. Thus, VSOR activated by ROS in mouse astrocytes in response to stimulation with bradykinin, serves as the pathway for glutamate release to mediate astrocyte-to-neuron signalling. Since bradykinin is an initial mediator of inflammation, VSOR might play a role in glia-neuron communication in the brain during inflammation.

Further characteristics of hyposmotically activated K loss in human lens epithelial cells (hLECs)

Regulatory volume decrease of hLECs in hyposmotic (150 mOsM) media involves KCa3.1 (IK) channel activation and cell water loss. High external K and clotrimazole (CTZ), but not a variety of anion channel blockers, were inhibitory. Extending these findings, we studied the selective IK channel blocker (TRAM‐34 [1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole]), and additional anion channel blockers used in other systems, especially DIOA {[(dihydro‐indenyl)oxy]alkanoic acid]}, phloretin and DCPIB [4‐2(butyl‐6,7‐dichloro‐2‐cyclopentylindan‐1‐on‐5‐yl)oxybutyric acid]. Ouabain‐ and bumetanide‐insensitive K loss and Rb influx were measured in hLECs (FHL124) by atomic absorption spectrophometry. TRAM‐34 (>50 μM) reduced K loss by ~ 30% with little effects on Rb influx, an asymmetry contrasting with the total inhibition of both fluxes by CTZ, also an inhibitor of P450, suggesting at least two different mechanisms either at the K channel and/or regulatory levels. Phloretin (0.25–1 mM) reduced Rb influx by ~60%, less affecting K loss. In Cl or NO3, 300 μM DIOA effectively inhibited K loss, but not Rb influx, a concentration tenfold higher than used to block K‐Cl cotransport. DCPIB (50 μM) mainly reduced K‐loss. Hence, hyposmotic K loss, through putative IK channels in hLECs, is associated with and/or rate‐limited by Cl loss through DIOA‐and phloretin‐sensitive anion channels whose nature awaits elucidation.

Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.

Characterization of selective estrogen receptor modulator (SERM) activity in two triarylethylene oxybutyric acids

Previously we identified 4-[1-(4-hydroxyphenyl)-2-phenylbuten-1-yl]phenoxy- n-butyric acid (4HBA) and its des-hydroxy analog (BA) as potential selective estrogen receptor modulators (SERMs) in the ovariectomized (OVX) rat. The aim of the present study was to characterize comprehensively the effects of 4HBA and BA in both the OVX rat and in estrogen-responsive cells. Thus, 4HBA was found to be an estrogen antagonist with partial agonist efficacy in estrogen-responsive reporter gene and estrogen-dependent proliferation assays (MVLN cells and MCF-7 human breast cancer cells, respectively). In the OVX rat, 4HBA and BA were equally effective and comparable to other known SERMs regarding (a) serum cholesterol reduction and suppression of serum markers of excessive bone metabolism, and (b) partial agonist efficacy in reproductive tissue relative to steroidal estrogens. Like steroidal estrogens, both compounds increased serum triglyceride levels, with BA being more effective in this regard. The maximal effects of 4HBA on all of these parameters except cholesterol lowering were seen at oral doses of 0.4 μmol/kg/day; maximal cholesterol lowering required doses of 10 μmol/kg/day. In OVX rat liver 9S fraction, BA was found to be efficiently converted to a single hydroxylated metabolite, 4HBA. These results suggest that the effects of BA in the OVX rat might, in part, be a consequence of biotransformation to 4HBA, and that those of 4HBA and BA in the OVX rat and in estrogen-responsive cells are qualitatively similar to those of SERMs such as tamoxifen and raloxifene.

