Nucleotide sequence and expression inE. coli of the complete P4 type VP4 from a G2 serotype human rotavirus

The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 inE. coli has not been achieved. In this study we present successful expression inE. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.

Regulation of Osteoblast Differentiation and Gene Expression by Phosphodiesterases and Bioactive Peptides

Bone is a mineralized tissue that enables multiple mechanical and metabolic functions to be carried out in the skeleton. Bone contains distinct cell types: osteoblasts (bone-forming cells), osteocytes (mature osteoblast that embedded in mineralized bone matrix) and the osteoclasts (bone-resorbing cells). Remodelling of bone begins early in foetal life, and once the skeleton is fully formed in young adults, almost all of the metabolic activity is in this form. Bone is constantly destroyed or resorbed by osteoclasts and then replaced by osteoblasts. Many bone diseases, i.e. osteoporosis, also known as bone loss, typically reflect an imbalance in skeletal turnover. The cyclic adenosine monophosphate (cAMP) and the cyclic guanosine monophosphate (cGMP) are second messengers involved in a variety of cellular responses to such extracellular agents as hormones and neurotransmitters. In the hormonal regulation of bone metabolism, i.e. via parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) and prostaglandin E2 signal via cAMP. cAMP and cGMP are formed by adenylate and guanylate cyclases and are degraded by phosphodiesterases (PDEs). PDEs determine the amplitudes of cyclic nucleotide-mediated hormonal responses and modulate the duration of the signal. The activities of the PDEs are regulated by multiple inputs from other signalling systems and are crucial points of cross-talk between the pathways. Food-derived bioactive peptides are reported to express a variety of functions in vivo. The angiotensin-converting enzymes (ACEs) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. The bioactive peptides offer also a nutriceutical and a nutrigenomic aspect to bone cell biology. The aim of this study was to investigate the influence of PDEs and bioactive peptides on the activation and the differentiation of human osteoblast cells. The profile of PDEs in human osteoblast-like cells and the effect of glucocorticoids on the function of cAMP PDEs, were investigated at the mRNA and enzyme levels. The effects of PDEs on bone formation and osteoblast gene expression were determined with chemical inhibitors and siRNAs (short interfering RNAs). The influence of bioactive peptides on osteoblast gene expression and proliferation was studied at the mRNA and cellular levels. This work provides information on how PDEs are involved in the function and the differentiation of osteoblasts. The findings illustrate that gene-specific silencing with an RNA interference (RNAi) method is useful in inhibiting, the gene expression of specific PDEs and further, PDE7 inhibition upregulates several osteogenic genes and increases bALP activity and mineralization in human mesenchymal stem cells-derived osteoblasts. PDEs appear to be involved in a mechanism by which glucocorticoids affect cAMP signaling. This may provide a potential route in the formation of glucocorticoid-induced bone loss, involving the down-regulation of cAMP-PDE. PDEs may play an important role in the regulation of osteoblastic differentiation. Isoleucine-proline-proline (IPP), a bioactive peptide, possesses the potential to increase osteoblast proliferation, differentiation and signalling.

Pheo-type: A diagnostic gene-expression assay for the classification of pheochromocytoma and paraganglioma

Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion

The suitability of human mesenchymal stem cells (hMSCs) in regenerative medicine relies on retention of their proliferative expansion potential in conjunction with the ability to differentiate toward multiple lineages. Successful utilisation of these cells in clinical applications linked to tissue regeneration requires consideration of biomarker expression, time in culture and donor age, as well as their ability to differentiate towards mesenchymal (bone, cartilage, fat) or non-mesenchymal (e.g., neural) lineages. To identify potential therapeutic suitability we examined hMSCs after extended expansion including morphological changes, potency (stemness) and multilineage potential. Commercially available hMSC populations were expanded in vitro for > 20 passages, equating to > 60 days and > 50 population doublings. Distinct growth phases (A-C) were observed during serial passaging and cells were characterised for stemness and lineage markers at representative stages (Phase A: P+5, approximately 13 days in culture; Phase B: P+7, approximately 20 days in culture; and Phase C: P+13, approximately 43 days in culture). Cell surface markers, stem cell markers and lineage-specific markers were characterised by FACS, ICC and Q-PCR revealing MSCs maintained their multilineage potential, including neural lineages throughout expansion. Co-expression of multiple lineage markers along with continued CD45 expression in MSCs did not affect completion of osteogenic and adipogenic specification or the formation of neurospheres. Improved standardised isolation and characterisation of MSCs may facilitate the identification of biomarkers to improve therapeutic efficacy to ensure increased reproducibility and routine production of MSCs for therapeutic applications including neural repair.

