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Aims/hypothesis Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. Methods The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption–ionisation-time of flight (MALDI–TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. Results Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. Conclusions/interpretation We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes. [ABSTRACT FROM AUTHOR]

Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1β, TNF-α, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1β signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor κB (NF-κB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-κB activation similar to that occurring with palmitate. Saturated-fatty-acid-induced NF-κB activation and endoplasmic reticulum stress may contribute to IL-1β production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release. [ABSTRACT FROM AUTHOR]

A full-length cDNA clone of olive latent virus 1 (OLV-1), a member of the genus Necrovirus, family Tombusviridae, was subjected to site-directed mutagenesis, and coat protein gene mutants were constructed. A mutant clone, denoted Δ3297, was obtained by deleting the nucleotide in position 3297, thus inducing a frameshift and replacing the last 49 amino acids of the viral coat protein (CP) by a shorter sequence of 39 amino acids. This mutant was viable, stable, able to synthesize a smaller CP, and able to give rise to the formation of apparently intact virus particles. Cell-to-cell movement of Δ3297 in Nicotiana benthamiana leaves was not affected, but, contrary to wild type OLV-1, it failed to spread systemically. These results indicate that virion formation is necessary but not sufficient for long-distance movement for OLV-1 and highlights the role of the CP carboxy-terminal domain in systemic infection. [ABSTRACT FROM AUTHOR]

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was oberved in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata. [ABSTRACT FROM AUTHOR]

Summary. The complete nucleotide sequence of Olive latent ringspot virus (OLRSV) RNA-2 was determined. This RNA is 3969 nucleotides in length and contains a single open reading frame of 3448 nt, that encodes a polypeptide of 1146 amino acids, with a calculated Mr of 126,044. OLRSV RNA-2 has a structural organization typical of nepoviruses, with the coat protein (CP) cistron located in the C-terminal and the putative movement protein (MP) in the N-terminal regions of the polyprotein. Computer-assisted comparison of coat proteins of OLRSV and other nepoviruses disclosed relationships that tally with subgrouping based onphysicochemical properties. [ABSTRACT FROM AUTHOR]

Genomic RNA of olive latent virus 1 (OLV-1) contains five open reading frames (ORFs) encoding proteins of 23, 82, 8, 6 and 30 (CP) kDa. A full-length cDNA copy of OLV-1 RNA was prepared and cloned in a low-copy-number vector (pMUC-19) downstream of or T7 RNA polymerase promoter. Transcripts derived from this template, denoted pMUC-OLV, were highly infectious when inoculated in local and systemic hosts and infected tissues contained virus-like particles. Genes required for replication and virus movement were mapped by site-directed and deletion mutogenesis of the pMUC-OLV. ORF1 and ORF2 mutants were not viable, suggesting that replication requires the 23 and 82 kDa proteins. The 8 and 6 kDa polypeptides were involved in cell-to-cell movement, since their absence did not interfere with RNA replication but prevented systemic infection of inoculated plants. Mutant clones in R and S domains of the CP gene could replicate, but they did not systemically infect Nicotiana benthamiana, indicating that the CP gene is required for OLV-1 long-distance translocation. Mutant clones with large deletions in the CP gene were not viable, probably due to loss of 3′-proximal sequences required for RNA replication. [ABSTRACT FROM AUTHOR]

The 3699 nt genome of olive latent virus 1 (OLV-1), described years ago from Southern Italy as a putative sobemovirus, was completely sequenced. OLV-1 genomic RNA was not polyadenylated and had a structure virtually identical to that of species of the Necrovirus rather than the Sobemovirus genus. Five open reading frames (ORFs) were identified, of which the 5′-proximal encoded a 23 K protein and ended with an amber codon whose readthrough could yield a putative 82 K product. This polypeptide had extensive sequence similarity with polymerases of serotypes A and D of tobacco necrosis necrovirus (TNV-A and TNV-D) and species of the family Tombusviridae and related genera ( Dianthovirus and Machlomovirus). Two small ORFs followed, which encoded polypeptides of 8 K and 6 K, respectively. The 6 K product had extensive homology with the comparable 6 K protein of TNV-A and was also related to the 11 K protein of shallot latent carlavirus, one of the 'triple block' polypeptides involved in cell-to-cell virus movement. The 3′-proximal ORF was in the same position as the coat protein (CP) cistron of necroviruses and encoded a 30 K product related to CP of both TNV-A and -D. Computer-assisted comparative analysis of structural and non-structural proteins of OLV-1, TNV-A and TNV-D disclosed an overall distant relationship between OLV-1 and TNV-D. OLV-1 genome appeared homologous to that of TNV-A, but differences from TNV-A were the absence of the small ORF downstream of the CP cistron and in the low degree of sequence identity in CP (39% aa identity). OLV-1 is serologically distantly related to TNV-A and even more distantly related to TNV-D. We propose that OLV-1 is a necrovirus species in its own right. [ABSTRACT FROM AUTHOR]

The 3′ terminal region of grapevine virus A (GVA) and grapevine virus B (GVB), encompassing 1883 and 2136 nucleotides, respectively, was sequenced by the deoxynucleotide chain termination method. Three putative open reading frames (ORF) were identified in both genomic viral RNAs, denoted 1 to 3 in the 5′ to 3′ direction. ORF 1 encoded a polypeptide with estimated M of 31 kDa (GVA) and 36.5 kDa (GVB), possessing the G/D motif of the '30 K superfamily' movement proteins, and showing good alignments with putative movement proteins of trichoviruses and capilloviruses. ORF 2 was identified as the coat protein (CP) cistron, coding for polypeptides with an estimated M of 21.5 kDa (GVA) and 21.6 kDa (GVB). These CPs showed substantial sequence homology with one another and with CPs of tricho- and capilloviruses, but not of closteroviruses. ORF 3 potentially coded for two small polypeptides with estimated M of 10 kDa (GVA) and 14 kDa (GVB). The ORF 3 product of GVB (14 K), but not that of GVA, shared some homology with the 3′ terminal polypeptides of different plant viruses that exhibit the 'zinc finger domain' of proteins with nucleic acid-binding properties. GVA and GVB have many properties in common with trichoviruses but possess an extra open reading frame (ORF 3). Whether this finding may have a bearing on the classification of these viruses is unclear. However, until the taxonomic significance of this difference in genome structure is established, it seems plausible to include GVA and GVB as tentative species in the Trichovirus genus. [ABSTRACT FROM AUTHOR]