Identification of new triarylethylene oxyalkanoic acid analogues as bone selective estrogen mimetics

Previously, the estrogen receptor (ER) ligand 4-[1-( p-hydroxyphenyl)-2-phenylethyl]phenoxyacetic acid ( 5) was found to have differential bone loss suppressive effects in the ovariectomized (OVX) rat approaching those of selective ER modulators (SERMs) such as tamoxifen. In an effort to improve efficacy, analogues of this compound were prepared which incorporated features designed to reduce polarity/ionizability. Thus, the acetic acid side chain of 5 was replaced by n-butanoic acid and 1 H-tetrazol-4-ylmethyl moieties, to give 8 and 10, respectively. Also, the phenolic hydroxyl of 5 was replaced, giving deoxy analogue 9. We also developed new methods for the synthesis of triarylethylene variants of 5 and 9, namely 4-{[1-( p-hydroxyphenyl)-2-phenyl-1-butenyl]phenoxy}- n-butanoic acid ( 6) and its des-hydroxy counterpart ( 7), because the former of these had in vitro antiestrogenic effects characteristic of known SERMs. In the OVX rat, 6 and 7 were as effective as 17β-estradiol in suppressing serum markers of bone resorption/turnover, namely osteocalcin and deoxypyridinoline, but had only 30% of the uterotrophic efficacy of 17β-estradiol. This study has thus identified two triarylethylene oxybutyric acids, 6 and 7, that have differential bone/uterus effects like those of known SERMs.

Oxidation of glycosphingolipids under basic conditions: synthesis of glycosyl "serine acids" as opposed to "ceramide acids". Precursors for neoglycoconjugates with increased ligand binding affinity.

Two types of oxidative cleavage of the double bond of glycosphingolipids (GSLs) are described. Oxidation of peracetylated GSL precursors with stoichiometric proportions of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system, gave nearly quantitative yields of the glycosyl ceramide acid, 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)oxybutyric acid [Mylvaganam, M., and Lingwood, C. A. (1999) J. Biol. Chem. 274, 20725-20732]. However, if the reaction medium was made alkaline, the hydroxyallylic function of the sphingolipid, as a whole, was oxidized and the glycosyl serine acid, 2-(N-acyl)-3-(O-glycosyl)oxypropionic acid, was obtained in good yield. This represents a new type of oxidation reaction. Optimized conditions gave glycosyl ceramide or serine acids with greater than 90% selectivity and in good yields (90%). Oxidation of dGSLs gave serine and ceramide oligosaccharides, devoid of hydrocarbon chains. An intriguing glycosyl species containing 5-hydroxy-4-oxo-3-hydroxy-2-(N-acyl)sphingosine (hydroxy-acyl intermediate) was identified via ESMS analyses. We propose that further oxidation of this intermediate is pH-dependent and will be oxidized to either serine or ceramide acids. On the basis of MS-MS analysis of specific homologues of serine and ceramide acids, two types of collision-induced dissociation (CID) patterns have been established. These CID patterns were then used in the identification of serine and ceramide acids synthesized from natural GSL samples. Also, on a qualitative basis, this oxidation protocol, in conjunction with ESMS, provides a novel method for characterizing the aglycone composition (acyl chain length, unsaturation position, dihydrosphingosine content, etc.) of natural GSLs. A novel class of neohydrocarbon conjugates were synthesized by coupling the acids to rigid hydrocarbon frames such as 2-aminoadamantane. Preliminary studies with conjugates derived from globotriaosyl ceramide (Gb3C), lactosyl ceramide (LC), and galactosyl ceramide (GalC) bound verotoxin with the expected specificity but with affinities much greater than that of the natural glycolipid. Also, the ceramide acid-based conjugates were better ligands than serine acid conjugates.

Carboxylic acid analogues of tamoxifen: (Z)-2-[p-(1, 2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine. Estrogen receptor affinity and estrogen antagonist effects in MCF-7 cells.

