Effects of Mechanical Stress on Endothelial Cells In Situ and In Vitro.

Endothelial cells lining blood vessels are essential for maintaining vascular homeostasis and mediate several pathological and physiological processes. Mechanical stresses generated by blood flow and other biomechanical factors significantly affect endothelial cell activity. Here, we review how mechanical stresses, both in situ and in vitro, affect endothelial cells. We review the basic principles underlying the cellular response to mechanical stresses. We also consider the implications of these findings for understanding the mechanisms of mechanotransducer and mechano-signal transduction systems by cytoskeletal components.

Recent Applications of Deconvolution Microscopy in Medicine.

Deconvolution microscopy is a computational image-processing technique used in conjunction with fluorescence microscopy to increase the resolution and contrast of three-dimensional images. Fluorescence microscopy is a widely used technique in biology and medicine that involves labeling specific molecules or structures within a sample with fluorescent dyes and then electronically photographing the sample through a microscope. However, the resolution of conventional fluorescence microscopy is limited by diffraction within the microscope's optical path, which causes blurring of the image and reduces the ability to resolve structures in close proximity with one another. Deconvolution microscopy overcomes this limitation by means of computer-based image processing whereby mathematical algorithms are used to eliminate the blurring caused by the microscope's optics and thus obtain a higher-resolution image that reveals the fine details of the sample with greater accuracy. Deconvolution microscopy, which can be applied to a range of image acquisition modalities, including widefield, confocal, and super-resolution microscopy, has become an essential tool for studying the structure and function of biological systems at the cellular and molecular levels. In this perspective, the latest deconvolution techniques have been introduced and image-processing methods for medical purposes have been presented.

Effects of Electrical Stimulation of the Cell: Wound Healing, Cell Proliferation, Apoptosis, and Signal Transduction.

Electrical stimulation of the cell can have a number of different effects depending on the type of cell being stimulated. In general, electrical stimulation can cause the cell to become more active, increase its metabolism, and change its gene expression. For example, if the electrical stimulation is of low intensity and short duration, it may simply cause the cell to depolarize. However, if the electrical stimulation is of high intensity or long duration, it may cause the cell to become hyperpolarized. The electrical stimulation of cells is a process by which an electrical current is applied to cells in order to change their function or behavior. This process can be used to treat various medical conditions and has been shown to be effective in a number of studies. In this perspective, the effects of electrical stimulation on the cell are summarized.

Effects of Electrical Stimulation on the Signal Transduction-Related Proteins, c-Src and Focal Adhesion Kinase, in Fibroblasts.

Electrical stimulation of the skin and muscles, e.g., in the fields of rehabilitation medicine and acupuncture, is known to locally increase blood flow and metabolism, and thus have beneficial health effects. However, little is known about the changes in cellular morphology or regulation of the localization of specific proteins in response to electrical stimuli. The present study was performed to examine the effects of electrical stimulation on the cytoskeletal system of cultured fibroblasts. Following application of electrical stimulation to cultured fibroblastic cells for a period of about 2 h, the stress fibers in the cells became thicker and the cells showed a contracted appearance. Cells were subjected to periodic electrical stimulation for 0 (unstimulated control), 2, 5, or 20 h. The stress fibers showed an increase in thickness within 2 h, and became gradually thicker until 20 h. In addition, the focal adhesions and stress fibers were enlarged after 2 h of continuous stimulation, and both stress fibers and focal adhesions became larger and thicker after 20 h of periodic stimulation. Cells showed increased staining of focal adhesions with anti-phosphotyrosine antibody (PY-20) after electrical stimulation. Cells also showed increased staining of tyrosine-phosphorylated focal adhesion kinase (FAK) (pY397) and tyrosine-phosphorylated c-Src (pY418), indicating that electrical stimulation affected signal transduction-related proteins.

Impact of insulin-like growth factor 1, immunoglobulin G and vitamin A in colostrum on growth of newborn Black Bengal goats and its crossbred.