PIM2 Induced COX-2 and MMP-9 Expression in Macrophages Requires PI3K and Notch1 Signaling

Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor-kappa B (NF-kappa B) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of uppressor of Hairless (CSL) and NF-kappa B to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

Regulation of Kinin receptor Expression in Heart Failure with a Focus on Vascular Endothelium

Accumulating evidence show that kinins, notably bradykinin (BK) and kallidin, have cardioprotective effects. To these include reduction of left ventricular hypertrophy (LVH) and progression of heart failure. The effects are mediated through two G protein-coupled receptors- bradykinin type-2 receptor (BK-2R) and bradykinin type -1 receptor (BK-1R). The widely accepted cardioprotective effects of BK-receptors relate to triggering the production and release of vasodilating nitric oxide (NO) by endothelial cells. They also exert anti-proliferative effects on fibroblasts and anti-hypertrophic effects on myocytes, and thus may play an essential role in the cardioprotective response to myocardial injury. The role for BK-1Rs in HF is based on experimental animal models, where the receptors have been linked to cardioprotective- but also to cardiotoxic -effects. The BK-1Rs are induced under inflammatory and ischemic conditions, shown in animal models; no previous reports, concerning BK-1Rs in human heart failure, have been presented. The expression of BK-2Rs is down-regulated in human end-stage heart failure. Present results showed that, in these patients, the BK-1Rs were up-regulated, suggesting that also BK-1Rs are involved in the pathogenesis of human heart failure. The receptors were localized mainly in the endothelium of intramyocardial coronary vessels, and correlated with the increased TNF-α expression in the myocardial coronary vessels. Moreover, in cultured endothelial cells, TNF-α was a potent trigger of BK-1Rs. These results suggest that cytokines may be responsible for the up-regulation of BK-1Rs in human heart failure. A linear relationship between BK-2R mRNA and protein expression in normal and failing human left ventricles implies that the BK-2Rs are regulated on the transcriptional level, at least in human myocardium. The expression of BK-2Rs correlated positively with age in normal and dilated hearts (IDC). The results suggest that human hearts adapts to age-related changes, by up-regulating the expression of cardioprotective BK-2Rs. Also, in the BK-2R promoter polymorphism -58 T/C, the C-allele was accumulated in cardiomyopathy patients which may partially explain the reduced number of BK-2Rs. Statins reduce the level of plasma cholesterol, but also exert several non-cholesterol-dependent effects. These effects were studied in human coronary arterial endothelial cells (hCAEC) and incubation with lovastatin induced both BK-1 and BK-2Rs in a time and concentration-dependent way. The induced BK-2Rs were functionally active, thus NO production and cGMP signaling was increased. Induction was abrogated by mevalonate, a direct HMG-CoA metabolite. Lovastatin is known to inhibit Rho activation, and by a selective RhoA kinase inhibitor (Y27632), a similar induction of BK-2R expression as with lovastatin. Interestingly a COX-2-inhibitor (NS398) inhibited this lovastatin-induction of BK-2Rs, suggesting that COX-2 inhibitors may affect the endothelial BK-2Rs, in a negative fashion. Hypoxia is a common denominator in HF but also in other cardiovascular diseases. An induction of BK-2Rs in mild hypoxic conditions was shown in cultured hCAECs, which was abolished by a specific BK-2R inhibitor Icatibant. These receptors were functionally active, thus BK increased and Icatibant inhibited the production of NO. In rat myocardium the expression of BK-2R was increased in the endothelium of vessels, forming at the border zone, between the scar tissue and the healthy myocardium. Moreover, in in vitro wound-healing assay, endothelial cells were cultured under hypoxic conditions and BK significantly increased the migration of these cells and as Icatibant inhibited it. These results show, that mild hypoxia triggers a temporal expression of functionally active BK-2Rs in human and rat endothelial cells, supporting a role for BK-2Rs, in hypoxia induced angiogenesis. Our and previous results show, that BK-Rs have an impact on the cardiovascular diseases. In humans, at the end stage of heart failure, the BK-2Rs are down-regulated and BK-1Rs induced. Whether the up-regulation of BK-1Rs, is a compensatory mechanism against the down-regulation of BK-2Rs, or merely reflects the end point of heart failure, remains to bee seen. In a clinical point of view, the up-regulation of BK-2Rs, under hypoxic conditions or statin treatment, suggests that, the induction of BK-2Rs is protective in cardiovascular pathologies and those treatments activating BK-2Rs, might give additional tools in treating heart failure.

Distress call-induced gene expression in the brain of the Indian short-nosed fruit bat, Cynopterus sphinx

Individuals in distress emit audible vocalizations to either warn or inform conspecifics. The Indian short-nosed fruit bat, Cynopterus sphinx, emits distress calls soon after becoming entangled in mist nets, which appear to attract conspecifics. Phase I of these distress calls is longer and louder, and includes a secondary peak, compared to phase II. Activity-dependent expression of egr-1 was examined in free-ranging C. sphinx following the emissions and responses to a distress call. We found that the level of expression of egr-1 was higher in bats that emitted a distress call, in adults that responded, and in pups than in silent bats. Up-regulated cDNA was amplified to identify the target gene (TOE1) of the protein Egr-1. The observed expression pattern Toe1 was similar to that of egr-1. These findings suggest that the neuronal activity related to recognition of a distress call and an auditory feedback mechanism induces the expression of Egr-1. Co-expression of egr-1 with Toe1 may play a role in initial triggering of the genetic mechanism that could be involved in the consolidation or stabilization of distress call memories.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light.

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA-treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had no detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These data indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.