A thermolytic route to a polysilyne.

We report a safe and convenient method to prepare a new class of network polysilane, or polysilyne ([RSi]n). Simple thermolysis of a readily accessible linear poly(phenylsilane), [PhSiH]n, affords polysilyne [PhSi]n with concomitant evolution of monosilanes. This new polymer shows a hyperbranched structure with unique features not observed in known polysilynes prepared via hazardous Wurtz coupling routes. Despite these differences, our soluble, yellow polysilyne exhibits some important properties associated with the traditional random network structure: it absorbs up to 400 nm in the UV spectrum, yet is stable to photolysis under inert atmosphere. This efficient new synthetic route opens the door to exciting applications for these hyperbranched polymers in materials and device technologies.

Coordination, reactivity, and structural properties of electron-rich ethoxy- and dimethylamino-substituted 1,3-diketiminate ligands and their complexes.

In this paper we describe the synthesis, characterization, and X-ray crystal structures of two ligands, diethyl-N,N'-bis(p-tolyl)malonimidate and 1,3-bis(dimethylamino)-N,N'-bis(p-tolyl)propanediimidate. Their corresponding rhodium(i) dicarbonyl, dimethylaluminium, and bis-ligated zinc complexes have also been prepared and characterized. The donor properties of the ligands have been studied and have been compared to those of the traditional anionic N,N'-chelating ligand nacnac.

Carbon-centered strong bases in phosphonium ionic liquids.

Phosphonium ionic liquids (PhosILs), most notably tetradecyl(trihexyl)phosphonium decanoate (PhosIL-C(9)H(1)9COO), are solvents for bases such as Grignard reagents, isocyanides, Wittig reagents (phosphoranes), and N-heterocyclic carbenes (NHCs). The stability of the organometallic species in PhosIL solution is anion dependent. Small bases, such as hydroxide, react with the phosphonium ions and promote C-H exchange as suggested by deuterium-labeling studies. A method to dry and purify the ionic liquids is described and this step is important for the successful use of basic reagents in PhosIL. NHCs have been generated in PhosIL, and these persistent solutions catalyze organic transformations such as the benzoin condensation and the Kumada-Corriu cross-coupling reaction. Phosphoranes were generated in PhosIL, and their reactivity with various organic reagents was also tested. Inter-ion contacts involving tetraalkylphosphonium ions have been assessed, and the crystal structure of [(n-C(4)H(90)(4)P][CH(3)CO(2).CH(3)CO(2)H] has been determined to aid the discussion. Decomposition of organometallic compounds may also proceed through electron-transfer processes that, inter alia, may lead to decomposition of the IL, and hence the electrochemistry of some representative phosphonium and imidazolium ions has been studied. A radical derived from the electrochemical reduction of an imidazolium ion has been characterized by electron paramagnetic resonance spectroscopy.

Flexible coordination of the carboxylate ligand in tin(II) amides and a 1,3-diaza-2,4-distannacyclobutanediyl.

A series of tin(II) amido complexes possessing m-terphenyl carboxylate ligands have been prepared. These complexes, namely [(Me(3)Si)(2)NSn(mu-O(2)CC(6)H(2)Ph(3))](2), [(Me(3)Si)(2)NSn(mu-O(2)CC(6)H(3)Mes(2))](2), and [(Me(3)Si)(2)NSn(mu-O(2)CC(6)H(2)Mes(2)Me)](2) [Mes = 2,4,6-trimethylphenyl], are the first structurally characterized examples of tin(II) carboxylate complexes exhibiting discrete Sn(2)O(4)C(2) heterocyclic cores. Initial reactivity studies led to the isolation of a 1,3-diaza-2,4-distannacyclobutanediyl, [(Mes(2)C(6)H(3)CO(2))Sn(mu-NSiMe(3))](2). This molecule possesses a Sn(2)N(2) heterocyclic core and it was crystallised as both the CH(2)Cl(2) and Et(2)O solvates. Although the tin atoms in this molecule have a formal oxidation state of 3+, preliminary computational studies on this molecule suggest that it is best described as a ground state singlet. Finally, the X-ray crystal structure of (CH(2)Cl)(Cl)Sn[N(SiMe(3))(2)](2), the product of oxidative addition of CH(2)Cl(2) to Sn[N(SiMe(3))(2)](2), is also presented herein.

