Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.     

Apoptosis,cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Background

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods

Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results

The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions

In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

     

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.     

Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Chronic sequelae complicate convalescence from both dengue and acute viral respiratory illness

Long Covid has raised awareness of the potentially disabling chronic sequelae that afflicts patients after acute viral infection. Similar syndromes of post-infectious sequelae have also been observed after other viral infections such as dengue, but their true prevalence and functional impact remain poorly defined. We prospectively enrolled 209 patients with acute dengue (n = 48; one with severe dengue) and other acute viral respiratory infections (ARI) (n = 161), and followed them up for chronic sequelae up to one year post-enrolment, prior to the onset of the Covid-19 pandemic. Baseline demographics and co-morbidities were balanced between both groups except for gender, with more males in the dengue cohort (63% vs 29%, p<0.001). Except for the first visit, data on symptoms were collected remotely using a purpose-built mobile phone application. Mental health outcomes were evaluated using the validated SF-12v2 Health Survey. Almost all patients (95.8% of dengue and 94.4% of ARI patients) experienced at least one symptom of fatigue, somnolence, headache, concentration impairment or memory impairment within the first week of enrolment. Amongst patients with at least 3-months of follow-up, 18.0% in the dengue cohort and 14.6% in the ARI cohort experienced persistent symptoms. The median month-3 SF-12v2 Mental Component Summary Score was lower in patients who remained symptomatic at 3 months and beyond, compared to those whose symptoms fully resolved (47.7 vs. 56.0, p<0.001), indicating that patients who self-reported persistence of symptoms also experienced functionally worse mental health. No statistically significant difference in age, gender distribution or hospitalisation status was observed between those with and without chronic sequelae. Our findings reveal an under-appreciated burden of post-infection chronic sequelae in dengue and ARI patients. They call for studies to define the pathophysiology of this condition, and determine the efficacy of both vaccines as well as antiviral drugs in preventing such sequelae.     

Plasminogen activator activity is inhibited while neuroserpin is up‐regulated in the Alzheimer disease brain

Amyloid‐beta plaques are a pathological hallmark of Alzheimer’s disease. Several proteases are known to cleave/remove amyloid‐beta, including plasmin, the product of tissue plasminogen activator cleavage of the pro‐enzyme plasminogen. Although plasmin levels are lower in Alzheimer brain, there has been little analysis of the plasminogen activator/plasmin system in the brains of Alzheimer patients. In this study, zymography, immunocapture, and ELISAs were utilized to show that tissue plasminogen activator activity in frontal cortex tissue of Alzheimer patients is dramatically reduced compared with age‐matched controls, while tissue plasminogen activator and plasminogen protein levels are unchanged; suggesting that plasminogen activator activity is inhibited in the Alzheimer brain. Analysis of endogenous plasminogen activator inhibitors shows that while plasminogen activator inhibitor‐1 and protease nexin‐1 levels are unchanged, the neuroserpin levels are significantly elevated in brains of Alzheimer patients. Furthermore, elevated amounts of tissue plasminogen activator‐neuroserpin complexes are seen in the Alzheimer brain, and immunohistochemical studies demonstrate that both tissue plasminogen activator and neuroserpin are associated with amyloid‐beta plaques in Alzheimer brain tissue. Thus, neuroserpin inhibition of tissue plasminogen activator activity leads to reduced plasmin and may be responsible for reduced clearance of amyloid‐beta in the Alzheimer disease brain. Furthermore, decreased tissue plasminogen activator activity in the Alzheimer brain may directly influence synaptic activity and impair cognitive function.     

Tumor suppression by chromosome 11 is not due to cellular senescence.

Previous hybrid studies involving fusion of normal with immortal human cells indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in normal growth regulatory genes. We have further assigned 28 different immortal human cell lines to at least four complementation groups for indefinite division. In order to identify the chromosomes involved in regulating cell proliferation, we have introduced single human chromosomes by microcell fusion into immortal human cells representative of the different complementation groups. Our results demonstrate that the introduction of chromosome 11, implicated in tumor suppression, does not cause cellular senescence in three different immortal human cell lines tested.     

Flight Performance and Dispersal Potential of Red Palm Weevil Estimated by Repeated Flights on Flight Mill

The red palm weevil (Rhynchophorus ferrugineus; Olivier, 1790) (Coleoptera: Curculionidae) has recently become the most severe palm pest in the Mediterranean basin. Its dispersal was initially supported mainly by the acquisition of infested trees, but was further facilitated by the weevils’ flight. Therefore, knowledge of weevils’ flight capacity is a key element in evaluating their dispersal capability and setting preventive actions in advance. We tested the weevils’ flight ability in repeated flights that were 7–10 days apart by computer-monitored flight mill with a seesaw design. Tested flight parameters were: flight distance, duration, and velocity, number of flights, and cumulative flight distance, of virgin and mated weevils of both sexes. Our tests found no differences in flight distance between virgin and mated individuals or between sexes. Weevils showed flight capability between the ages of 2 and 97 days, and covered up to 315 km of cumulative distance during this time. In addition, we tested the effect of age of flight initiation and found that old starters perform fewer flights than young starters, and are thus assumed to possess inferior dispersal capabilities.