The study aims to investigate the relationships between colostral concentrations of insulin-like growth factor 1 (IGF-1), immunoglobulin G (IgG) and vitamin A (Vit A) and growth (body weight and average daily gain) in Black Bengal (BB) and its crossbred. The colostrum from dams (n = 16) was collected at parturition to measure the concentrations of IGF-1, IgG and Vit A. The kid weight at birth (W-0), day 14 (W-14) and day 28 (W-28) were measured and the average daily gain during day 1-14 (ADG1-14) and day 14-28 (ADG14-28) were calculated. The average concentrations of IGF-1, IgG and Vit A in colostrum were 504.6 ± 74.9 ng/ml, 9.7 ± 0.6 mg/ml and 549.1 ± 72.5 µg/100 g, respectively. The average body weight of kids at birth, day 14 and 28 were 1.72 ± 0.08, 2.95 ± 0.11 and 3.94 ± 0.13 kg respectively. Kid's breed, IGF-1, IgG and Vit A had significant positive effects on ADG14-28 while parity, litter size and sex had no effect. The growth factors that were classified into 2 classes based upon the mean values of colostral contents in all kids showed that the kids receiving the higher concentrations of IGF-1, IgG and Vit A in colostrum had higher body weight gain than those receiving the lower concentrations (92.1 ± 7.8 vs. 59.8 ± 5.7 g/day; p = 0.002, 88.3 ± 7.8 vs. 60.3 ± 6.1 g/day; p = 0.009 and 91.1 ± 6.8 vs. 56.7 ± 5.8 g/day; p < 0.001 respectively). It is concluded that IGF-1, IgG and Vit A concentrations in colostrum of dams were associated with increased kid's body weight gain at the end of first month in BB and BB crossbred goats.

Growth performance of Black Bengal, Saanen, and their crossbred F1 as affected by sex, litter size, and season of kidding.

The objective of the present study was to determine the factors that influenced growth performance of the goat kids of Black Bengal (BB), Saanen (SA), and their crossbred F1 (male Bengal × female Saanen [BBSA] and male Saanen × female Black Bengal [SABB]). Data for 674 kids were analyzed from 316 litters and 134 does. All kids were weekly measured on their characteristics (body weight, length, height at the withers, and chest girth) from birth to 11 weeks old. The kid's breed and sex, litter size, and season of kidding influenced birth weight and other characteristics through the experiment. The SA and BBSA kids showed similar performance, which were higher than BB and SABB kids. Male kids had higher performance than female kids, and kids from a single litter showed the highest performance. Kids born during rainy season showed lower performance than those born in hot and cool seasons. In conclusion, the crossbred BBSA is superior to SABB or BB to raise in tropical climate Moreover, sex, litter size, and kidding season also affected growth performance during the preweaning period up to 11 weeks old.

Effects of Litter Size and Parity Number on Mammary Secretions Including, Insulin-Like Growth Factor-1, Immunoglobulin G and Vitamin A of Black Bengal, Saanen and Their Crossbred Goats in Thailand.

The present study aims to investigate the composition including concentrations of IGF-1, IgG and Vit A in colostrum and their effects by litter size and goat parity in 3 groups of goats; Black Bengal (BB), Saanen (SA) and their crossbred (BBSA). Thirty-eight goats were used (23 BB, 7 BBSA and 8 SA). The composition (fat, protein, lactose and total solid; TS) in colostrum (Day 0; D0) and milk (Day 4; D4 and Day 7; D7) were measured. The IGF-1, IgG concentrations were analysed in some samples collected at D0, D4 and D7 while Vit A was analysed only in colostrum. The results showed that colostrum components were similar among experimental groups. However, the colostral IGF-1 concentration of BBSA (983.0 ± 163.6 ng/mL) was higher than that of BB (340.7 ± 85.5 ng/mL, p < 0.01) and SA (417.1 ± 93.9 ng/mL, p < 0.01). The colostral IgG concentration of BB (8.2 ± 0.9 mg/mL) was lower than that of BBSA (12.9 ± 1.7 mg/mL, p < 0.05) and SA (12.9 ± 1.0 mg/mL, p < 0.01). Colostral Vit A concentration in BBSA (787.2 ± 152.6 µg/100 gm) was higher than that in BB (388.9 ± 84.3 µg/100 gm, p < 0.05) but was not different from SA (522.8 ± 96.9 µg/100 gm). Colostrum from all groups contained higher protein and TS but was lower in lactose concentration than milk. The IGF-1 and IgG concentrations in colostrum were much higher than in milk both D4 and D7 (p < 0.001). Additionally, litter size had no effects on colostrum contents but colostrum from goats with a higher parity number had higher IgG concentration. It is concluded that colostrum from BBSA may be superior when fed to BB newborn goats in terms of higher IGF-1, IgG and Vit A contents. Moreover, colostrum from goats with a high parity number contained more IgG content.

Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase.

Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.

Validation of Brix refractometer to estimate immunoglobulin G concentration in goat colostrum.

