Deletion of the Hsp70 chaperone gene SSB causes hypersensitivity to guanidine toxicity and curing of the [PSI+] prion by increasing guanidine uptake in yeast.

Yeast Ssb proteins (Ssbp) are ribosome-associated Hsp70 chaperones that function in translation. Elevated levels of Ssbp enhance the ability of over-expressed Hsp104 chaperone to eliminate the yeast [PSI+] prion, while depletion of Ssbp reduces this effect. Millimolar concentrations of guanidine in the growth medium cure yeast cells of prions by inactivating Hsp104.

Nuclear pore complexes exceeding eightfold rotational symmetry.

Nuclear pore complexes are rotationally symmetric structures that span the nuclear envelope and provide channels for nucleocytoplasmic traffic. These large complexes normally consist of eight spokes arranged around a central channel, although, occasionally, 9- and 10-fold nuclear pore complexes are found in preparations of Xenopus oocyte macronuclei. Here we examine these unusual nuclear pore complexes by negative stain electron microscopy and image analysis and compare the results with data previously obtained from 8-fold structures.

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review.

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors.

Practical and high-yield syntheses of dihydromorphine from tetrahydrothebaine and efficient syntheses of (8S)-8-bromomorphide.

A practical method for the conversion of tetrahydrothebaine to dihydromorphine in 92% yield is described. The procedure should allow more efficient production of opium products and may be easily modified for large-scale synthesis. The conversion of codeine to (8S)-8-bromomorphide, a potentially valuable intermediate to 6-demethoxyoripavine and derivatives, is also described.

Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene.

O-Linked N-acetylglucosamine (GlcNAc) transferase (OGT) mediates a novel hexosamine-dependent signal transduction pathway. Yet, little is known about the regulation of ogt gene expression in mammals. We report the sequence and characterization of the mouse ogt locus and provide a comparison with the human and rat ogt genes.

Loss of interleukin 6 results in delayed mammary gland involution: a possible role for mitogen-activated protein kinase and not signal transducer and activator of transcription 3.

We have investigated the role of IL-6 in the initiation and progression of mouse mammary gland involution in IL-6-null mice. This study was based on the hypothesis that IL-6 is the activating cytokine for signal transducer and activator of transcription 3 (Stat), the transcription factor whose presence is required for controlled mammary gland involution. We now show that expression of IL-6 is low during lactation but increases at the onset of involution in parallel with the activation of Stat3 and p44/42 MAPK.

Mammary epithelial cells are not able to undergo pregnancy-dependent differentiation in the absence of the helix-loop-helix inhibitor Id2.

Mammary alveolar development during pregnancy is triggered by hormone signals. The prolactin receptor/Jak2/signal transducer and activator of transcription (Stat) 5 signal transduction pathway is the principal mediator of these cues and alveolar development is abrogated in its absence. The loss of the basic helix-loop-helix protein inhibitor of differentiation (Id)2 results in a similar defect.

Chromosome cohesion: ring around the sisters?

Sister chromatid cohesion is a key aspect of accurate chromosome transmission during mitosis, yet little is known about the structure of cohesin, the protein complex that links the two sister chromatids. Recent studies shed light on the structure of the cohesin complex, leading to intriguing models that could explain how sister chromatids are held together.

Second generation adeno-associated virus type 2-based gene therapy systems with the potential for preferential integration into AAVS1.

Adeno-associated virus type 2 (AAV-2) is a non-pathogenic human parvovirus that is being developed as a gene therapy vector for the treatment of numerous diseases. One property of wild-type AAV-2, that is highly desirable in a gene therapy vector, is its ability to preferentially integrate its DNA into a 4 kilobase region of human chromosome 19, designated AAVS1. One disadvantage of AAV-2 is its relatively small packaging capacity, approximately 4.

Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor.

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII.

CRHR1 Receptor binding and lipophilicity of pyrrolopyrimidines, potential nonpeptide corticotropin-releasing hormone type 1 receptor antagonists.

A series of compounds related to N-butyl-N-ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amine (1, antalarmin) have been prepared and evaluated for their CRHR1 binding affinity as the initial step in the development of selective high affinity hydrophilic nonpeptide corticotropin-releasing hormone type 1 receptor (CRHR1) antagonists. Calculated log P (Clog P) values were used to evaluate the rank order of hydrophilicity for these analogues. Introducing oxygenated functionalities (delta-hydroxy or bis-beta-ethereal) into 1 gave more hydrophilic compounds, which had good affinity for the receptor.

