2-Methoxy-6-methyl-3-nitro-4-(2-nitroprop-1-enyl)phenyl acetate

In the title compound, C13H14N2O7, steric crowding around the aromatic ring results in significant out-of-plane twisting of the nitro, methoxy, acetoxy and 2-nitropropenyl functional groups. These distortions are explained by comparison with less congested substituted benzene analogues.

Immobilisation of electroactive macrocyclic complexes within titania films

The 4-carboxyphenyl-appended macrocyclic ligand trans-6,13-dimethyl-6-((4-carboxybenzyl)amino)-1,4,8,11-tetraazacyclotetradecane-6-amine (HL10) has been synthesised and complexed with Co-III. The mononuclear complexes [Co(HL10)(CN)](2+) and [CoL10(OH)](+) have been prepared and the crystal structures of their perchlorate salts are presented, where the ligand is bound in a pentadentate mode in each case while the 4-carboxybenzyl-substituted pendent amine remains free from the metal. The cyano-bridged dinuclear complex [CoL10-mu-NC-Fe(CN)(5)](2-) was also prepared and chemisorbed on titania-coated ITO conducting glass. The adsorbed complex is electrochemically active and cyclic voltammetry of the modified ITO working electrode in both water and MeCN solution was undertaken with simultaneous optical spectroscopy. This experiment demonstrates that reversible electrochemical oxidation of the Fe-II centre is coupled with rapid changes in the optical absorbance of the film.

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-D-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GuR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity. (C) 2005 Elsevier Inc. All rights reserved.

Interferon-gamma is required for killing keratinocytes in vitro and in vivo

Epithelial malignancies are common in immunosuppressed individuals and the general population. However the mechanisms by which the adaptive immune system can eliminate immunogenic epithelial cells remain undefined. The aim of this project was to determine the effector molecules required for induction of apoptosis in murine epidermal keratinocytes (MEKs) in vitro and in vivo. HPV16E7-specific CTL lines and T cell receptor transgenic (E7TCRtg) effector cells were obtained from wild type (wt)-C57 and syngeneic mice rendered functionally inactive for perforin (Pfp), interferon-g (IFN-g) or FasL. CTLs or E7TCRtg spleen cells were co-cultured with primary MEKs in vitro or transferred into skin graft recipients. Inhibition of colony formation and skin graft rejection were used as indicators of T cell:KC interaction. Wt E7-specific CTLs and CTLs deficient in perforin, FasL or IFN-g produced mean reductions in colony formation of 67% (62.4–71.3%), 72% (71.1–72%), 76% (73–78%) and 21.5% (14– 34%) respectively. Wt, perforin deficient or FasL deficient CTLs all induced rejection of skin grafts (wt: 6/12; Pfp: 9/15; FasL: 3/13 survival). Transfer and immunisation of wt E7TCRtg spleen cells induces rejection of 50% of grafts (4/8 survival). In contrast, perforin or IFN-g deficient E7TCRtg failed to induce graft rejection (5/6; 4/4 survival). FasL deficient E7TCRtg induced nonspecific rejection of grafts (E7- 2/6 survival; C57- 4/7 survival). Therefore IFN-g production by CTL is necessary and sufficient in vitro and in vivo to kill epithelial cells which express a nonself antigen. Assessment of immunotherapies directed against epithelial tissues may be more effectively achieved by assaying the amount of IFN-g production by CD8 T cells, and the number and affinity of those cells, in conjunction with quantitation of perforin mediated effects in short term assays.

