The objective of this study was to evaluate the effect of Lipopolysaccharide (LPS) and peptidoglycan (PGN) on galectin-3 expression and secretion in goat blood. Meat goats are exposed to various pathogens while grazing on pasture. Pathogen-associated molecular patterns (PAMPs) from the outer membrane of gram-negative and gram-positive bacteria such as E. coli and S. aureus cause inflammatory diseases in goats. The importance of host and pathogen galectins in health is recognized. Galectin-3 is a β-galactoside-binding protein that plays an important role in pathogen recognition and hosts inflammatory diseases. Three (3) BoerXSpanish goats from the North Carolina Agricultural and Technical State University farm were used. Whole blood (5ml) was collected from the jugular vein into tubes containing acid citrate dextrose. One mL of blood was treated with 10µg/ml of LPS, 10µg/ml of PGN in 200µl of PBS. Treatment with PBS alone served as control. Total plasma was collected and secretion of galectin-3 was detected using goat Gal-3 specific ELISA. Total RNA was isolated using Trizol and converted to cDNA. Specific primers for goat LGAL-3 was used for real-time PCR. Beta-actin and GAPDH housekeeping genes were used as internal controls. Fold changes(FC) in transcript abundance was compared to control and calculated using the Livak method. Data were analyzed using PROC GLM model in SAS 9.4 version (p<0.05). Treatment with LPS decreased transcription of LGal-3 (FC=-4.47) and increased secretion of Gals-3(p<0.01). Treatment with PGN increased expression LGAL-3 (FC=2) and decreased the secretion of Gals-3 (p<0.01). Distinct patterns of modulation of galectin-3 were observed in response to PAMPS from gram-negative and gram-positive bacteria. These results have implications for the use of galectin-3 based diagnostics and interventions in goats and their role in inflammatory disease warrants further study.
Pathogenesis and lack of effective therapeutics pose a threat to animal and public health. Recognition of pathogen-associated molecular patterns (PAMPS) by Toll-like receptors is associated with gene activation. Galectins are sugar-binding proteins that play a role in pathogen-recognition and immune modulation. The objective of this study was to investigate the effect of different TLR agonists on galectin gene expression and secretion in goat blood. Blood was collected from the jugular vein of BoerXSpanish goats (n=3). Blood was treated with 10 µg/ml Lipopolysaccharide, Peptidoglycan, 12.5 µg/ml of Polyinosinic-polycytidylic acid, 10 µg/ml CpG ODN (2216) and 10 µg/ml CpG ODN (2006) or PBS control. Secretion of galectin-1, -3 and -9 was measured by ELISA. Expression of LGALS-1, -2, -3, -4, -7, -8, -9, -11, -12, -14, -15, -16 was assessed using real-time PCR. Beta-actin and GAPDH served as controls. Fold change in gene expression was calculated using the 2−ΔΔCt method. Data were analyzed using SAS 9.4 statistical analysis software (p < 0.05). Expression and secretion galectins was modulated by different PAMPS (p < 0.05) and may have implications for design of therapeutics based on TLR agonists.
ABSTRACT Neuronal mitochondria play important roles beyond ATP generation, including Ca 2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly in the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer- specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Camkk2- dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a new activity- dependent molecular mechanism underlying the extreme subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise regulation of mitochondria fission/fusion balance.