The triarylethylene estrogen mimetic (E, Z)-4-[1-(p-hydroxyphenyl)-2-phenyl-1-butenyl]phenoxyacetic acid (4) represents a novel class of estrogen receptor (ER) ligands which, like tamoxifen (1), can elicit estrogen agonist and antagonist effects, in turn, in nonreproductive and reproductive tissues. Analogues of 4, incorporating structural features shown previously in triarylethylenes to improve ER affinity and estrogen antagonist properties, were prepared with the ultimate aim of identifying substances with improved estrogenicity exclusive of reproductive tissues. Thus, the side chain of 4 was elongated to give oxybutyric acid 13, which was further altered by (a) repositioning of its p-hydroxyl to the neighboring m-position (12) and (b) ethylenic bond reduction (14). Also, the p-hydroxyl group and oxyacetic acid groups of 4 were, in turn, shifted to the neighboring m-positions, affording 8 and 9. Oxybutyric acid analogue 13 had about 2 times the affinity for human ERalpha as 4, and its antiproliferative effect in MCF-7 cells was greater than that of 1. Dihydro analogue 14, which was conformationally similar to cis-13, had very low ER affinity and antiestrogenicity, and m-hydroxy analogue 12 also had reduced ER affinity and potency, but its MCF-7 cell antiproliferative efficacy was retained. Modest ER affinity and antiproliferative potency were seen with 8, in which phenolic and phenyl rings were trans to one another, but 9, in which these rings were cis, was inactive. Our findings indicate that two-carbon side-chain elongation and/or m-positioning of the hydroxyl group in 4 affords analogues with dominant estrogen antagonist effects in MCF-7 cells.

A Convenient Oxidation of Natural Glycosphingolipids to Their “Ceramide Acids” for Neoglycoconjugation: BOVINE SERUM ALBUMIN-GLYCOSYLCERAMIDE ACID CONJUGATES AS INVESTIGATIVE PROBES FOR HIV gp120 COAT PROTEIN-GLYCOSPHINGOLIPID INTERACTIONS

A new method to cleave the double bond of sphingolipids has been developed. Using limited concentrations of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system gave nearly quantitative yields of the oxidized product. A variety of natural glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb3C, Gb4C, Gg4C, Gb5C, and GM1C, gave the corresponding acids: 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. "glycosyl ceramide acids" (GSL.CCOOH) in excellent yields (80-90%). Deacyl GSLs (dGSLs) were oxidized to acids containing the oligosaccharides devoid of hydrocarbon chains, i.e. "ceramide oligosaccharides" (dGSL. NRR1CCOOH, where R = R1 = H; R = H, R1 = CH3CO; or R = R1 = Me). The efficacy of this method was demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC, Gb3C, and Gb4C into neoglycoproteins via coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of GalC-BSA conjugates, (GalC.CONH)nBSA and (GalS.NHCO)nBSA gave a value of 9 +/- 1 and 16 +/- 2 for n. Neoglycoconjugates derived from GlcC, GalC (type I and II and the behenic analog), SGC, LC, and Gb3C were recognized by the recombinant human immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate showed significantly reduced binding, and the Gb4C conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires a terminal galactose and/or glucose residue. Terminal N-acetylgalactosamine containing GSLs are not bound. The ceramide acid conjugates provide a more effective scaffold for presentation of glycone for rgp120 binding than those derived from dGSLs. The retention of receptor specificity of the glycoconjugates was validated by retention of the expected binding specificity of VT1 and VT2e for Gb3C and Gb4C conjugates, respectively. These studies open a new vista in the generation of glycoconjugates from GSLs and further emphasize the role of aglycone in glycolipid recognition.

CHEMOTHERAPY IN NONSPECIFIC INFECTIONS OF THE URINARY TRACT

During the last ten years there has been remarkable advancement in diagnostic and operative procedures in urology. Revolutionary changes in the treatment of infections of the urinary tract now command our attention. Ten years ago the urologist was content to treat these infections expectantly or by administering drugs now believed to be of questionable efficacy. Intravenous neoarsphenamine in the treatment of coccic infection was perhaps the only specific drug therapy. In 1932 the ketogenic diet with the discovery of the bactericidal action of oxybutyric acid was the first step toward specific therapy in bacillus infections. However, many patients could not tolerate this unappetizing, nauseating diet. The following year specific bacteriophage was tried, but this met with very little success. In 1936 the first specific drug therapy, mandelic acid, was instituted in the treatment of infections of the urinary tract. It is bactericidal for most infections with Streptococcus faecalis and from

