100 research outputs found
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OpMNPV p32, a baculovirus polyhedral envelope-associated protein : genetic location, nucleotide sequence, transcriptional mapping and immunocytochemical characterization
Baculoviruses comprise a diverse group of pathogens
infectious for members of the insect orders Lepidoptera, Hymenoptera
and Diptera. Of the three subgroups, two (subgroup A, nuclear
polyhedrosis virus (NPV) and subgroup B, granulosis virus (GV) are
occluded in a crystalline protein structure while the remaining
subgroup (subgroup C, non-occluded virus) is not. These viruses
have a large double-stranded, supercoiled DNA genome (88-160 kb)
packaged within a rod-shaped, enveloped nucleocapsid. Two viral
phenotypes are observed during the life cycle of the virus. The
polyhedra-derived virus (PDV) is packaged in polyhedra within the
cell nucleus late in infection and is responsible for primary
infection of the insect. The nucleocapsid of the budded virus (BV)
is assembled in the nucleus and acquires an envelope as it is budded
from the cell early in infection. This form of the virus is
responsible for spread of infection from cell to cell in the insect.
Very few baculovirus structural proteins have been well
characterized as to their genetic location, expression and function.
Characterization of viral and virally-associated structural proteins
will lead to a better understanding of virus structure and function
and determination of phenotype specific proteins will allow a better
understanding of phenotype regulation. The purpose of this study
was to determine the genetic location of a structural protein and
charcterize its expression and location during infection and
possibly elucidate its function.
Using a polyclonal mouse antiserum produced against purified
virions of the multicapsid nuclear polyhedrosis virus of Orgyia
pseudotsugata (OpMNPV), I identified two immunoreactive lambda gtll
clones containing non-overlapping insert DNAs which mapped to a
single open reading frame (ORF) in the HindIII-M fragment. Analysis
of nucleotide sequence data indicates that this ORF encodes a
protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing
the entire p32 ORF was constructed, and the fusion protein was
purified and used to immunize rabbits. Western blot analysis and
immunofluorescence studies using the anti-TrpE-p32 antiserum
detected a polyhedra-derived virus (PDV)-associated protein of 32
kDa at 24 hours post infection (hpi). The protein was observed in
the cytoplasm and nucleus at 24 hpi and became concentrated in the
cytoplasm late in infection. Western blot analysis and
immunofluorescent microscopy of polyhedra solubilized under various
conditions, indicated that p32 is associated with the polyhedral
envelope. The predicted amino acid sequence for p32 showed 58%
amino acid identity with the predicted amino acid sequence for an
ORF (ORF 3) in a similar region of the genome of the MNPV of
Autographa californica (AcMNPV) (Oellig et al., 1987). The
solubility properties of the p32 protein and reciprocal
immunoblotting experiments indicate the OpMNPV p32 gene encodes a
protein which is homologous to the polyhedral envelope-associated
phosphoprotein, pp34, recently reported by Whitt and Manning (1988).
In addition, the sequenced HindIII-M region of the OpMNPV
genome was transcriptionally mapped and compared to the homologous
region in the AcMNPV genome. Five ORFs (including the above
mentioned ORF 3) were identified and compared between AcMNPV and
OpMNPV and amino acid homologies of 25-70% were observed. The
comparison revealed a number of major differences in the genomes of
the two viruses. Discontinuous homology between the two viruses of
the ORF 1 gene led to the identification of a putative transposable
element in the AcMNPV ORF 1 sequence reported by Oellig et al
(1987). In addition, it was found that a region corresponding to
the 4 kb HindIII-K/EcoRl-S region of AcMNPV was not present in the
OpMNPV genome. Although the ORFs characterized in the OpMNPV
HindIII-M region all were expressed as late genes and all contained
the conserved ATAAG putative promoter/mRNA start site sequence,
primer extension analysis indicated that use of this signal for
transciptional initiation may vary between different ORFs
Exaptation of an ancient Alu short interspersed element provides a highly conserved vitamin D-mediated innate immune response in humans and primates