Coq3 and Coq4 define a polypeptide complex in yeast mitochondria for the biosynthesis of coenzyme Q.

Coenzyme Q (Q) is a redox active lipid essential for aerobic respiration in eukaryotes. In Saccharomyces cerevisiae at least eight mitochondrial polypeptides, designated Coq1-Coq8, are required for Q biosynthesis. Here we present physical evidence for a coenzyme Q-biosynthetic polypeptide complex in isolated mitochondria. Separation of digitonin-solubilized mitochondrial extracts in one- and two-dimensional Blue Native PAGE analyses shows that Coq3 and Coq4 polypeptides co-migrate as high molecular mass complexes. Similarly, gel filtration chromatography shows that Coq1p, Coq3p, Coq4p, Coq5p, and Coq6p elute in fractions higher than expected for their respective subunit molecular masses. Coq3p, Coq4p, and Coq6p coelute with an apparent molecular mass exceeding 700 kDa. Coq3 O-methyltransferase activity, a surrogate for Q biosynthesis and complex activity, also elutes at this high molecular mass. We have determined the quinone content in lipid extracts of gel filtration fractions by liquid chromatography-tandem mass spectrometry and find that demethoxy-Q(6) is enriched in fractions with Coq3p. Co-precipitation of biotinylated-Coq3 and Coq4 polypeptide from digitonin-solubilized mitochondrial extracts shows their physical association. This study identifies Coq3p and Coq4p as defining members of a Q-biosynthetic Coq polypeptide complex.

Yeast Coq5 C-methyltransferase is required for stability of other polypeptides involved in coenzyme Q biosynthesis.

Coenzyme Q (Q) functions in the electron transport chain of both prokaryotes and eukaryotes. The biosynthesis of Q requires a number of steps involving at least eight Coq polypeptides. Coq5p is required for the C-methyltransferase step in Q biosynthesis. In this study we demonstrate that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Phenotypic characterization of a collection of coq5 mutant yeast strains indicates that while each of the coq5 mutant strains are rescued by the Saccharomyces cerevisiae COQ5 gene, only the coq5-2 and coq5-5 mutants are rescued by expression of Escherichia coli ubiE, a homolog of COQ5. The coq5-2 and coq5-5 mutants contain mutations within or adjacent to conserved methyltransferase motifs that would be expected to disrupt the catalysis of C-methylation. The steady state levels of the Coq5-2 and Coq5-5 mutant polypeptides are not decreased relative to wild type Coq5p. Two other polypeptides required for Q biosynthesis, Coq3p and Coq4p, are detected in the wild type parent and in the coq5-2 and coq5-5 mutants, but are not detected in the coq5-null mutant, or in the coq5-4 or coq5-3 mutants. The effect of the coq5-4 mutation is similar to a null, since it results in a stop codon at position 93. However, the coq5-3 mutation (G304D) is located just four amino acids away from the C terminus. While C-methyltransferase activity is detectable in mitochondria isolated from this mutant, the steady state level of Coq5p is dramatically decreased. These studies show that at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis and second, it is involved in stabilizing the Coq3 and Coq4 polypeptides required for Q biosynthesis.

Project management system for structural and functional proteomics: Sesame.

