A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure

A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.     

The mechanism of the angiotensin-converting enzyme inhibitor quinapril is not related to bradykinin level in heart tissue

In order to examine the effect of the angiotensin-converting enzyme inhibitor (ACEi) quinapril, we performed a sensitive and specific radioimmunoassay (RIA) to quantify bradykinin, BK-(1-9), in heart and kidney tissues. The BK-(1-9) level was unaffected in the heart of sham and water-deprived rats treated for 2h with quinapril (10mg/kg), but was significantly higher in the kidneys in the two groups. In these conditions, circulating and tissue angiotensin II (Ang II) levels were significantly decreased by quinapril. Moreover, our results indicated that acute treatment with this dose of quinapril induced kinin-mediated effects which were not related to its action on bradykinin degradation in rat hearts.     

Mapping QTLs for resistance against <Emphasis Type="Italic">Globodera pallida</Emphasis> (Stone) Pa2/3 in a diploid potato progeny originating from <Emphasis Type="Italic">Solanum spegazzinii</Emphasis>

A "F1" diploid population between Solanum tuberosum 2 x and the wild Solanum spegazzinii was studied. It segregated for resistance against the potato cyst nematode Globodera pallida derived from the wild species. The inheritance had a quantitative nature. Linkage maps of AFLP and RFLP markers were constructed for both parents. Three QTLs were identified on the map of the resistant parent on chromosomes V, VI and XII, respectively. The first one had a major effect and explained more than 50% of the total variance of resistance. It is located in a cluster of resistance genes and may be the same locus as Gpa which has been described formerly. The two others explained about 20% of the total variance each. The QTL on chromosome XII is also in a cluster of resistance genes, and in an orthologous position with resistance genes against nematodes in tomato and pepper.     

Development of an Equine Groove Model to Induce Metacarpophalangeal Osteoarthritis: A Pilot Study on 6 Horses

The aim of this work was to develop an equine metacarpophalangeal joint model that induces osteoarthritis that is not primarily mediated by instability or inflammation. The study involved six Standardbred horses. Standardized cartilage surface damage or “grooves” were created arthroscopically on the distal dorsal aspect of the lateral and medial metacarpal condyles of a randomly chosen limb. The contralateral limb was sham operated. After 2 weeks of stall rest, horses were trotted 30 minutes every other day for 8 weeks, then evaluated for lameness and radiographed. Synovial fluid was analyzed for cytology and biomarkers. At 10 weeks post-surgery, horses were euthanized for macroscopic and histologic joint evaluation. Arthroscopic grooving allowed precise and identical damage to the cartilage of all animals. Under the controlled exercise regime, this osteoarthritis groove model displayed significant radiographic, macroscopic, and microscopic degenerative and reactive changes. Histology demonstrated consistent surgically induced grooves limited to non-calcified cartilage and accompanied by secondary adjacent cartilage lesions, chondrocyte necrosis, chondrocyte clusters, cartilage matrix softening, fissuring, mild subchondral bone inflammation, edema, and osteoblastic margination. Synovial fluid biochemistry and cytology demonstrated significantly elevated total protein without an increase in prostaglandin E2, neutrophils, or chondrocytes. This equine metacarpophalangeal groove model demonstrated that standardized non-calcified cartilage damage accompanied by exercise triggered altered osteochondral morphology and cartilage degeneration with minimal or inefficient repair and little inflammatory response. This model, if validated, would allow for assessment of disease processes and the effects of therapy.     

Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.     

Loss of α1β1 Soluble Guanylate Cyclase,the Major Nitric Oxide Receptor,Leads to Moyamoya and Achalasia

Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal “moyamoya” vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya.     

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor has various cellular effects such as cell growth and apoptosis. In the present study, we evaluated the effects of SBHA on the growth and death of A549 lung cancer cells. SBHA inhibited the growth of A549 cells with an IC₅₀ of approximately 50 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that SBHA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, as evidenced by sub-G1 cells and annexin V-FITC staining cells. SBHA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, Bax increase, and the activation of caspase-3. All of the tested caspase inhibitors significantly rescued some cells from SBHA-induced A549 cell death. However, none of the caspase inhibitors prevented the loss of MMP (ΔΨ_m) induced by SBHA. Intracellular reactive oxygen species (ROS) levels including O ₂ ^?? were increased in 50 μM SBHA-treated A549 cells. None of the caspase inhibitors attenuated ROS levels in these cells. SBHA also elevated the number of glutathione (GSH)-depleted cells in A549 cells, which was reduced by treatment with caspase inhibitors. In conclusion, this is the first report that SBHA inhibited the growth of A549 lung cancer cells via caspase-dependent apoptosis, which was related to GSH depletion rather than changes in ROS level.     

