Oxygen-bound Hell's gate globin I by classical versus LB nanotemplate method

X‐ray atomic structure of recombinant Hell's gate globin I (HGbI) from Methylacidophilum infernorum was calculated from the X‐ray diffraction data of two different types of crystals: obtained by classical hanging drop and by LB nanotemplate method under the same crystallization conditions. After the accurate comparison of crystallographic parameters and electron density maps of two structures they appears to be quite similar, while the quality of the crystals grown by LB nanotemplate method was higher then of those grown by classical method. Indeed, the resolution of the LB crystal structure was 1.65 Å, while classical crystals showed only 3.2 Å resolution. Moreover, the reproducibility of this result in the case of LB crystals was much better—nine crystals from 10 gave the same structural results, while only two of 10 classical crystals were appropriate for the X‐ray structure resolution. J. Cell. Biochem. 113: 2543–2548, 2012. © 2012 Wiley Periodicals, Inc.     

Crystal structure of the Japanese encephalitis virus envelope protein

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.     

Serotonergic mechanisms of the lateral parabrachial nucleus and cholinergic-induced sodium appetite

Central cholinergic mechanisms are suggested to participate in osmoreceptor-induced water intake. Therefore, central injections of the cholinergic agonist carbachol usually produce water intake (i.e., thirst) and are ineffective in inducing the intake of hypertonic saline solutions (i.e., the operational definition of sodium appetite). Recent studies have indicated that bilateral injections of the serotonin receptor antagonist methysergide into the lateral parabrachial nucleus (LPBN) markedly increases salt intake in models involving the activation of the renin-angiotensin system or mineralocorticoid hormones. The present studies investigated whether sodium appetite could be induced by central cholinergic activation with carbachol (an experimental condition where only water is typically ingested) after the blockade of LPBN serotonergic mechanisms with methysergide treatment in rats. When administered intracerebroventricularly in combination with injections of vehicle into both LPBN, carbachol (4 nmol) caused water drinking but insignificant intake of hypertonic saline. In contrast, after bilateral LPBN injections of methysergide (4 microg), intracerebroventricular carbachol induced the intake of 0.3 M NaCl. Water intake stimulated by intracerebroventricular carbachol was not changed by LPBN methysergide injections. The results indicate that central cholinergic activation can induce marked intake of hypertonic NaCl if the inhibitory serotonergic mechanisms of the LPBN are attenuated.     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

An integrated assessment of histopathological changes of the enteric neuromuscular compartment in experimental colitis

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4‐dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c‐Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS‐treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA‐positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c‐Kit‐ and TMEM16A/ANO1‐positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS‐induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.     

Cerebral Circulation Time is Prolonged and Not Correlated with EDSS in Multiple Sclerosis Patients: A Study Using Digital Subtracted Angiography

Literature has suggested that changes in brain flow circulation occur in patients with multiple sclerosis. In this study, digital subtraction angiography (DSA) was used to measure the absolute CCT value in MS patients and to correlate its value to age at disease onset and duration, and to expand disability status scale (EDSS). DSA assessment was performed on eighty MS patients and on a control group of forty-four age-matched patients. CCT in MS and control groups was calculated by analyzing the angiographic images. Lesion and brain volumes were calculated in a representative group of MS patients. Statistical correlations among CCT and disease duration, age at disease onset, lesion load, brain volumes and EDSS were considered. A significant difference between CCT in MS patients (mean = 4.9s; sd = 1.27s) and control group (mean = 2.8s; sd = 0.51s) was demonstrated. No significant statistical correlation was found between CCT and the other parameters in all MS patients. Significantly increased CCT value in MS patients suggests the presence of microvascular dysfunctions, which do not depend on clinical and MRI findings. Hemodynamic changes may not be exclusively the result of a late chronic inflammatory process.     

The interleukin-1 receptor antagonist gene and the inhibitor of kappa B-like protein gene polymorphisms are not associated with myocardial infarction in Slovene population with type 2 diabetes

In this study we investigated the association of the interleukin-1 receptor antagonist gene variable number tandem repeat (IL1RN VNTR) polymorphism and of the inhibitor of kappa B-like protein (IKBL) gene polymorphism with myocardial infarction (MI) in a group of patients with type 2 diabetes. The IL1RN VNTR and the IKBL+ 738T > C gene polymorphisms were tested in 374 Caucasians: 151 cases with MI and 223 subjects with no history of coronary artery disease. The IL1RN VNTR polymorphism was not a risk factor for MI in Caucasians with type 2 diabetes (genotype 22 vs. the rest: odds ratio (OR) 1.6; 95% confidence interval (CI) = 0.8-3.5; p = 0.2). We also failed to demonstrate that IKBL+ 738T > C gene polymorphism was associated with MI in patients with type 2 diabetes (OR = 0.9; 95% CI = 0.3-2.6; p = 0.9). We provide evidence that the IL1RN VNTR and the IKBL + 738T > C gene polymorphisms are not risk factors for MI in Caucasians with type 2 diabetes.