The aim of this study was to evaluate the expression of galectins in cow blood and to evaluate their modulation in periparturient cows at different stages of lactation. Galectins are multipotent, evolutionarily conserved, carbohydrate- binding proteins that, by crosslinking cell surface glycoconjugates, trigger a cascade of transmembrane signaling events such as cell activation, cytokine secretion, migration, and apoptosis There are 15 galectin protein subtypes that all share the shared characteristic of AA sequences and affinity for β-galactoside sugars. Galectins are known to have an impact on immunomodulation and are involved in uterine immunoregulation during pregnancy. The cows were grouped into 3 lactation periods (first, second, and third lactations). Blood was taken 2 wk close to parturition (close up) and 7 d after parturition(c+7) at Michigan State University dairy farm and shipped in Paxgene tubes for analysis. Total RNA was isolated, reverse transcribed to cDNA, and then used in real-time PCR experiments. With the use of Primer-Blast from the National Center for Biotechnology Information, specific primers for galectins 1, 2, 3, 4, 7, 8, 9, and 12 and β-actin (forward and reverse) were sequenced and used for this project. β-actin was used as internal control. Fold change in transcript abundance was calculated using the Livak method. In first-lactation cows, Galectin 1 was turned off after parturition. Galectin 2 was absent in both close up and c+7 cows. Galectins 3 and 7 were present in both close up and c+7 cows but levels did not change after parturition (fold change < 2). Galectin 4 was present before parturition but absent a week after parturition, Galectin 9 expression increased after parturition (fold change = 2). Galectin 12 was turned off after parturition. In the second-lactation cows, Galectin 1 was turned off after parturition, and Galectins 2, 3, 7, and 9 increased in transcription after parturition (fold change > 2). Galectins 1, 4, 8, and 12 were present before and after parturition but their fold changes were not significant. In third-lactation cows, all 8 galectins except for Galectin 8 were detected in both close ups and c+7 cows. Expression levels for these galectin genes did not change (fold change < 2). All genes tested were expressed in cow blood at varying levels. It is clear from this study that galectin gene expression is affected by stage of lactation and parturition. Further studies are needed to determine the factors that contribute to the different galectin expressions in cow blood during the periparturient period.
The objective of this study was to investigate the effect of crude cowpea polyphenol extract (CPE) on the expression of genes involved in the inflammatory response in bovine blood in vitro. Plant-derived polyphenols in animal feeds are being used as alternatives to antibiotics to treat and prevent invading microbes. These compounds have antioxidant and anti-inflammatory properties and are able to modulate immune and inflammation responses. Whole blood collected from lactating Holstein-Friesian cows (n = 10) were incubated with 10 µg of CPE for 60 min at 37°C and 5% CO2.Total RNA was extracted from whole blood after incubation and reverse transcribed to cDNA, and quantitative PCR (qPCR) was performed using the cow inflammatory cytokines and receptor array (Qiagen) with 84 genes. The qPCR data were analyzed using Livak's method to calculate fold change in gene expression between CPE-treated and control cows. Normalization of data was performed with GAPDH as an internal control. Out of the 84 genes tested, 81 were expressed, 13 were upregulated and 68 were downregulated, in response to CPE. Treatment with CPE downregulated the expression of proinflammatory cytokine TNFα (fold change [FC; treatment/control] = −43.39), IL1α (FC = −6.19), ILβ (FC = -3.62), and IL8 (FC = −1.25). Expression of chemokines such as CXCL10, CXCL12, and CXCR2 was not altered by treatment with CPE. Interestingly, expression of IL10RA (FC = 3.39), a receptor for IL10, a well-known anti-inflammatory cytokine, was upregulated in blood incubated with CPE. IL15, a cytokine that regulates T and natural killer cell activation and proliferation, was upregulated (FC = 2.08) by CPE treatment. The study results demonstrate that polyphenols derived from cowpea have an anti-inflammatory effect in cow blood, and target genes modulated by CPE have been identified for further characterization.
ABSTRACT HELB is a human helicase involved in initiation of DNA replication, the replication stress response, and regulation of double-strand DNA break repair. rs75770066 is a rare SNP in the HELB gene that affects age at natural menopause. rs75770066 results in a D506G substitution in an acidic patch within the 1A domain of the helicase that is known to interact with RPA. We found that this amino acid change dramatically impairs the cellular function of HELB. D506G-HELB exhibits impaired interaction with RPA, which likely results in the effects of rs75770066 as this reduces recruitment of HELB to sites of DNA damage. Reduced recruitment of D506G-HELB to double-strand DNA breaks and the concomitant increase in homologous recombination likely alters the levels of meiotic recombination, which affects the viability of gametes. Because menopause occurs when oocyte levels drop below a minimum threshold, altered repair of meiotic double-stranded DNA breaks has the potential to directly affect the age at natural menopause.