A novel Rtg2p activity regulates nitrogen catabolism in yeast.

The inactivity of Ure2p, caused by either a ure2 mutation or the presence of the [URE3] prion, increases DAL5 transcription and thus enables Saccharomyces cerevisiae to take up ureidosuccinate (USA+). Rtg2p regulates transcription of glutamate-repressible genes by facilitation of the nuclear entry of the Rtg1 and Rtg3 proteins. We find that rtg2 Delta cells take up USA even without the presence of [URE3].

Three-dimensional reconstruction of dynamin in the constricted state.

Members of the dynamin family of GTPases have unique structural properties that might reveal a general mechanochemical basis for membrane constriction. Receptor-mediated endocytosis, caveolae internalization and certain trafficking events in the Golgi all require dynamin for vesiculation. The dynamin-related protein Drp1 (Dlp1) has been implicated in mitochondria fission and a plant dynamin-like protein phragmoplastin is involved in the vesicular events leading to cell wall formation.

Dynamin family of mechanoenzymes.

The dynamin family of proteins is continually growing, and in recent years members have been localized to areas of mitochondrial fission, plant phragmoplasts and chloroplasts, and viral ribonucleoprotein complexes. All the dynamin-like proteins examined to-date appear to assemble into oligomers, such as rings or spirals; however, it remains to be determined if a global mechanism of action exists. Even the role of dynamin in vesicle formation remains controversial as to whether it behaves as a molecular switch or as a mechanochemical enzyme.

A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins.

The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs).

Sister chromatid separation: falling apart at the seams.

Cohesion between sister chromatids must be dissolved at the time of chromosome segregation. Recent studies reveal that the principles of cohesion dissolution in mitosis and meiosis are the same, but that there are important differences that stem from the distinct natures of these two processes.

Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis and regulation.

Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mutations in the human enzyme. The mutations, which lead to accumulation of L-homocysteine, are linked to homocystinuria and cardiovascular diseases. Here we characterize the domain architecture of the heme-independent yeast cystathionine beta-synthase.

3' poly(A) is dispensable for translation.

In wild-type cells, the 3' poly(A) structure is necessary for translation of mRNA and for mRNA stability. The superkiller 2 (ski2), ski3, ski6, ski7, and ski8 mutations enhance the expression of the poly(A)(-) mRNAs of yeast RNA viruses. Ski2p is a DEVH-box RNA helicase and Slh1p resembles Ski2p.

A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein.

Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt-Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system.

Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from Arrhenius plots, absorption spectra, and primary kinetic isotope effects.

To investigate the linkage between enzyme conformation and catalysis, we have determined the effects of temperature on catalytic properties of the tryptophan synthase alpha(2)beta(2) complex and beta(2) subunit in the absence or presence of different monovalent cations (Cs(+), Na(+), and GuH(+)) and of an allosteric ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data between 5 and 50 degrees C are nonlinear in the presence of certain ligands but not others. The conditions that yield nonlinear Arrhenius plots also yield temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects.

Characterization of novel N,N'-disubstituted piperazines as sigma receptor ligands.

sigma Receptors have been the focus of extensive studies because of their potential functional role in several important physiological and biochemical processes. To further evaluate the properties of sigma receptors, especially sigma-1 and sigma-2 subtypes, we have synthesized a series of N,N'-disubstituted piperazine compounds (1-32). The design of these compounds was based upon the early structure-activity relationship (SAR) studies of the minimum structural requirements of a molecule necessary to elicit sigma receptor binding activity.

Nuclear glycogen and glycogen synthase kinase 3.

Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS.

The prion model for [URE3] of yeast: spontaneous generation and requirements for propagation.

The genetic properties of the non-Mendelian element, [URE3], suggest that it is a prion (infectious protein) form of Ure2p, a mediator of nitrogen regulation in Saccharomyces cerevisiae. Into a ure2Delta strain (necessarily lacking [URE3]), we introduced a plasmid overproducing Ure2p. This induced the frequent "spontaneous generation" of [URE3], with properties identical to the original [URE3].