BackgroundAbout 45% of the human genome is comprised of mobile transposable elements or "junk DNA". The exaptation or co-option of these elements to provide important cellular functions is hypothesized to have played a powerful force in evolution; however, proven examples are rare. An ancient primate-specific Alu short interspersed element (SINE) put the human CAMP gene under the regulation of the vitamin D pathway by providing a perfect vitamin D receptor binding element (VDRE) in its promoter. Subsequent studies demonstrated that the vitamin D-cathelicidin pathway may be a key component of a novel innate immune response of human to infection. The lack of evolutionary conservation in non-primate mammals suggested that this is a primate-specific adaptation. Evidence for evolutionary conservation of this regulation in additional primate lineages would provide strong evidence that the TLR2/1-vitamin D-cathelicidin pathway evolved as a biologically important immune response mechanism protecting human and non-human primates against infection.ResultsPCR-based amplification of the Alu SINE from human and non-human primate genomic DNA and subsequent sequence analysis, revealed perfect structural conservation of the VDRE in all primates examined. Reporter gene studies and induction of the endogenous CAMP gene in Rhesus macaque peripheral blood mononuclear cells demonstrated that the VDREs were conserved functionally. In addition, New World monkeys (NWMs) have maintained additional, functional steroid-hormone receptor binding sites in the AluSx SINE that confer retinoic acid responsiveness and provide potential thyroid hormone receptor binding sites. These sites were less well-conserved during human, ape and Old World monkey (OWM) evolution and the human CAMP gene does not respond to either retinoic acid or thyroid hormone.ConclusionWe demonstrated that the VDRE in the CAMP gene originated from the exaptation of an AluSx SINE in the lineage leading to humans, apes, OWMs and NWMs and remained under purifying selection for the last 55-60 million years. We present convincing evidence of an evolutionarily fixed, Alu-mediated divergence in steroid hormone nuclear receptor gene regulation between humans/primates and other mammals. Evolutionary selection to place the primate CAMP gene under regulation of the vitamin D pathway potentiates the innate immune response and may counter the anti-inflammatory properties of vitamin D
Reply to "Overstated Claims of Efficacy and Safety. Comment On:Optimal Nutritional Status for a Well-Functioning Immune System Is an Important Factor to Protect against Viral Infections. Nutrients 2020, 12, 1181"
We thank Vorland et al [...
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Changes in gene expression related to the decline of the aging immune system
In this study, the gene expression levels between 6 month old mice and 26 month
old mice were compared. The purpose of this research was to identify genes that are
differentially regulated due to aging. The results showed 148 significantly differentiated
genes in the analysis of a total of 44,170 genes with 16 genes found to be related to
immunity and inflammation
Micronutrients to Support Vaccine Immunogenicity and Efficacy
The world has entered the third year of the coronavirus disease 2019 (COVID-19) pan-demic. Vaccination is the primary public health strategy to protect against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in addition to other measures, such as mask wearing and social distancing. Vaccination has reduced COVID-19 severity and mortality dramatically. Nevertheless, incidence globally remains high, and certain populations are still at risk for severe outcomes. Additional strategies to support immunity, including potentially enhancing the response to vaccination, are needed. Many vitamins and trace minerals have recognized immunomodulatory actions, and their status and/or supplementation have been reported to corre-spond to the incidence and severity of infection. Furthermore, a variety of observational and some interventional studies report that adequate micronutrient status or micronutrient supplementation is associated with enhanced vaccine responses, including to COVID-19 vaccination. Such data suggest that micronutrient supplementation may hold the potential to improve vaccine immunogenicity and effectiveness, although additional interventional studies to further strengthen the existing evidence are needed. Positive findings from such research could have important implications for global public health, since deficiencies in several micronutrients that support immune function are prevalent in numerous settings, and supplementation can be implemented safely and inexpensively
MyD88 and TRIF mediate the cyclic adenosine monophosphate (cAMP) induced corticotropin releasing hormone (CRH) expression in JEG3 choriocarcinoma cell line
Background: Classically protein kinase A (PKA) and transcription factor activator protein 1 (AP-1) mediate the cyclic AMP (cAMP) induced-corticotrophin releasing hormone (CRH) expression in the placenta. However enteric Gram (-) bacterial cell wall component lipopolysaccharide (LPS) may also induce-CRH expression via Toll like receptor (TLR)4 and its adaptor molecule Myd88. Here we investigated the role of MyD88, TRIF and IRAK2 on cAMP-induced CRH promoter activation in JEG3 cells in the absence of LPS/TLR4 stimulation. Methods: JEG3 cells were transfected with CRH-luciferase and Beta-galactosidase expression vectors and either empty or dominant-negative (DN)-MyD88, DN-TRIF or DN-IRAK2 vectors using Fugene6 (Roche). cAMP-induced CRH promoter activation was examined by using a luminometer and luciferase assay. Calorimetric Beta-galactosidase assays were