Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation.

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.

Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites.

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.

Effect of Otoconial Proteins Fetuin A, Osteopontin, and Otoconin 90 on the Nucleation and Growth of Calcite.

We investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became an inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution-substrate interfacial energy.

In vitro calcite crystal morphology is modulated by otoconial proteins otolin-1 and otoconin-90.

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.

Cyclooxygenase (COX)-1 and COX-2 participate in 5,6-epoxyeicosatrienoic acid-induced contraction of rabbit intralobar pulmonary arteries.

Epoxyeicosatrienoic acids (EETs) have been reported to contract intralobar pulmonary arteries (PA) of the rabbit in a cyclooxygenase (COX)-dependent manner. In the present study, we observed that COX-1 and COX-2 isoforms were expressed in freshly isolated PA of healthy rabbits. We examined the hypothesis that both COX isoforms participate in 5,6-EET-induced contraction of rabbit intralobar PA. Selective inhibition of COX-1 with 300 nM 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560) prevented 5,6-EET (1x10(-8)-1x10(-5) M)-induced contractions of isolated intralobar rabbit PA rings in a manner similar to that observed with the nonselective cyclooxygenase inhibitor indomethacin at 10 microM. Selective inhibition of COX-2 with either 100 nM 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DUP-697) or 3 microM N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) shifted the EC50 value of 5,6-EET-induced PA contraction to the right but with considerably lower efficacy than SC-560. In rabbit PA, 5,6-EET-induced contraction was primarily dependent on COX-1 activity. Differential metabolism of 5,6-EET by COX-1 and COX-2 does not explain the primary dependence of PA contraction on COX-1 activity because 5,6-EET was metabolized similarly by both COX isoforms. COX-1 and -2 were expressed primarily in PA endothelium where COX-1 expression was dense and uniform, whereas COX-2 expression was sparse and nonuniform. 5,6-EET-induced PA contraction was endothelium-dependent. These results suggest that 5,6-EET-induced contraction is primarily dependent on COX-1 activity.

A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors.

Cyclo-oxygenase (COX) enzymes are the targets for non-steroidal anti-inflammatory drugs (NSAIDs). These drugs demonstrate a variety of inhibitory mechanisms, which include simple competitive, as well as slow binding and irreversible inhibition. In general, most NSAIDs inhibit COX-1 and -2 by similar mechanisms. A unique class of diarylheterocyclic inhibitors has been developed that is highly selective for COX-2 by virtue of distinct inhibitory mechanisms for each isoenzyme. Several of these inhibitors, with varying selectivity, have been utilized to probe the mechanisms of COX inhibition. Results from analysis of both steady-state and time-dependent inhibition were compared. A generalized mechanism for inhibition, consisting of three sequential reversible steps, can account for the various types of kinetic behaviour observed with these inhibitors.

Psychological testing and psychological assessment. A review of evidence and issues.

This article summarizes evidence and issues associated with psychological assessment. Data from more than 125 meta-analyses on test validity and 800 samples examining multimethod assessment suggest 4 general conclusions: (a) Psychological test validity is strong and compelling, (b) psychological test validity is comparable to medical test validity, (c) distinct assessment methods provide unique sources of information, and (d) clinicians who rely exclusively on interviews are prone to incomplete understandings. Following principles for optimal nomothetic research, the authors suggest that a multimethod assessment battery provides a structured means for skilled clinicians to maximize the validity of individualized assessments. Future investigations should move beyond an examination of test scales to focus more on the role of psychologists who use tests as helpful tools to furnish patients and referral sources with professional consultation.

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

In vitro fertilisation. A review of drug therapy and clinical management.

Since the first in vitro fertilisation (IVF) pregnancy was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for the infertile couple. Pregnancy rates with IVF improve as the number of high quality embryos available for transfer increases; therefore, ovarian stimulation agents to produce multiple oocysts for IVF are advantageous. Clomifene (clomiphene citrate), human menopausal gonadotrophin (hMG; menotropins), and subsequent generations of products are commonly used as stimulation agents. In conjunction with the stimulation agents, gonadotrophin-releasing hormone (GnRH) agonists and human chorionic gonadotrophin (hCG) serve as adjuvants for successful control of all events in the induction process. Clomifene, an estrogen agonists/antagonist, occupies the estrogen receptor for a longer period of time than estrogen (weeks versus hours). Because this signal is interpreted as low estrogen, GnRH is released, which produces a rise in circulating levels of follicle-stimulating hormone (FSH) and luteinising hormone (LH) and subsequent ovarian follicular development. Menotropins is collected by passing urine from menopausal donors over a Sepharose column, followed by removal of high molecular weight impurities by chromatography. The mixture of FSH and LH is biologically standardised. This product stimulates multiple ovarian follicular development. Urofollitrophin is produced using antibodies to hCG anchored to a separation column. LH then can be excluded from the eluate by binding to the hCG antibodies (LH immunoaffinity column). Highly purified FSH is obtained by passing menopausal urine over a column with monoclonal antibodies to FSH. The isolated FSH is then eluted from the column by a highly basic solution and crystallised. This product delivers FSH at a 90% purity and can be administered subcutaneously rather than intramuscularly. Dosage is standardised on a mg/kg basis. Recombinant human FSH is completely free of LH and offers the advantages of better batch consistency, greater purity, and absence of any human contaminants. It may be given both subcutaneously and intravenously. Genetically engineered FSH combines portions of the native protein with another protein (hCG) which enhances its potency and extends the half-life compared with wild-type FSH. Short, medium and ultra-long activity analogues of genetically engineered FSH may be used to tailor stimulation protocols in various clinical situations. Growth hormone is an adjuvant to ovarian stimulation which results in a decreased number of ampoules of menotropins being required to achieve ovulation in poor responders. Ovulation triggers include both hCG and GnRH agonists. Progesterone supplementation is generally used in the luteal phase of the IVF cycle and is administered by intramuscular injection or vaginal suppository. It appears that conscious sedation with midazolam, pethidine (meperidine) and fentanyl is nontoxic for oocyte recovery. If full anaesthesia is required for gamete intrafallopian tube transfer (GIFT) or zygote intrafallopian tube transfer (ZIFT), balanced anaesthesia with nitrous oxide and an opioid appears to be the most appealing option. Appropriate information on the clinical use of the drugs used in IVF greatly reduces patient stress associated with the complex multidrug regimens associated with the procedure.