Pathogen-associated Molecular Patterns (PAMPs) are highly conserved structural motifs that are recognized by Pathogen Recognition receptors (PRRs) to initiate immune responses. Infection by these pathogens and the immune response to PAMPS such as lipopolysaccharide (LPS), Peptidoglycan (PGN), bacterial oligodeoxynucleotides [CpG oligodeoxynucleotides 2006 (CpG ODN2006) and CpG oligodeoxynucleotides 2216 (CpG ODN2216)], and viral RNA Polyinosinic-Polycytidylic Acid (Poly I:C), are associated with infectious and metabolic diseases in animals impacting health and production. It is established that PAMPs mediate the production of cytokines by binding to PRRs such as Toll-like receptors (TLR) on immune cells. Galectins (Gal) are carbohydrate-binding proteins that when expressed play essential roles in the resolution of infectious and metabolic diseases. Thus it is important to determine if the expression of galectin gene (LGALS) and Gal secretion in blood are affected by exposure to LPS and PGN, PolyI:C and bacterial CpG ODNs. LPS increased transcription of LGALS4 and 12 (2.5 and 2.02 folds respectively) and decreased secretion of Gal 4 (p < 0.05). PGN increased transcription of LGALS-1, -2, -3, -4, -7, and -12 (3.0, 2.3, 2.0, 4.1, 3.3, and 2.4 folds respectively) and secretion of Gal-8 and Gal-9 (p < 0.05). Poly I:C tended to increase the transcription of LGALS1, LGALS4, and LGALS8 (1.78, 1.88, and 1.73 folds respectively). Secretion of Gal-1, -3, -8 and nine were significantly increased in treated samples compared to control (p < 0.05). CpG ODN2006 did not cause any significant fold changes in LGALS transcription (FC < 2) but increased secretion of Gal-1, and-3 (p < 0.05) in plasma compared to control. Gal-4 was however reduced in plasma (p < 0.05). CpG ODN2216 increased transcription of LGALS1 and LGALS3 (3.8 and 1.6 folds respectively), but reduced LGALS2, LGALS4, LGALS7, and LGALS12 (-1.9, -2.0, -2.0 and; -2.7 folds respectively). Secretion of Gal-2 and -3 in plasma was increased compared to control (p < 0.05). Gal-4 secretion was reduced in plasma (p < 0.05). The results demonstrate that PAMPs differentially modulate galectin transcription and translation of galectins in cow blood.
<p>Programs based on antibiotics are failing to control diseases due to increase in resistance of pathogens to antibiotics. Food safety, animal welfare and public health concerns have fueled interest in the use of plant-based alternatives. This study was conducted to evaluate the effect of a plant (Sericea Lespedeza, SL), and pathogen associated molecular patterns (PAMPs) (Lipopolysaccharide (LPS) and peptidoglycan (PGN)) on gene activation in ruminant blood. A water extract of SL, was used as a source of plant-derived tannins. Blood was collected from Holstein-Friesian cows (N = 4), Spanish × Boer goats (N = 4), St Croix sheep (N = 4) and incubated with 100 ng/mL of SL in the presence or absence of LPS or PGN. Samples maintained in Phosphate-buffered saline (PBS) served as negative control. The total protein concentration, WNT5a, and prostaglandin E2 in plasma were determined. Total RNA was isolated, reverse transcribed and Real time-PCR was performed using gene specific primers for TLR2, TLR4, WNT5a, and FZD. TLR2 and FZD were up-regulated in response to PAMPs. WNT5a and TLR4 genes were undetected in PAMP treated blood. SL regulated protein and prostaglandin concentration in all species. SL reduced PGE2 in sheep and cow blood. WNT5a was only secreted in LPS treated cow blood. Transcription and translation of genes involved in innate and adaptive immunity and the WNT signaling pathway in ruminant blood were responsive to diverse PAMPS, and can be modulated by SL. This suggests that dietary tannins may promote the health of ruminants. Further studies are needed to determine the significance of these changes in immune gene expression on ruminant health.</p>
ABSTRACT Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs) dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating ATP and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stresses such as oxidative stress, or in various neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address the pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show a difference between mitochondrial morphology when samples were fixed with PFA/glutaraldehyde or PFA/sucrose containing solutions, but not PFA alone. Our study provides optimized methods for examining mitochondrial morphology in neurons, and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.