RATE OF EXCRETION AND BACTERICIDAL POWER OF MANDELIC ACID IN THE URINE

Last year we1presented data to show the minimal concentration of oxybutyric acid in urine and the optimalpHof urine that were necessary to cause sterilization of urine that had been inoculated with various strains of organisms isolated from urine. This acid results from the incomplete combustion of fats when only extremely small amounts of carbohydrate are given. The so-called ketogenic diet is difficult for many people to take. Recent work originating in England and pioneered by Rosenheim2has resulted in the knowledge that mandelic acid, when administered orally, escapes metabolism or conjugation in the animal organism and is excreted in the urine by the kidney in a concentration sufficient for bactericidal action, provided thepHof the urine is lowered simultaneously. This acid was chosen as a result of experiments with many organic acids. Methods for sterilization of urine in cases of infection of

Alternating Behavior of Fatty Acids in Rubber Compounds

Abstract It has been shown (Bayer, Ber., 10, 1286 (1877); Garner and Randall, J. Chem. Soc., 125, 881 (1924)) that the alternations in the melting points and heats of crystallization of normal fatty acids were due to differences in the crystalline structure. Two distinct curves of behavior corresponding to the acids with odd and even numbers of carbon atoms have been shown for the heats of crystallization. McKee (Naturwissenschaften, 11, 938 (1923)) discussing the fats used in the diet of diabetics, states that in the case of glycerides of acids with an uneven number of carbon atoms these are tolerated and their use leads to gradual reduction in acidosis. Glycerides having an even number of carbon atoms break down during metabolism to form butyric, oxybutyric, and acetoacetic acids. Smith and Boone (Ind. Eng. Chem., 19, 398 (1927)) have experimented with the addition of purified fatty acids to standard mixings of different kinds of raw rubber. They employed molar proportions of the acid to conform with the recommendations of Kratz, Flower, and Coolidge (Ind. Eng. Chem., 12, 317 (1920)) that comparisons of organic accelerators should be made with molecularly equivalent amounts of the substances in question. They determined the tensile strength and elongation of the vulcanized products and found no relations between the elongation and the number of carbon atoms present in the acids, but in the case of the tensile strength an alternating effect was evident, the extent of the differences being affected by cure. The tests were carried out on two kinds of rubber, namely, blanket crepe and pale crepe, and the alternating effect of the acids on the tensile strength was more marked in the former case. As pointed out in the paper, the difference in behavior of the two kinds of rubber when treated with fatty acids was presumably due to the difference in the kind and proportions of acids which they originally contained. In fact, in the case of pale crepe the fatty acids already present in the raw rubber masked the alternating behavior to a marked extent, and the curve obtained was in most respects quite different from that obtained with blanket crepe.

SIMPLE DIET CHARTS FOR DIABETES OF ALL GRADES OF SEVERITY

Diabetes is characterized by decreased glucose-oxidizing capacity. Treatment consists in diminishing the glucose supply and increasing the glucose-oxidizing capacity until equilibrium is reestablished between the work to be done and the capacity to do it. Decreased glucose metabolism lowers fat-oxidizing capacity, excess fat being oxidized only as far as oxybutyric acid, diacetic acid, and acetone; treatment includes, therefore, limiting the fat intake to the fat-oxidizing capacity. Diabetic therapeutics implies quantitative understanding of glucose and fat sources; and a knowledge of the glucose and fat oxidizing capacity and the requirements for maintenance in the case under treatment. GLUCOSE SOURCES A totally diabetic animal—after pancreatectomy, or phlorizin administration—cannot oxidize any glucose; all that is formed is excreted. Any carbohydrate administered to such an animal appears in the urine as glucose: G = C, where G indicates the amount of glucose formed, and C the amount of carbohydrate administered. When such an animal is