Neuro-endocrine basis for altered plasma glucose homeostasis in the Fragile X mouse

Background

The fragile X mouse model shows an increase in seizure susceptibility, indicating an involvement of the GABAergic system via an alteration in cellular excitability. In the brain, we have previously described a reduction in GABA_A receptor expression as a likely basis for this susceptibility. In the brains of fragile X mice, this reduction in receptor expression culminates with a concomitant increase in the expression of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis. Further, voltage-sensitive calcium channel expression is reduced in the pancreas of the fragile X mouse. Since there are considerable similarities in the GABAergic system in the brain and pancreas, we evaluated the protective role of taurine in pancreatic islet development in both wild type (WT) and fragile X mice (KO).

Methods

One-month-old FVB/NJ males or age-matched fmr1-knockout (KO) mice were supplemented with taurine in drinking water (0.05% w/v) for four weeks. Age-matched controls were fed water only for the same duration. At four weeks, mice were sacrificed and pancreases processed for histology and immunohistochemical studies on changes of insulin, glucagon and somatostatin expression. Additional mice were subjected to a glucose tolerance test.

Results

Taurine treatment resulted in a significant increase in the number and size of islets. WT taurine-fed mice, slightly hypoglycemic prior to glucose injection, showed significantly reduced plasma glucose at 30 min post-injection when compared to control mice. KO mice had normal baseline plasma glucose concentration; however, following glucose injection they had higher plasma glucose levels at 30 min when compared to controls. Supplementation of taurine to KO mice resulted in reduced baseline levels of plasma glucose. After glucose injection, the taurine-fed KO mice had reduced plasma glucose at 30 min compared to KO. Concomitant with the increased islets size and glucose tolerance observed in taurine-fed mice there was an increase in insulin, glucagon and somatostatin immunoreactivity in the islets of WT mice. In the KO mice however, insulin levels were not affected whereas glucagon and somatostatin levels were reduced. Exocytosis of these hormones is calcium-dependent, therefore any exacerbation of calcium homeostasis could affect hormone release. We found the expression of the voltage sensitive calcium channels (VSCC) is drastically reduced in the pancreas of fragile X mice.

Conclusions

During early development, the VSCC play an important role in calcium-dependent gene expression. Since these channels are also involved in depolarization and calcium-mediated vesicular release of neurotransmitters and pancreatic hormones, alterations in the expression of VSCC not only will affect calcium-mediated gene expression but also hormonal and neurotransmitter release creating therefore a neuroendocrine perturbation in the fragile X that may potentially affect other organ systems. We find that in the fragile X mouse, taurine treatment may partially restore functionality of the neuro-endocrine pancreas.

     

The effect of rFVIIa on pro- and anti-inflammatory cytokines in serum and cerebrospinal fluid in a swine model of traumatic brain injury

Traumatic brain injury (TBI) is associated with significant infectious and inflammatory complications. Though increasing evidence suggests that rFVIIa administration may be efficacious for the pre-hospital treatment of TBI, the FVIIa-tissue factor complex has been shown to be immunologically active. To date the cytokine response to rFVIIa administration for the treatment of TBI has not been evaluated. Twenty anesthetized immature Yorkshire swine underwent fluid percussion TBI. At 15 min following injury, animals were randomized to receive either 90 μg/kg rFVIIa (rFVIIa) or nothing. Animals were observed for 6 h and then euthanized. Plasma and cerebrospinal (CSF) samples were collected at 0 min and 360 min, and ELISA analysis of TNF-α, IL-1β and IL-10 was performed. Survival in both groups was 100%. Baseline cytokine concentrations were not statistically different between rFVIIa and control animals in plasma or CSF. Animals in both groups did not have significant changes in plasma cytokine concentrations following TBI. Control animals did not demonstrate significant changes from baseline of CSF cytokine concentrations following TBI. The administration of rFVIIa however, resulted in significant increases in CSF TNF-α concentration (232.0 pg/ml ± 75.9 vs 36.4 pg/ml ± 10.4, p = 0.036) and IL-10 concentration (10.7 pg/ml ± 0.6 vs 8.8 pg/ml ± 0.1, p = 0.015). IL-1β concentrations were not significantly changed over the experimental time course. These results suggest that rFVIIa administration for the treatment of TBI is not immunologically inert, and is associated with increased CSF concentrations of TNF-α and IL-10.