A computing infrastructure (Sesame) has been designed to manage and link individual steps in complex projects. Sesame is being developed to support a large-scale structural proteomics pilot project. When complete, the system is expected to manage all steps from target selection to data-bank deposition and report writing. We report here on the design criteria of the Sesame system and on results demonstrating successful achievement of the basic goals of its architecture. The Sesame software package, which follows the client/server paradigm, consists of a framework, which supports secure interactions among the three tiers of the system (the client, server, and database tiers), and application modules that carry out specific tasks. The framework utilizes industry standards. The client tier is written in Java2 and can be accessed anywhere through the Internet. All the development on the server tier is also carried out in Java2 so as to accommodate a wide variety of computer platforms. The database tier employs a commercial database management system. Each Sesame application module consists of a simple user interface in the client tier, corresponding objects in the server tier, and relevant data stored in the centralized database. For security, access to stored data is controlled by access privileges. The system facilitates both local and remote collaborations. Because users interact with the system using Java Web Start or through a web browser, access is limited only by the availability of an Internet connection. We describe several Sesame modules that have been developed to the point where they are being utilized routinely to support steps involved in structural and functional proteomics. This software is available to parties interested in using it and assisting to guide its further development.

The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis.

Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.

Development of interleukin-12-producing capacity throughout childhood.

Increasing evidence indicates that the capacity to induce protective Th1 immune responses is impaired in early childhood, an observation that can be partially attributed to deficiencies in antigen-presenting-cell function. Synthesis of interleukin 12 (IL-12), a key Th1-trophic cytokine, is markedly reduced in the neonatal period, though there is a paucity of knowledge concerning the ontogeny of IL-12-synthetic capacity throughout the childhood years. Hence, we examined the production of bioactive IL-12 p70 by circulating mononuclear cells in a population of healthy individuals. As expected, the capacity to synthesize IL-12 p70 in response to either lipopolysaccharide or heat-killed Staphylococcus aureus was markedly impaired at birth, even after priming of cells with gamma interferon. Surprisingly however, IL-12 p70 synthesis by peripheral blood mononuclear cells from both 5- and 12-year-old children was still substantially below that seen in adults, and this did not appear to be related to excessive production of IL-10. In contrast, dendritic cells from adults and neonates, derived from monocytes with granulocyte-macrophage colony-stimulating factor and IL-4, synthesized equivalent amounts of IL-12 p70 in response to microbial stimulation. This indicates that the impaired capacity for IL-12 synthesis in childhood is not an intrinsic property of circulating mononuclear cells but rather can be readily overcome in response to appropriate maturational stimuli. Because IL-12 arose predominantly from circulating HLA-DR(+) cells that lacked B-cell- and monocyte-specific markers, we propose that the slow maturation of IL-12-synthetic capacity in the childhood years can be attributed to deficiencies in the number and/or function of dendritic cells.

Testing the NKT cell hypothesis of human IDDM pathogenesis.

Defects in IL-4-producing CD1d-autoreactive NKT cells have been implicated in numerous Th1-mediated autoimmune diseases, including diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and systemic sclerosis. Particular attention has been focused on autoimmune insulin-dependent diabetes mellitus (IDDM) because nonobese diabetic (NOD) mice and humans with IDDM are both reported to express severe deficiencies in the frequency and Th2 functions of NKT cells. Furthermore, experimental manipulations of the NKT defect in the NOD mouse induced corresponding changes in disease. Taken together, these converging studies suggested a general role of NKT cells in natural protection against destructive autoimmunity. However, in previous reports the identification of NKT cells was based on indirect methods. We have now devised a direct, highly specific CD1d tetramer-based methodology to test whether humans with IDDM have associated NKT cell defects. Surprisingly, although we find marked and stable differences in NKT cells between individuals, our study of IDDM patients and healthy controls, including discordant twin pairs, demonstrates that NKT cell frequency and IL-4 production are conserved during the course of IDDM. These results contradict previous conclusions and refute the hypothesis that NKT cell defects underlie most autoimmune diseases.

Distinct functional lineages of human V(alpha)24 natural killer T cells.

CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy subjects express <1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents alpha-galactosylceramide (alphaGalCer) loaded CD1d-tetramers and anti-V(alpha)24 monoclonal antibody, to specifically identify and characterize these rare cells in fresh PBLs. We report here that CD4(+) and CD4(-)CD8(-) (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4(+) NKT cells were the exclusive producers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases.