Pheromone binding proteins of the mouse,Mus musculus

Proteins of the Major Urinary Complex of the adult male mouse (Mus musculus) selectively bind the male pheromones 2-(sec-butyl)thiazoline and dehydro-exo-brevicomin, and concentrate them in urine.     

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11)

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.Received 8 December 2004; received after revision 27 January 2005; accepted 8 March 2004The nucleotide sequence for the cDNA to mouse TFIP11 (previously known as TIP39 and TIP39kDa) has been submitted to Gen- Bank^TM/ EBI Data Bank with accession numbers AF290474 and NM_018783. The accession number for the human TFIP11 homologueis NM_012143.     

Upregulation of rat P23 (a member of the YjgF protein family) by fasting, glucose diet and fatty acid feeding

In a previous study, we identified and purified a 99-amino-acid rat liver-kidney perchloric-acid-soluble 23-kDa protein (P23) which displays 30% identity with a highly conserved domain of heat shock proteins (HSPs), as well as an AT-rich 3 untranslated region, which has also been described to play a role in H70 mRNA life span and protein expression. An identical perchloric-acid-soluble protein inhibiting protein synthesis in a rabbit reticulocyte lysate system was also found 2 years later by another group. More recently, the novel, the YjgF, protein family has been described, comprising, 24 full-length homologues, including P23, highly conserved through evolution, and consisting of approximately 130 residues each and sharing a common ternary structure. Independent studies from different laboratories have provided various hypothetical functions for each of these proteins. The high degree of evolutionary conservation may suggest that these proteins play an important role in cellular regulation. Although the function of none of these proteins is known precisely, we present experimental evidence which, combined with the relationship to glucose-regulating protein revealed here, and the relationship to fatty-acid-binding protein revealed by others, allow us to propose a role for P23. In rat liver, P23 expression is developmentally regulated and modulated by dietary glucose, and its mRNA is induced by starvation, in the presence of fatty-acids and in 3-MeDAB-induced hepatomas. The mRNA encoding mouse liver P23 is also hormonally modulated in a mouse line AT1F8. These data indicate that P23 protein might be a key controller of intermediary metabolism during fasting.Received 7 June 2003; received after revision 8 September 2004; accepted 10 October 2004     

FSH receptor binding inhibiting activity associated with low molecular weight inhibin from sheep,human, rat and chicken

Summary Low molecular weight inhibin (1500 daltons) was obtained from sheep, human, rat and chicken testes by sequential chromatography on Sephadex G-100 and G-25. In addition to its ability to suppress circulating FSH levels in adult castrated male rats, it also exhibits binding inhibition of I¹²⁵hFSH to rat testicular receptors.     

Structure-function relationships of the polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation. Received 16 August 2007; received after revision 18 September 2007; accepted 2 October 2007     

Interaction of volkensin with HeLa cells: binding,uptake, intracellular localization,degradation and exocytosis

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10^-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.Received 21 April 2004; received after revision 26 May 2004; accepted 9 June 2004     

Comparison of the effects of different isomers of bicuculline infused in the preoptic area on male rat sexual behavior

Summary Intracerebral infusion of (+) bicuculline methiodide, but not of its (–) isomer, in the preoptic area, stimulated masculine sexual behavior in rat as evidenced by a decrease in the number of intromissions preceding ejaculation and a shortening of the ejaculation latency and postejaculatory interval. Data suggest a role of the GABAergic system in mediating masculine sexual behavior.Acknowledgments. Authors wish to thank Ms Elisabeth Wallin for excellent technical assistance and Ms Madelene Kröning for preparing the figures.     

The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors

Summary The mechanism of the in vitro inhibition of Ca²⁺-, phosphatidylserine-dependent protein kinase C (PK-C)² by the purifiedholo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that i) the PK-C-inhibitory action ofholo-cRBP andholo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; ii) the reduced phosphorylation of theholo-retinoid-binding proteins and brain cytosolic proteins is not the result of a retinoid-induced soluble phosphatase or protease activity; iii) retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and iv) the structure-function activity of the retinoids and the specific interaction of these effect of retinoids on plasma membrane-associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins.We would like to thank Dr L.M. De Luca (NIH, USA) for his contribution of retinylphosphate, Dr H.N. Bhagavan (Hoffmann-La Roche) for his contribution of the arotinoids, and Merrill-Dow Corp. for their contribution of difluoromethylornithine. This work was supported by NIH Grants CA-34968, CA-07175, CA-22484, and CA-09020.     