How psychological testing can reinstate its value in an era of cost containment.

Managed care methods, and the cost containment concerns from which they arose, have dramatically reduced the use of psychological testing dramatically. This article critiques that trend, and proposes how selected tests can be used cost-effectively in specific situations. The author has conducted research and written extensively on this subject, including a chapter in "The Computerization of Behavioral Healthcare: How to Enhance Clinical Practice, Management and Communications."

The Rorschach: projective technique or psychometric test?

Various approaches to the Rorschach Technique are described in terms of the idiographic-nomothetic axis and the perceptual-content axis. It is suggested that it is most productive to view the Rorschach as a projective tool, with perceptual scoring a secondary factor. Current efforts at objectification of the Rorschach are not seen as useful as efforts to enhance its projective qualities. Some possible ways are discussed in which the projective value of the instrument can be maximized.

Clinical correlates of short- and long-term weight loss.

This study was designed to identify psychological, behavioral, and physiological correlates of short- and long-term weight loss. Measures of psychological functioning, body composition, fat cell size and number, and attendance were evaluated in 76 obese women for their relationship to weight loss at the end of treatment and at a 1-y follow-up evaluation. Losing more weight during the first month of treatment and attending a higher percentage of treatment sessions were strongly associated with greater weight loss at the end of treatment and at 1-y follow-up. In addition, patients with the highest initial weights lost the most weight both at the end of treatment and at 1-y follow-up. Easily obtained measures are as successful in predicting weight loss as are more expensive and complicated measures.

Sclerosing encapsulating peritonitis in three dogs.

A condition resembling human sclerosing encapsulating peritonitis was identified in 3 young large-breed dogs. All 3 dogs had similar clinical signs: ascites, vomiting, and excessive borborygmus. The condition was discovered at laparotomy and confirmed histologically. Laparotomy revealed a sac-like formation totally or partially occupying the peritoneal cavity. The predominant histologic abnormality was marked peritoneal and serosal fibrosis. None of the dogs had a favorable response to treatment, and euthanasia was performed because of development of abdominal distention.

Hypophosphatemia. Causes and clinical consequences.

Severe hypophosphatemia (i.e., serum phosphorus concentration below 1 mg/dl) occurs infrequently in veterinary patients. It is most often associated with diabetic ketoacidosis in small animals. Phosphate is necessary for the production of 2,3 diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP); both are important for normal cellular metabolism. Consequences of severe hypophosphatemia may include hemolytic anemia, seizures, altered mentation, cardiomyopathy, and skeletal muscle weakness. Parenteral phosphate therapy is necessary in most cases of severe hypophosphatemia.

Monoaminergic substances in the teleost brain: Catecholamine levels in male and female winter flounder,Pseudopleuronectes americanus Walbaum, associated with gonadal recrudescence.

High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to quantitate catecholamine (CA) levels in the winter flounder brain following perchloric acid extraction/alumina purification of CNS tissues. Greater concentrations of norepinephrine (NE) and dopamine (DA) were present in the hypothalamus compared with the CA levels in whole brain. A seasonal study of CA brain levels in reproductively active male and female flounder demonstrated that monoamine levels reach their maxima in October in association with the rapid increases in gonadosomatic index. When perchloric acid extracts of the teleost and rat hypothalamus were submitted to direct HPLC-EC analysis, without alumina purification of CA neurotransmitters, similar hypothalamic profiles were obtained indicating the presence of identifiable biogenic amine neurotransmitters substances including NE, DA and serotonin (5-HT).

Comparative validity of the MMPI and two short forms: psychiatric ratings.

Minnesota Multiphasic Personality Inventory (MMPI) scales were used to predict psychiatric ratings in two male and two female groups of adult state psychiatric hospital inpatients. The resulting regression coefficients were cross-validated on the other same-sex group using MMPI, MMPI-168, and Faschingbauer Abbreviated MMPI (FAM) scales as predictors. The validity coefficients obtained in this manner were generally small and similar across MMPI forms. There was a trend for MMPI-based predictions to be superior to those of the short forms as the validity coefficients grew larger. This finding suggested caution in the use of the MMPI-168 and FAM as substitutes for the full MMPI. A suggestive for blending realism and rigor in future research is offered.

Conversion equations for two modified MMPI short forms.

Extracted the MMPI-168 and Faschingbauer Abbreviated MMPI (FAM), along with the K scale items normally excluded from those MMPI short forms, from the full MMPIs of about 535 psychiatric inpatients. Those scales augmented by K items were used to estimate full MMPI scores via regression and substitution equations. The equations were cross-validated on samples of about 545 from the same population. Shrinkage was minimal and cross-validation estimates compared favorably with derivation estimates from other short-form studies.