Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing

Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells

Background: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. Results: We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. Conclusion: The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux

Axonal injury in asymptomatic individuals preceding onset of multiple sclerosis

Axonal loss is the main cause of irreversible disability in multiple sclerosis (MS). Serum neurofilament light (sNfL) is a biomarker of axonal disintegration. In this nested case-control study, blood samples from 519 presymptomatic persons (age range 4-39 years) who later received an MS diagnosis showed higher sNfL concentrations than 519 matched controls (p < 0.0001), noticeable at least 10 years before clinical MS onset. Mean values for pre-MS and control groups were 9.6 pg/mL versus 7.4 pg/mL 0-5 years before onset, and 6.4 pg/mL versus 5.8 pg/mL 5-10 years before onset. These results support that axonal injury occurs early in MS pathogenesis

Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana

Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation.QC 20170320</p

Russia in a changing climate

Climate change will shape the future of Russia, and vice versa, regardless of who rules in the Kremlin. The world\u27s largest country is warming faster than Earth as a whole, occupies more than half the Arctic Ocean coastline, and is waging a carbon-intensive war while increasingly isolated from the international community and its efforts to reduce greenhouse gas emissions. Officially, the Russian government argues that, as a major exporter of hydrocarbons, Russia benefits from maintaining global reliance on fossil fuels and from climate change itself, because warming may increase the extent and quality of its arable land, open a new year-round Arctic sea route, and make its harsh climate more livable. Drawing on the collective expertise of a large group of Russia-focused social scientists and a comprehensive literature review, we challenge this narrative. We find that Russia suffers from a variety of impacts due to climate change and is poorly prepared to adapt to these impacts. The literature review reveals that the fates of Russia\u27s hydrocarbon-dependent economy, centralized political system, and climate-impacted population are intertwined and that research is needed on this evolving interrelationship, as global temperatures rise and the international economy decarbonizes in response. This article is categorized under: Policy and Governance \u3e National Climate Change Policy Trans-disciplinary Perspectives \u3e National Reviews Trans-disciplinary Perspectives \u3e Regional Reviews. © 2023 The Authors. WIREs Climate Change published by Wiley Periodicals LLC

Greater impairment of postprandial triacylglycerol than glucose response in metabolic syndrome subjects with fasting hyperglycaemia

Abstract Objective: Studies have started to question whether a specific component or combinations of metabolic syndrome (MetS) components may be more important in relation to cardiovascular disease risk. Our aim was to examine the impact of the presence of raised fasting glucose as a MetS component on postprandial lipaemia. Methods: Men classified with the MetS underwent a sequential test meal investigation, in which blood samples were taken at regular intervals after a test breakfast (t=0 min) and lunch (t=330 min). Lipids, glucose and insulin were measured in the fasting and postprandial samples. Results: MetS subjects with 3 or 4 components were subdivided into those without (n=34) and with (n=23) fasting hyperglycaemia (≥ 5.6 mmol/l), irrespective of the combination of components. Fasting lipids and insulin were similar in the two groups, with glucose significantly higher in the men with glucose as a MetS component (P<0.001). Following the test meals, there was a higher maximum concentration (maxC), area under the curve (AUC) and incremental AUC (P≤0.016) for the postprandial triacylglycerol (TAG) response in men with fasting hyperglycaemia. Greater glucose AUC (P<0.001) and insulin maxC (P=0.010) was also observed in these individuals after the test meals. Multivariate regression analysis revealed fasting glucose to be an important predictor of the postprandial TAG and glucose response. Conclusion: Our data analysis has revealed a greater impairment of postprandial TAG than glucose response in MetS subjects with raised fasting glucose. The worsening of postprandial lipaemic control may contribute to the greater CVD risk reported in individuals with MetS component combinations which include hyperglycaemia

Effect of natalizumab on disease progression in secondary progressive multiple sclerosis (ASCEND). a phase 3, randomised, double-blind, placebo-controlled trial with an open-label extension

Background: Although several disease-modifying treatments are available for relapsing multiple sclerosis, treatment effects have been more modest in progressive multiple sclerosis and have been observed particularly in actively relapsing subgroups or those with lesion activity on imaging. We sought to assess whether natalizumab slows disease progression in secondary progressive multiple sclerosis, independent of relapses. Methods: ASCEND was a phase 3, randomised, double-blind, placebo-controlled trial (part 1) with an optional 2 year open-label extension (part 2). Enrolled patients aged 18–58 years were natalizumab-naive and had secondary progressive multiple sclerosis for 2 years or more, disability progression unrelated to relapses in the previous year, and Expanded Disability Status Scale (EDSS) scores of 3·0–6·5. In part 1, patients from 163 sites in 17 countries were randomly assigned (1:1) to receive 300 mg intravenous natalizumab or placebo every 4 weeks for 2 years. Patients were stratified by site and by EDSS score (3·0–5·5 vs 6·0–6·5). Patients completing part 1 could enrol in part 2, in which all patients received natalizumab every 4 weeks until the end of the study. Throughout both parts, patients and staff were masked to the treatment received in part 1. The primary outcome in part 1 was the proportion of patients with sustained disability progression, assessed by one or more of three measures: the EDSS, Timed 25-Foot Walk (T25FW), and 9-Hole Peg Test (9HPT). The primary outcome in part 2 was the incidence of adverse events and serious adverse events. Efficacy and safety analyses were done in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01416181. Findings: Between Sept 13, 2011, and July 16, 2015, 889 patients were randomly assigned (n=440 to the natalizumab group, n=449 to the placebo group). In part 1, 195 (44%) of 439 natalizumab-treated patients and 214 (48%) of 448 placebo-treated patients had confirmed disability progression (odds ratio [OR] 0·86; 95% CI 0·66–1·13; p=0·287). No treatment effect was observed on the EDSS (OR 1·06, 95% CI 0·74–1·53; nominal p=0·753) or the T25FW (0·98, 0·74–1·30; nominal p=0·914) components of the primary outcome. However, natalizumab treatment reduced 9HPT progression (OR 0·56, 95% CI 0·40–0·80; nominal p=0·001). In part 1, 100 (22%) placebo-treated and 90 (20%) natalizumab-treated patients had serious adverse events. In part 2, 291 natalizumab-continuing patients and 274 natalizumab-naive patients received natalizumab (median follow-up 160 weeks [range 108–221]). Serious adverse events occurred in 39 (13%) patients continuing natalizumab and in 24 (9%) patients initiating natalizumab. Two deaths occurred in part 1, neither of which was considered related to study treatment. No progressive multifocal leukoencephalopathy occurred. Interpretation: Natalizumab treatment for secondary progressive multiple sclerosis did not reduce progression on the primary multicomponent disability endpoint in part 1, but it did reduce progression on its upper-limb component. Longer-term trials are needed to assess whether treatment of secondary progressive multiple sclerosis might produce benefits on additional disability components. Funding: Biogen