Quantitative differences in the pharmacological effects of (+)- and (−)-cathinone

Summary The optically pure isomers of cathinone were prepared by separating synthetic cathinone racemate and used to study central and peripheral effects of these indirect sympathomimetics in rats and guinea pigs. The (–)-isomer was significantly more potent than the (+)-isomer in stimulating locomotor activity whereas no difference was observed with respect to their cardiac effects. In analogy to observations with (+)- and (–)-amphetamine such variable isomer discrimination may be due to different stereoselectivities of amine uptake mechanisms in the target tissues.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Differential binding of conA and WGA on the cell surface,the role of sialic acid in their expression and the increased activity of sialidase after cis-Platin treatment

Summary It is reported that concanavalin A (conA) and wheat germ agglutinin (WGA) have a differential binding pattern on normal mouse spleen lymphocytes and the surface of Dalton's lymphoma cells. It is suggested that sialic acid on the cell surface controls the expression of lectin binding sites. Further, it has been observed that the increased release of sialic acid from cell surfaces aftercis-dichlorodiammine platinum (II) (cis-Platin) treatment is due to the increased activity of sialidase.To whom reprint requests should be addressed.     

Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT₁ and MT₂ membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT₁ and ROR receptor mRNA and protein are expressed in both thymus and spleen, while MT₂ receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.Received 2 June 2003; received after revision 6 August 2003; accepted 14 August 2003     

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes

We have previously shown that the protein kinase C (PKC) system plays a pivotal role in regulation of proliferation and differentiation of the human keratinocyte line HaCaT which is often used to assess processes of immortalization, transformation, and tumorigenesis in human skin. In this paper, using pharmacological and molecular biology approaches, we investigated the isoform-specific roles of certain PKC isoenzymes (conventional cPKC and ; novel nPKC and ) in the regulation of various keratinocyte functions. cPKC and nPKC stimulated cellular differentiation and increased susceptibility of cells to actions of inducers of apoptosis, and they markedly inhibited cellular proliferation and tumor growth in immunodeficient mice. In marked contrast, cPKC and nPKC increased both in vitro and in vivo growth of cells and inhibited differentiation and apoptosis. Our data present clear evidence for the specific, antagonistic roles of certain cPKC and nPKC isoforms in regulating the above processes in human HaCaT keratinocytes.Received 13 January 2004; received after revision 18 February 2004; accepted 25 February 2004     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Chemical reductionism revisited: Lewis, Pauling and the physico-chemical nature of the chemical bond

The wave-mechanical treatment of the valence bond, by Walter Heitler and Fritz London, and its ensuing foundational importance in quantum chemistry has been traditionally regarded as the basis for the argument that chemistry may be theoretically reduced to physics. Modern analyses of the reductionist claim focuses on the limitations to achieving full reduction in practice because of the approximations used in modern quantum chemical methods, but neglect the historical importance of the chemical bond as a chemical entity. This paper re-examines these arguments with a study of the development of the valence bond by chemist Gilbert Lewis within a chemically autonomous framework, and its extension by Linus Pauling using Heitler and London’s methods. Here, we see that the chemical bond is best described as a theoretical synthesis or physico-chemical entity, to represent its full interdisciplinary importance from the philosophical and historical perspectives.     

Specific interactions of the adenovirus proteinase with the viral DNA,an 11-amino-acid viral peptide,and the cellular protein actin

The adenovirus proteinase (AVP) is synthesized in an inactive form that requires cofactors for activation. The interaction of AVP with two viral cofactors and with a cellular cofactor, actin, is characterized by quantitative analyses. The results are consistent with a specific model for the regulation of AVP. Late in adenovirus infection, inside nascent virions, AVP becomes partially activated by binding to the viral DNA, allowing it to cleave out an 11-amino-acid viral peptide, pVIc, that binds to AVP and fully activates it. Then, about 70 AVP-pVIc complexes move along the viral DNA, via one-dimensional diffusion, cleaving virion precursor proteins 3200 times to render a virus particle infectious. Late in adenovirus infection, in the cytoplasm, the cytoskeleton is destroyed. The amino acid sequence of the C terminus of actin is homologous to that of pVIc, and actin, like pVIc, can act as a cofactor for AVP in the cleavage of cytokeratin 18 and of actin itself. Thus, AVP may also play a role in cell lysis.Received 14 November 2002; received after revision 28 April 2003; accepted 30 April 2003     

Localization and the interface between quantum mechanics, quantum field theory and quantum gravity II: The search of the interface between QFT and QG

The main topics of this second part of a two-part essay are some consequences of the phenomenon of vacuum polarization as the most important physical manifestation of modular localization. Besides philosophically unexpected consequences, it has led to a new constructive “outside-inwards approach” in which the pointlike fields and the compactly localized operator algebras which they generate only appear from intersecting much simpler algebras localized in noncompact wedge regions whose generators have extremely mild almost free field behavior.Another consequence of vacuum polarization presented in this essay is the localization entropy near a causal horizon which follows a logarithmically modified area law in which a dimensionless area (the area divided by the square of dR where dR is the thickness of a light-sheet) appears. There are arguments that this logarithmically modified area law corresponds to the volume law of the standard heat bath thermal behavior. We also explain the symmetry enhancing effect of holographic projections onto the causal horizon of a region and show that the resulting infinite dimensional symmetry groups contain the Bondi–Metzner–Sachs group. This essay is the second part of a partitioned longer paper.