A seroprevalence survey of Toxoplasma gondii in common wombats (Vombatus ursinus)

Serum was collected from 23 wild common wombats (Vombatus ursinus) on a pastoral property in the Southern Tablelands of New South Wales, Australia (231N 616E) between 3rd August 2001 and 25th March 2002. The serum was tested using three serological methods for antibodies to Toxoplasma gondii. Six animals (26.1%) were shown to have antibodies to T. gondii. The latex agglutination test proved to be less sensitive than the direct agglutination test or the modified agglutination test. This is the first serological survey of T. gondii in wombats. This is the first recorded use of the latex agglutination test on wombat serum. This study demonstrated the highest percentage of seropositive animals in any serological survey for T. gondii in marsupials.     

Dynamics of Rhodobacter capsulatus [2FE-2S] ferredoxin VI and Aquifex aeolicus ferredoxin 5 via nuclear resonance vibrational spectroscopy (NRVS) and resonance Raman spectroscopy

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(2)S(2)(Cys)(4) sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm(-1), and additional features observed as high as the B(2u) mode at approximately 421 cm(-1). Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B(3u) mode at approximately 288 cm(-1), attributed to an asymmetric combination of the two FeS(4) breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS(4) breathing. NRVS and RR analysis of isotope shifts with (36)S-substituted into bridging S(2-) ions in Rc FdVI allowed quantitation of S(2-) motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at approximately 190 cm(-1) in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields. Progressively more complex D(2h) Fe(2)S(2)S'(4), C(2h) Fe(2)S(2)(SCC)(4), and C(1) Fe(2)S(2)(Cys)(4) models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.     

Design of 11-residue peptides with unusual biophysical properties: induced secondary structure in the absence of water

A series of oligopeptides with β-forming and adhesive properties, were synthesized and analyzed for adhesion shear strength, secondary structure, and association properties. The sequences contained related hydrophobic core segments varying in length from 5 to 12 residues and flanked by di- or tri-lysine segments. Three remarkable peptides consisting of just 11 residues with hydrophobic core sequences of FLIVI, IGSII, and IVIGS flanked by three lysine residues gave the highest dry adhesion shear strength and displayed unusual biophysical properties in the presence and absence of water. KKKFLIVIKKK had its highest adhesion strength at 2% (w/v) at pH 12.0 and showed the highest adhesion strength after exposure to water (water resistance). Both KKKIGSIIKKK and KKKIVIGSKKK, at 4% (w/v) at pH 12.0, displayed nearly identical dry shear strength values to that with the FLIVI core sequence. The peptide with IGSII core, however, displayed a lower water resistance and the latter, IVIGS, showed no water resistance, completely delaminating upon soaking in water. These are the smallest peptides with adhesive properties reported to date and show remarkable adhesion strength even at lower concentrations of 0.2% (w/v), which corresponds to 1.6 mM. The FLIVI containing peptide adopted a β-sheet secondary structure in water while the IGSII- and IVIGS-containing sequences folded similarly only in the absence of water. Analytical ultracentrifugation studies showed that when the FLIVI sequence adopts β-structure in aqueous solution, it associates into a large molecular weight assembly. The random coils of IGSII and IVIGS showed no tendency to associate at any pH.     

Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.     

IMMUTANS does not act as a stress-induced safety valve in the protection of the photosynthetic apparatus of Arabidopsis during steady-state photosynthesis

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O(2) to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P(700) to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6 x) and 16 (OE-16 x) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1)). Similar results were observed either after 3-d cold stress at 5 degrees C or after full-leaf expansion at 5 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1). Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25 degrees C or 5 degrees C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P(700)(+) during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.     

Implementing Gaussian process inference with neural networks

Gaussian processes compare favourably with backpropagation neural networks as a tool for regression, and Bayesian neural networks have Gaussian process behaviour when the number of hidden neurons tends to infinity. We describe a simple recurrent neural network with connection weights trained by one-shot Hebbian learning. This network amounts to a dynamical system which relaxes to a stable state in which it generates predictions identical to those of Gaussian process regression. In effect an infinite number of hidden units in a feed-forward architecture can be replaced by a merely finite number, together with recurrent connections.     

β-Phenylethyl and 8-methylsulphinyloctyl isothiocyanates, constituents of watercress, suppress LPS induced production of nitric oxide and prostaglandin E2 in RAW 264.7 macrophages

Beta-phenylethyl (PEITC) and 8-methylsulphinyloctyl isothiocyanates (MSO) represent two phytochemical constituents present in watercress Rorripa nasturtium aquaticum, with known chemopreventative properties. In the present investigation, we examined whether PEITC and MSO could modulate the inflammatory response of Raw 264.7 macrophages to bacterial lipopolysaccharide (LPS) by assessment of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Overproduction of both nitric oxide (NO) and prostaglandins (PGE) has been associated with numerous pathological conditions including chronic inflammation and cancer. Our results demonstrate that LPS (1 microg/ml approximately 24 h) induced nitrite and prostaglandin E2 (PGE-2) synthesis in Raw 264.7 cells was attenuated by both isothiocyanates (ITCs) in a concentration-dependent manner. Both PEITC and MSO decreased (iNOS) and (COX-2) protein expression levels leading to reduced secretion of both pro-inflammatory mediators. Interestingly, the reduction in both iNOS and COX-2 expression were associated with the inactivation of nuclear factor-kappaB and stabilization of IkappaBalpha. Taken together our data gives further insight into the possible chemopreventative properties of two dietary derived isothiocyanates from watercress.     

The effects of caloric restriction- and exercise-induced weight loss on left ventricular diastolic function

Little is known about the effects of weight loss on diastolic function. Furthermore, it is not known whether both caloric restriction (CR)- and exercise (Ex)-induced weight loss have salutary effects on diastolic function. Therefore, we assessed the effects of yearlong CR (n = 12) and Ex (n = 13) interventions, which induced approximately 12% weight loss, on diastolic function in healthy, nonobese (body mass index = 23.5-29.9 kg/m2) men and women aged 50 to 60 yr. Recordings of Doppler transmitral flow and Doppler tissue imaging were acquired and analyzed by conventional approaches and a validated parameterized diastolic filling (PDF) formalism. Isovolumic relaxation time decreased after weight loss in both groups (P < 0.05). Septal peak early mitral annular velocity (E') increased (P < 0.01) and peak E-wave velocity/E' decreased (P < 0.05) after weight loss in the CR group. Based on the PDF-derived indexes, CR resulted in a decrease in global ventricular stiffness (k) and increases in longitudinal (septal annulus motion) stored elastic strain (chi'o), peak force (k'chi'o), and peak stored strain energy (1/2k'chi'o2). In the Ex group, k was unchanged, although septal chi'o and 1/2k'chi'o2 increased significantly and k'chi'o (P = 0.13) tended to increase. We conclude that weight loss, whether induced by CR or Ex, has salutary effects on diastolic function.     

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.