Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

The extrusion of continuous lengths of YBa2Cu3O7-x wire using hydroxypropyl methylcellulose

Wires of YBa2Cu3O7-x were fabricated by extrusion using a hydroxypropyl methylcellulose (HPMC) binder. As little as 2 wt.% binder was added to an oxide prepared by a novel co-precipitation process, to produce a plastic mass which readily gave continuous extrusion of long lengths of wire in a reproducible fashion. Critical temperatures of 92K were obtained for wires given optimum high-temperature heat treatments. Critical current densities greater than 1000 A cm-1 were measured at 77.3K using heat treatments at around 910°C for 10h. These transport critical current densities, measured on centimeter-long wires, were obtained with microstructures showing a relatively dense and uniform distribution of randomly oriented, small YBa2Cu3O7-x grains. © 1993.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

Association between a 19 bp deletion polymorphism at the dopamine beta-hydroxylase (DBH) locus and migraine with aura

Migraine is a debilitating neurological disorder, affecting 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is unclear. Our previous work has investigated dopamine related migraine candidate genes and has reported a significant allelic association with migraine of a microsatellite localised to the promoter region of the dopamine beta-hydroxylase (DBH) gene. The present study performed an association analysis in a larger population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls) examining two different genetic DBH polymorphisms (a functional insertion/deletion promoter and a coding SNP A444G polymorphism). Although no significant association was found for the SNP polymorphism, the results showed a significant association between the insertion/deletion variant and disease (chi(2)=8.92, P=0.011), in particular in migraine with aura (chi(2)=11.53, P=0.003) compared to the control group. Furthermore, the analysis of this polymorphism stratified by gender, revealed that male individuals with the homozygote deletion genotype had three times the risk of developing migraine, compared to females. The DBH insertion/deletion polymorphism is in linkage disequilibrium with the previously reported migraine associated DBH microsatellite and this insertion/deletion polymorphism is functional, which may explain a potential role in susceptibility to migraine.

Detection of mRNA levels for the estrogen alpha, estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue

Previous studies in our laboratory have shown association of nuclear receptor expression and histological breast cancer grade. To further investigate these findings, it was the objective of this study to determine if expression levels of the estrogen alpha, estrogen beta and androgen nuclear receptor genes varied in different breast cancer grades. RNA extracted from paraffin embedded archival breast tumour tissue was converted into cDNA and cDNA underwent PCR to enable quantitation of mRNA expression. Expression data was normalised against the 18S ribosomal gene multiplex and analysed using ANOVA. Analysis indicated a significant alteration of expression for the androgen receptor in different cancer grades (P=0.014), as well as in tissues that no longer possess estrogen receptor alpha proteins (P=0.025). However, expression of estrogen receptors alpha and beta did not vary significantly with cancer grade (P=0.057 and 0.622, respectively). Also, the expression of estrogen receptor alpha or beta did not change, regardless of the presence of estrogen receptor alpha protein in the tissue (P=0.794 and 0.716, respectively). Post-hoc tests indicate that the expression of the androgen receptor is increased in estrogen receptor negative tissue as well as in grade 2 and grade 3 tumours, compared to control tissue. This increased expression in late stage breast tumours may have implications to the treatment of breast tumours, particularly those lacking expression of other nuclear receptor genes.

Gas-phase reactions of aryl radicals with 2-butyne : an experimental and theoretical investigation employing the N-methyl-pyridinium-4-yl radical cation

