Expression of transient receptor potential vanilloid (TRPV) channels in different passages of articular chondrocytes

Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV) subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0) and cells from passages 1–3 (P1, P2 and P3) by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes

Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes

The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca2+-activated K+ (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca2+ and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P < 0.001)

Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes.

The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca(2+) and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P < 0.001)

Vitamin D mitigates adult onset diseases in male and female mice induced by early-life exposure to endocrine disruptor BPA

Background: During early development, environmental compounds can induce adult onset diseases and disrupt thecirculating vitamin D (VitD) levels.Aim: This study aimed to examine the protective role of VitD against the adverse effects of BPA on male and female mice.Methods: A total of 60 male and female Swiss Albino mice (3 weeks old) were randomly divided into 5 groups; each consisted of 12 mice (6 males and 6 females) and was treated as follows: Group I received no treatment (sham control); Group II, sterile corn oil only (vehicle control); Group III, BPA (400 μg/kg); Group IV, VitD (2,195 IU/kg); and Group V, BPA + VitD. At 10.5 weeks, the animals were sacrificed to conduct histological examinations.Results: BPA-exposed mice were found to have neurobehavioral abnormalities, heart, kidney, and lung diseases with increased apoptotic indices in both sexes. On the other hand, the treatment of BPA mice with VitD altered this scenario with modulated motor activity, enhanced body and organ weights, and preserved the heart, kidney, and lung architecture, alongside a decreased percent apoptotic index.Conclusion: Our findings illustrate that VitD protects mice against BPA-induced heart, kidney, and lung abnormalities

Expression of Transient Receptor Potential Vanilloid (TRPV) channels in different passages of articular chondrocytes.

Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV) subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0) and cells from passages 1-3 (P1, P2 and P3) by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes

Technological Economic for an Aggregate During Modernization Parameters of Milk Traits Weights of Healthy Dairy Cattle

One of the conditions for increasing the efficiency of milk production is the modernization of farms with the introduction of technologies for loose maintenance and maintenance of livestock. Mainly the transition to a loose-fitting content allows to significantly reduce labour costs and the number of employees, to facilitate the work of operators directly servicing animals. At the same time, problems of mechanization and automation of processes are more easily solved. Productive longevity, expressed as the number of lactations per year or the milk yield per year during the lifetime, is a function of three or four variables: age at the first calving, calving interval, number of lactations during the lifetime and, respectively, milk / fat / protein yield in the standard lactation. Because these two functions are not linear, their expressions are approximated as linear functions of the considered variables. These functions express the phenotypic value of the trait, the number of lactation per year or the milk yield per year during the lifetime. Each of these functions is used to define an aggregate genotype.            The calculated aggregate genotypes put together usual traits, which are difficult to be used in an index-based selection. If these traits are genetically evaluated, the selection index for an animal results directly from the aggregate genotype by replacing the true breeding values with the estimated breeding values. The expected number of lactations may substitute the number of lactations of a cow, which did not finish its career

Genomic Technologies in Improving the Efficiency of Animal Health and Husbandry

One of the reasons for this lag in the practical application of the accumulated technologies seems to be the lack of a concept of successive stages of their application in order to increase efficiency and reduce the time for traditional selection work. The first stage of such a technological chain should be a set of methods that allows selecting and selecting animals with an increased likelihood of the desired manifestation of the characteristics of productivity and adaptive potential in their offspring. In order to forecast productivity at the present time, particularly in dairy cattle breeding, "genomic selection" methods are used when, based on the genomic scanning by mononucleotide substitutions (SNP), groups of polylocus genotypes are combined, combining bulls with high milk productivity of their daughters. Genetic engineering is the name of a group of techniques used to identify, replicate, modify and transfer the genetic material of cells, tissues or complete organisms. Important applications of genetic engineering in animal breeding are: 1) Marker-assisted selection (MAS). The objective of this technology is to increase disease resistance, productivity and product quality in economically important animals by adding information of DNA markers to phenotypes and genealogies for selection decisions. 2) Transgenesis, the direct transfer of specific genes/alleles between individuals, species, or even Kingdoms, in order to change their phenotypic expression in the recipients. Compared to the 'traditional' improvement techniques based on phenotypic information only, these gene-by-gene techniques allow theoretically a more complete management of animal genomes for animal breeding. In spite of high expectations and new technical developments, its actual efficiency is not always high, as they require a thorough knowledge of functional genomics, and pose additional technical, economical and ethical problems. The possible role for cloning adult animals in breeding is also discussed