performed to correct for transfection efficiency. Luciferase expression vectors of cAMP-downstream molecules, CRE and AP-1, were used to further examine the signaling cascades. Results: cAMP stimulation induced AP-1 and CRE promoter expression and led to dose-dependent CRH promoter activation in JEG3 cells. Inhibition of MyD88 signaling blocked cAMP-induced CRE and CRH promoter activation. Inhibition of TRIF signaling blocked cAMP-induced CRH but not CRE expression, while inhibition of IRAK2 did not have an effect on cAMP-induced CRH expression. Conclusion: MyD88 and TRIF exert direct regulatory effect on cAMP-induced CRH promoter activation in JEG3 cells in the absence of infection. MyD88 most likely interacts with molecules upstream of IRAK2 to regulate cAMP-induced CRH expression
Perspective: Role of Micronutrients and Omega-3 Long-Chain Polyunsaturated Fatty Acids for Immune Outcomes of Relevance to Infections in Older Adults:A Narrative Review and Call for Action
The immune system is weakened by advancing age, often referred to as immunosenescence, increasing the vulnerability to, and frequently the severity of, infectious diseases in older people. This has become very apparent in the current coronavirus disease 2019 (COVID-19) pandemic for which older people are at higher risk of severe outcomes, even those who are fully vaccinated. Aging affects both the innate and adaptive immune systems and is characterized by an imbalanced inflammatory response. Increasing evidence shows that optimal status of nutrients such as vitamins C, D, and E and selenium and zinc as well as the omega-3 (n-3) fatty acids DHA and EPA can help compensate for these age-related changes. While inadequate intakes of these nutrients are widespread in the general population, this is often more pronounced in older people. Maintaining adequate intakes is a challenge for them due to a range of factors such as physical, physiological, and cognitive changes; altered absorption; and the presence of noncommunicable diseases. While nutritional requirements are ideally covered by a balanced diet, this can be difficult to achieve, particularly for older people. Fortified foods and nutritional complements are effective in achieving adequate micronutrient intakes and should be considered as a safe and cost-effective means for older people to improve their nutritional status and hence support their defense against infections. Complementing the diet with a combination of micronutrients, particularly those playing a key role in the immune system such as vitamins C, D, and E and selenium and zinc as well as DHA and EPA, is recommended for older people. Optimal nutrition to support the immune system in older people will remain essential, particularly in the face of the current COVID-19 pandemic and, thus, developing strategies to ensure adequate nutrition for the growing number of older adults will be an important and cost-effective investment in the future
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Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway
The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1alpha,25
dihydroxy-vitamin D3. Recent in vitro studies suggested that curcumin and poly-unsaturated fatty
acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized
that curcumin and PUFAs would induce expression of known VDR target genes in cells. In this study,
we tested whether these compounds regulated two important VDR target genes - human cathelicidin
antimicrobial peptide (CAMP) and 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1)- in human
monocytic cell line U937, colon cancer cell line HT-29 and keratinocyte cell line HaCaT. We
demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines,
but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced
CAMP promoter activity from a luciferase reporter construct lacking the VDR binding site and did not
increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation
assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D
receptor-independent manner. We conclude that PUFAs and curcumin do not function as ligands for
the VDR.This is the authors' peer-reviewed accepted manuscript. The version of record is published and copyrighted by Elsevier and can be found at: http://www.elsevier.com/wps/find/journaldescription.cws_home/525013/description#descriptionKeywords: curcumin, vitamin D receptor, cathelicidin, poly-unsaturated fatty acid, innate immunit
In Vivo Deficiency of Both C/EBPβ and C/EBPε Results in Highly Defective Myeloid Differentiation and Lack of Cytokine Response
金沢大学医薬保健研究域医学系The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated C/EBPβ and C/EBPε double-knockout (bbee) mice and compared their phenotypes to those of single deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2–3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(−)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of LPS- and IFNγ-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct C/EBP-targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice
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