Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled α-Factor Receptor

The α-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the α-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the α-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser²⁵⁴ showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln²⁵³ and Ser²⁵⁴ are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln²⁵³ and Ser²⁵⁴ fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln²⁵³ and Ser²⁵⁴ face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser²⁸⁸ and Ser²⁹²) that are potential contact residues for Gln²⁵³ because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the α-factor receptor that regulates its ability to enter the activated conformation.     

Characterization of the oligosaccharide alditols from ovarian cyst mucin glycoproteins of blood group A using high pressure liquid chromatography (HPLC) and high field 1H NMR spectroscopy

A combination of reverse phase and normal phase high pressure liquid chromatography has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of a blood group A ovarian cyst mucin glycoproteins. Fourteen compounds, ranging in size from a monosaccharide to a decasaccharide, have been isolated preparatively using a Zorbax C-18 reverse phase column eluted with water and a MicroPak AX-5 normal phase column eluted with aqueous acetonitrile. The purity of the products and their structures were determined from the fully assigned high field proton NMR spectra. The resonances of exchangeable amide protons, observed by the Redfield selective pulse sequence in H2O, were assigned by decoupling to the resonances of H2 of the 2-acetamido sugars. Nuclear Overhauser effects were used to establish the relationship of the anomeric protons and those of the aglycone. In exception to earlier proposals that nuclear Overhauser effect on irradiation of the anomeric proton should always be observed at the proton attached to the aglycone carbon, we find that for the linkage of GalNAcp(1----3)Gal, nuclear Overhauser effect on irradiation of the alpha-anomeric proton resonance is observed not at H3 but at H4 of galactose. A combination of NMR methods and enzymatic degradation was employed to determine the structures of 13 different oligosaccharides of which seven have not previously been reported. These oligosaccharides, which terminate with beta-Gal, alpha-Fuc, beta-GlcNAc, and alpha-GalNAc, account for 75% of the total glycoprotein carbohydrate, the remainder being isolated as a mixture of glycopeptides and a high molecular weight polysaccharide whose NMR spectrum implies a simple repeating subunit structure closely related to that of the oligosaccharides.     

Interaction between transmembrane domains five and six of the alpha -factor receptor

The alpha-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae. Previous studies implicated the third intracellular loop of this receptor in G protein activation. Therefore, the roles of transmembrane domains five and six (TMD5 and -6) that bracket the third intracellular loop were analyzed by scanning mutagenesis in which each residue was substituted with cysteine. Out of 42 mutants examined, four constitutive mutants and two strong loss-of-function mutants were identified. Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes. Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C, L248C, or S251C mutations in TMD6. Also, the L226C mutation in TMD5 caused constitutive activity when combined with either the M250C or S251C mutations in TMD6. The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact. In support of this, cysteines substituted at position 223 in TMD5 and position 247 in TMD6 formed a disulfide bond, providing the first direct evidence of an interaction between these transmembrane domains in the alpha-factor receptor. Altogether, these results identify an important region of interaction between conserved hydrophobic regions at the base of TMD5 and TMD6 that is required for the proper regulation of receptor signaling.     

Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans

BackgroundApproximately 8% of the human genome consists of sequences of retroviral origin, a result of ancestral infections of the germ line over millions of years of evolution. The most recent of these infections is attributed to members of the human endogenous retrovirus type-K (HERV-K) (HML-2) family. We recently reported that a previously undetected, large group of HERV-K (HML-2) proviruses, which are descendants of the ancestral K111 infection, are spread throughout human centromeres.ResultsStudying the genomes of certain cell lines and the DNA of healthy individuals that seemingly lack K111, we discover new HERV-K (HML-2) members hidden in pericentromeres of several human chromosomes. All are related through a common ancestor, termed K222, which is a virus that infected the germ line approximately 25 million years ago. K222 exists as a single copy in the genomes of baboons and high order primates, but not New World monkeys, suggesting that progenitor K222 infected the primate germ line after the split between New and Old World monkeys. K222 exists in modern humans at multiple loci spread across the pericentromeres of nine chromosomes, indicating it was amplified during the evolution of modern humans.ConclusionsCopying of K222 may have occurred through recombination of the pericentromeres of different chromosomes during human evolution. Evidence of recombination between K111 and K222 suggests that these retroviral sequences have been templates for frequent cross-over events during the process of centromere recombination in humans.     