Selection of Healthy and Highly Productive Dairy Cattle

Dairy cow breeding has become highly complex as progress is made into the understanding of which genes are responsible for which traits and how well a particular trait is passed from one generation to the next. The impact of industrial use of cows on following genetic parameters: phenotypic variability, correlation, repeatability and heritability of economically useful traits in the high producing dairy cows herd of black-motley breed with the high level of Holsteins has been studied. After setting the breeding objectives it is important to decide which traits can be used to select individuals for breeding, and these traits being known as selection criteria. With the increasing the period of use cows up to 7 or more lactations, coefficients of variation of all monitored indicators declined in connection with the removal of cows with extreme indicators of productivity and longevity during first 3-4 lactations. The correlation coefficients of milk yields during the first, highest lactation and in lifetime, regardless of the period of use is closely related to the life-long milk yield, milk yield per day of productive life and milk yield per day of life. The heritability coefficients (h²) of the indicators of longevity with increasing period of use cows up to 5 or more lactations has tended to increase: by the age of first calving from 0.01 to 0.16, by the number of highest lactation from 0.18 to 0.33, by milking days from 0.10 to 0.30, by lifetime milk yields from 0.18 to 0.32, and by days of life from 0.01 up to 0.21. The highest repeatability of indicators of milk yield, milk fat, body weight of cows regardless of the duration of their use was for the adjacent lactations (1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6). With increasing distance of lactations from each other the coefficient of repeatability of these traits is lowered

Effects Of Molecular Markers Consequences On Genotyping Errors

Genotype error can greatly reduce the power of a genetic study. For family data, genotype error can be assessed by examining marker data for non-Mendelian inconsistencies, closely linked markers for double recombination events, and consistency of duplicate genotypes. Genetic markers are widely used to determine the parentage of individuals in studies of mating systems, reproductive success, dispersals, quantitative genetic parameters and in the management of conservation populations. These markers are, however, imperfect for parentage analyses because of the presence of genotyping errors and undetectable alleles, which may cause incompatible genotypes (mismatches) between parents and offspring and thus result in false exclusions of true parentage. Highly polymorphic markers widely used in parentage analyses, such as microsatellites, are especially prone to genotyping errors. Molecular markers based on a hybridization reaction or PCR stage that detect DNA polymorphism are currently used in various fields of biology, including the study and preservation of genetic diversity, identification of individuals, phylogenetic, mapping of useful traits of quality and resistance to stress factors. , in the breeding process, biotechnology, etc. Before starting the experiment, researchers must determine what type of markers to use based on the following criteria: the variability and number of required markers, the need for their codominance, the corresponding requirements for the extracted DNA; practical - efficiency, reproducibility of analysis, the necessary appropriate technical support and cost. For a correct interpretation of the results of genotyping, one should take into account the fact that the use of any type of molecular markers is associated with a number of genotyping errors, the main ones being the loss of larger alleles, “zero” alleles, “stutter” alleles due to the peculiarities of Tag polymerase, and non-homology of amplified sequences of the same size (homoplasia). Researchers formulate defining conditions to reduce genotyping errors and reduce their impact on the final analysis. These include the quality and quantity of analyzed DNA, the level of technical capabilities and professionalism of the staff, since the human factor is defined as one of the main reasons for incorrect results; conducting pilot experiments for a comparative assessment of the theoretical and real error rate. The minimization of errors is achieved by assessing the capabilities of a particular type and screening markers; optimization of experimental methods, proper use of controls, replicates, and development of statistical approaches to identify errors. The trade-off between culling error-generating loci and increasing the potential of the retained loci to amplify the genetic signal can be different in different studies, but the main thing is that this signal is not lost for the sake of an “acceptable” level of error and the researchers develop empirical approaches to achieve the desired compromise.so that this signal is not lost for the sake of an "acceptable" level of error and researchers develop empirical approaches to achieve the desired compromise.so that this signal is not lost for the sake of an "acceptable" level of error and researchers develop empirical approaches to achieve the desired compromise