Aromatic radicals form in a variety of reacting gas-phase systems, where their molecular weight growth reactions with unsaturated hydrocarbons are of considerable importance. We have investigated the ion-molecule reaction of the aromatic distonic N-methyl-pyridinium-4-yl (NMP) radical cation with 2-butyne (CH3C CCH3) using ion trap mass spectrometry. Comparison is made to high-level ab initio energy surfaces for the reaction of NMP and for the neutral phenyl radical system. The NMP radical cation reacts rapidly with 2-butyne at ambient temperature, due to the apparent absence of any barrier. The activated vinyl radical adduct predominantly dissociates via loss of a H atom, with lesser amounts of CH3 loss. High-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry allows us to identify small quantities of the collisionally deactivated reaction adduct. Statistical reaction rate theory calculations (master equation/RRKM theory) on the NMP + 2-butyne system support our experimental findings, and indicate a mechanism that predominantly involves an allylic resonance-stabilized radical formed via H atom shuttling between the aromatic ring and the C-4 side-chain, followed by cyclization and/or low-energy H atom beta-scission reactions. A similar mechanism is demonstrated for the neutral phenyl radical (Ph center dot)+2-butyne reaction, forming products that include 3-methylindene. The collisionally deactivated reaction adduct is predicted to be quenched in the form of a resonance-stabilized methylphenylallyl radical. Experiments using a 2,5-dichloro substituted methyl-pyridiniumyl radical cation revealed that in this case CH3 loss from the 2-butyne adduct is favoured over H atom loss, verifying the key role of ortho H atoms, and the shuttling mechanism, in the reactions of aromatic radicals with alkynes. As well as being useful phenyl radical analogues, pyridiniumyl radical cations may form in the ionosphere of Titan, where they could undergo rapid molecular weight growth reactions to yield polycyclic aromatic nitrogen hydrocarbons (PANHs).

Changes in lycopene and beta carotene contents in aril and oil of gac fruit during storage

Gac fruits were physically measured and stored under ambient conditions for up to 2 weeks to observe changes in carotenoid contents (lycopene and beta carotene) in its aril. Initial concentrations in the aril of lycopene were from 2.378 mg/g fresh weight (FW) to 3.728 mg/g FW and those of beta carotene were from 0.257 to 0.379 mg/g FW. Carotenoid concentrations in the aril remained stable after 1 week but sharply declined after 2 weeks of storage. Gac oil, pressed from gac aril, has similar concentrations of lycopene and beta carotene (2.436 and 2.592 mg/g, respectively). Oil was treated with 0.02% of butylated hydroxytoluene, or with a stream of nitrogen or untreated then stored in the dark for up to 15 or 19 weeks under different temperatures (5 °C, ambient, 45 and 60 °C). Lycopene and beta carotene in control gac oil degraded following the first-order kinetic model. The degradation rate of lycopene and beta carotene in the treated oil samples were lower than that in the control oil but the first-order kinetic was not always followed. However, both lycopene and beta carotene degraded quickly in gac oil with the first-order kinetic under high temperature conditions (45 and 60 °C) regardless of the treatments used. © 2009 Elsevier Ltd. All rights reserved.

Influence of interleukin-1 beta on platelet-poor plasma clot formation: A potential impact on early bone healing

Objectives Hematoma quality (especially the fibrin matrix) plays an important role in the bone healing process. Here, we investigated the effect of interleukin-1 beta (IL-1β) on fibrin clot formation from platelet-poor plasma (PPP). Methods Five-milliliter of rat whole-blood samples were collected from the hepatic portal vein. All blood samples were firstly standardized via a thrombelastograph (TEG), blood cell count, and the measurement of fibrinogen concentration. PPP was prepared by collecting the top two-fifths of the plasma after centrifugation under 400 × g for 10min at 20°C. The effects of IL-1β cytokines on artificial fibrin clot formation from PPP solutions were determined by scanning electronic microscopy (SEM), confocal microscopy (CM), turbidity, and clot lysis assays. Results The lag time for protofibril formation was markedly shortened in the IL-1β treatment groups (243.8 ± 76.85 in the 50 pg/mL of IL-1β and 97.5 ± 19.36 in the 500 pg/mL of IL-1β) compared to the control group without IL-1β (543.8 ± 205.8). Maximal turbidity was observed in the control group. IL-1β (500 pg/mL) treatment significantly decreased fiber diameters resulting in smaller pore sizes and increased density of the fibrin clot structure formed from PPP (P < 0.05). The clot lysis assay revealed that 500 pg/mL IL-1β induced a lower susceptibility to dissolution due to the formation of thinner and denser fibers. Conclusion IL-1β can significantly influence PPP fibrin clot structure, which may affect the early bone healing process.