An event-related fMRI study of cognitive deficits in Huntington's disease

The BOLD contrast signal history determined by lagged Unear correlation has a significant contribution to functional connectivity in activation data sets. It has been demonstrated that in resting state fMRI data, the major contribution to synchronous correlation between functionally connected areas arises from low frequency contributions (

Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited

Evaluation of fly ash as a component of potting substrates

A series of laboratory and glasshouse experiments were undertaken to assess the potential for incorporation of fly ash in soilless potting substrates. The physical and chemical properties of a commercially available bark based substrate, the University of California (UC) 1:1 peat:sand mix and a range uf test substrates containing fly ash were characterised. In test mixtures, fly ash was substituted for a portion of either the peat or sand component of the UC mix, at rates of 10, 20, 30 and 50% of the mix volume, Incorporation of fly ash greatly increased the plant available water capacity (10-1500 kPa) of the substrate. However, high pH, increased substrate strength and reduced air-filled porosity were considered adverse effects, particularly at ash rates > 20%. The growth of tomato (Lycopersicon esculentum), petunia (Petunia x hybrida grandiflora) and Boston fern (Nephrolepis exaltata) in the substrates was assessed. Two watering regimes, capillary watering and irregular hosing, were used to identify effects of available water capacity on plant growth, but no effect was identified. Test mixtures containing fly ash as 20% of the substrate volume produced growth equal to that in the UC mix, with substrates containing 10% ash producing significantly greater growth of tomato and petunia. At rates of incorporation > 20% reduced plant growth was attributed to both adverse physical and chemical characteristics of the substrate. As fly ash is available at low cost and can be successfully substituted for a considerable portion of the expensive peat component, its use at low application rates in potting substrates may be desirable from an economic viewpoint.

The chemistry of 5-oxodihydroisoxazoles .16. A new synthesis of pyrroles, furans, thiophens and their benzo analogues

Ethyl 5-oxo-2-phenyl-2,5-dihydroisoxazole-4-carboxylate (2) was photolysed at 300 mn in the presence of phenols, enols, anilines, enamines, aryl thiols and thioenols affording enamines. Treatment of these enamines with Lewis or protic acids gives the respective benzo and five-membered ring systems.

Functional CD40-ligand is expressed by T cells in rheumatoid arthritis

CD40-1igand (CD40-L), a member of the tumour necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. CD40 is expressed by B cells, monocytes and dendritic cells. Interactions between CD40-L and CD40 induce B cell proliferation, differentiation, immunoglobulin production and isotype switching as well as monocyte activation and dendritic cell differentiation. Since the rheumatoid synovium is characterized by T cell activation, B cell immunoglobulin production, monocyte cytokine production and dendritic cell differentiation, the expression and function of CD40-L in RA was examined. RA synovial fluid (SF) T ceils expressed CD40-L mRNA, as well as low level cell surface CD40-L. A subset of CD4+ RA synovial fluid T cells could express cell surface CD40-L within 15 rain of in vitro activation even in the presence of cycloheximide. CD40-L expressed by RA SF T cells was functional, since RA SF T cells, but not normal PB T cells, stimulated CD40-L dependent B cell immunoglobulin production in the absence of in vitro T cell activation. These data indicate that SF T cells express functionally significant levels of surface CD40-L, and have the potential for rapid upregulation of surface expression from preformed CD40-L stores. Thus, CD40-L is likely to play a central role in the perpetuation of RA by induction of Ig synthesis, cytokine production and dendritic cell differentiation. Moreover, the data provide important evidence of recent activation of RA synovial T cells. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Encephalitogenicity of murine, but not bovine, DM20 in SJL mice is due to a single amino acid difference in the immunodominant encephalitogenic epitope