High-Dose Benzodiazepine Dependence: A Qualitative Study of Patients’ Perceptions on Initiation,Reasons for Use,and Obtainment

Background

High-dose benzodiazepine (BZD) dependence is associated with a wide variety of negative health consequences. Affected individuals are reported to suffer from severe mental disorders and are often unable to achieve long-term abstinence via recommended discontinuation strategies. Although it is increasingly understood that treatment interventions should take subjective experiences and beliefs into account, the perceptions of this group of individuals remain under-investigated.

Methods

We conducted an exploratory qualitative study with 41 adult subjects meeting criteria for (high-dose) BZD-dependence, as defined by ICD-10. One-on-one in-depth interviews allowed for an exploration of this group’s views on the reasons behind their initial and then continued use of BZDs, as well as their procurement strategies. Mayring’s qualitative content analysis was used to evaluate our data.

Results

In this sample, all participants had developed explanatory models for why they began using BZDs. We identified a multitude of reasons that we grouped into four broad categories, as explaining continued BZD use: (1) to cope with symptoms of psychological distress or mental disorder other than substance use, (2) to manage symptoms of physical or psychological discomfort associated with somatic disorder, (3) to alleviate symptoms of substance-related disorders, and (4) for recreational purposes, that is, sensation-seeking and other social reasons. Subjects often considered BZDs less dangerous than other substances and associated their use more often with harm reduction than as recreational. Specific obtainment strategies varied widely: the majority of participants oscillated between legal and illegal methods, often relying on the black market when faced with treatment termination.

Conclusions

Irrespective of comorbidity, participants expressed a clear preference for medically related explanatory models for their BZD use. We therefore suggest that clinicians consider patients’ motives for long-term, high-dose BZD use when formulating treatment plans for this patient group, especially since it is known that individuals are more compliant with approaches they perceive to be manageable, tolerable, and effective.     

Perigestational suppression of weight gain with central leptin gene therapy results in lower weight F1 generation

The efficacy of central leptin therapy on weight homeostasis through various phases of reproduction, pregnancy outcome and postnatal, prepubertal and pubertal growth of offspring was assessed. Enhanced leptin transgene expression after a single intracerebroventricular injection of recombinant adeno-associated virus vector encoding the leptin gene (rAAV-lep) decreased calorie intake and weight in adult nulliparous female rats. rAAV-lep treated rats conceived normally, displayed unremarkable pregnancy rate, parturition and delivered normal sized litters. Significantly lower weight was maintained through gestation, lactation, and post-lactation periods. The maintenance of a modest weight reduction was accompanied by voluntarily reduced calorie intake, increased thermogenic energy expenditure, decreased adiposity as reflected by drastically reduced leptin levels, and suppressed insulin and insulin-like growth factor 1 levels through lactation and post-lactation in rAAV-lep treated dams. The offspring at birth weighed significantly less than those of controls and this lower weight range was sustained during postnatal, prepubertal, pubertal and adult (3 months old) periods, contemporaneous with metabolic circulating hormones in the normal range. For the first time we show the persistent efficacy of central leptin gene therapy to suppress weight gain through all phases of reproduction, lactation and post-lactation in dams and reveal the potential imprinting link to producing lower weight in the F1 generation.     

N-[(3S)-1-benzylpyrrolidin-3-yl]-(2-thienyl)benzamides: human dopamine D4 ligands with high affinity for the 5-HT2A receptor

A series of N-[(3S)-1-benzylpyrrolidin-3-yl]-(2-thienyl)benzamides 8 has been prepared and found to bind with high affinity to the human D(4) (hD(4)) and 5-HT(2A) receptors. Several compounds displayed selectivity for these receptors versus hD(2) and alpha(1) adrenergic receptors of over 500-fold.