Myelin proteolipid protein (PLP) contains 2 immunodominant encephalitogenic epitopes in SJL mice, namely PLP residues 139-151 and 178-191. DM20, a minor isoform of PLP, lacks residues 116-150 and consequently contains only the single major encephalitogenic epitope 178-191. However, it has been found previously that bovine DM20 is not encephalitogenic in SJL mice. Since residue 188 within peptide 178-191 is phenylalanine (F) in murine DM20 and alanine (A) in bovine DM20, we tested the effect of this difference on the immune responses and induction of EAE. SJL mice were immunized with either highly purified murine or bovine DM20. Residues 178-191 were found to be immunodominant for each, but only murine and not bovine DM20 was encephalitogenic. A synthetic peptide corresponding to the murine 178-191 sequence (F188) was also encephalitogenic, whereas the peptide corresponding to the bovine sequence (A188) was not. Both F188 and A188 bind with high affinity to I-A(s) and both are recognized by the SJL T cell repertoire. A188-specific T cell lines reacted to both A188 and F188, but F188-specific T cell lines were not stimulated by A188. F188-specific T cell lines produced mRNA for the Th1 cytokines IL2 and IFN gamma and, in passive transfer experiments, were encephalitogenic upon stimulation with F188, but not A188. In contrast, A188-specific T cell lines produced mRNA for IL4, IL5 and IL10, in addition to IL2 and IFN gamma, and were not encephalitogenic after stimulation with either F188 or A188. Cotransfer of A188-specific T cell lines with F188-specific T cell lines resulted in protection from EAE. Thus, A188 induces a functionally different phenotype of T cells from that induced by F188. Taken together these data suggest that the failure of bovine DM20 to induce EAE may be attributable to induction of protective rather than pathogenic T cells by the immunodominant epitope.

An investigation into the synthesis of polycarbazole squaraine derivatives

The polycondensation of squaric acid with 1,2-(9-Ethylcarbazol-3-yl)ethene and N-ethyliminostilbene in polyphosphoric acid yielded insoluble polymers which included substituted phosphate groups on the phenyl rings. The presence of phosphorus in these polymers was identified using solid-state P-31 NMR and EDAX techniques. Furthermore the phosphate groups were not ionic, hence no charge-balancing anions were present; Both polymers did not electrically conduct but exhibited dielectric breakdown values of 0.1 and 0.06 MV cm(-1) respectively.

Structural and electron self-exchange rate variations in isomeric (hexaamine)cobalt(III/II) complexes

The syntheses and characterisation of the new macrocyclic hexaamine trans-(5(S),7(S),12(R),14(R)-tetramethyl)-1,4,8,11-tetraazacyclotetradecane-6,13-diamine (L-6) and its Co-III complex are reported. The X-ray crystal structural analyses of [CoL6]Cl-2(ClO4) [monoclinic, space group C2/c, a = 16.468(3) Angstrom, b = 9.7156(7) Angstrom, c = 15.070(3) Angstrom, beta = 119.431(8)degrees, Z = 4] and the closely related cis-diamino-substituted macrocyclic complex [CoL2](ClO4)(3) . 2H(2)O (L-2 = cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine) [orthorhombic, space group Pna2(1), a = 16.8220(8) Angstrom, b = 10.416(2) Angstrom, c = 14.219(3) Angstrom, Z = 4] reveal significant variations in the observed Co-N bond lengths and coordination geometries, which may be attributed to the trans or cis disposition of the pendent primary amines. The Co-III/II self-exchange electron transfer rate constants for these and other closely related hexaamines have been determined, and variations of some 2 orders of magnitude are found between pairs of trans and cis isomeric Co-III complexes.

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

The chemistry of novolac resins .3. C-13 and N-15 nmr studies of curing with hexamethylenetetramine

The reactions between novolac resins and hexamethylenetetramine (HMTA) which occur on curing have been studied by C-13 and N-15 high-resolution n.m.r. in both solution and the solid state. Strong evidence for the existence of many curing intermediates is obtained. New curing intermediates are reported along with experimental data to support previously postulated intermediates. The initial curing reactions between novolac and HMTA produce various substituted benzoxazines and benzylamines. Thermal decomposition/oxidation and further reactions of these initial intermediates generate methylene linkages between phenolic rings for chain extension and cross-linking. Among the three kinds of methylene linkages, the para-para methylene linkages are formed at relatively lower temperatures. Various imine, amide and imide side-products also concurrently appear during the process. The initial amount of HMTA plays a critical role in the curing reactivity and chemical structures of the cured resins. The lower the amount of HMTA, the lower the temperature at which curing occurs, and the lower the amount of the nitrogen-containing side-products in the finally cured resins. The ortho-linked intermediates are relatively stable, and can remain in the cured resins up to higher temperatures. The study provides an extensive description of the curing reactions of novolac resins. (C) 1997 Elsevier Science Ltd.