BACKGROUND AND AIM: Immunoglobulin G (IgG) concentration is high in goat colostrum, particularly in the first few hours after parturition, and this is important for the kid's immunity and growth. IgG levels vary depending on several factors, including breed, disease status, colostrum management, handling, and collection time postpartum. A handheld optical refractometer, an affordable instrument that is simple to use in the field, is used widely in dairy farms to measure total solids. However, it can also be applied to estimate colostrum IgG content on the basis of comparison with standard measurement methods, usually radial immunodiffusion. Studies comparing %Brix values in relation to IgG concentration measured using enzyme-linked immunosorbent assay (ELISA) in goats are limited. The present study aimed to evaluate the use of a handheld optical Brix refractometer for the measurement of IgG concentration in goat colostrum, compare results with those using ELISA, and estimate the %Brix cutoff value equating to low-quality colostrum. MATERIALS AND METHODS: Colostrum samples were collected on day 0 from 21 goats (nine Black Bengal, six Saanen, and six of their crossbred offspring) and were frozen. Subsequently, they were analyzed for IgG concentration using a goat-specific ELISA test and Brix percentage using a handheld refractometer. The optimum %Brix cutoff value for the evaluation of colostrum quality was evaluated. RESULTS: The mean IgG concentration and %Brix in colostrum were 10.60±0.64 mg/mL and 25.0±0.9, respectively. There was a significant (p<0.01) correlation between %Brix and IgG concentration. For an IgG concentration of 6.9 mg/dL, the cutoff value for %Brix was 18.5, equating to high specificity (100%) but low sensitivity (50%). A higher %Brix cutoff value of 21.5 showed high specificity (95%) and high sensitivity (100%). CONCLUSION: A Brix refractometer can be used to estimate goat colostrum quality with a proposed %Brix cutoff value of <18.5%-21.5% for poor-quality colostrum.

IL-12p40 gene expression in lung and hilar lymph nodes of MPS-resistant pigs.

Mycoplasma pneumonia of swine (MPS) is caused by Mycoplasma hyopneumoniae (M.hp) and is a common chronic respiratory disease of pigs. Recently, a genetically selected variant of the Landrace pig (Miyagino L2) has a lower incidence of pulmonary MPS lesions. We investigated the pathological and immunological characteristics of MPS resistance in these pigs (n = 24) by comparing with the normal landrace pig (control: n = 24). The pathological MPS lung lesion score in MPS-selected landrace pigs was significantly lower than in the control. The gene expression of interleukin (IL)-12p40, which acts as a chemoattractant and a component of the bioactive cytokines IL-12 and IL-23, was significantly higher at the hilar lymph nodes, lung, and spleen in MPS-selected landrace pigs than in control landrace pigs, and these were negatively correlated with the macroscopic MPS lung lesion score. In summary, we demonstrate that resistance against MPS in Miyagino L2 pigs is associated with IL-12p40 up-regulation, in comparison with normal landrace pigs without the MPS vaccine. In addition, a comparative study of macroscopic MPS lung lesions and IL-12p40 gene expression in lung and hilar lymph nodes may lead to beneficial selection traits for the genetic selection for MPS resistance in pigs.

FAK-Dependent Cell Motility and Cell Elongation.

Fibroblastic cells show specific substrate selectivity for typical cell-substrate adhesion. However, focal adhesion kinase (FAK) contributes to controlling the regulation of orientation and polarity. When fibroblasts attach to micropatterns, tyrosine-phosphorylated proteins and FAK are both detected along the inner border between the adhesive micropatterns and the nonadhesive glass surface. FAK likely plays important roles in regulation of cell adhesion to the substrate, as FAK is a tyrosine-phosphorylated protein that acts as a signal transduction molecule at sites of cell-substrate attachment, called focal adhesions. FAK has been suggested to play a role in the attachment of cells at adhesive micropatterns by affecting cell polarity. Therefore, the localization of FAK might play a key role in recognition of the border of the cell with the adhesive micropattern, thus regulating cell polarity and the cell axis. This review discusses the regulation and molecular mechanism of cell proliferation and cell elongation by FAK and its associated signal transduction proteins.

Addition of Wakame seaweed (Undaria pinnatifida) stalk to animal feed enhances immune response and improves intestinal microflora in pigs.

This study was conducted to evaluate the effects of supplementation of Wakame seaweed stalks on the immunity and intestinal microflora of pigs. Three separate experiments were performed: Relatively young (start at 20-30 kg; Experiments 1 and 2) and fattening period (70 kg; Experiment 3). All pigs (including the control group) were fed the same commercial feed, free from antibiotic additives, but in the feed for the treatment groups, 1% seaweed powder was added. There were no group differences observed in daily weight gain and feed intake in Experiments 1 and 2 between groups; however, daily weight gain was significantly higher in the treatment group compared to the control group in Experiment 3. The percentage of peripheral blood natural killer cells of the treatment group was significantly higher than that of the control group in all experiments. Although addition of seaweed changed the gene expression of cytokine and toll-like receptors of the small intestinal Peyer's patches slightly, seaweed seems to alter intestinal microflora preferentially, for instance, there was an increase in Lactobacillus and a decrease of Escherichia coli observed. These results suggest that Wakame seaweed can be used as supplement for pig feed to improve the gut health and immunity of pigs.

Correlations between mitochondrial respiration activity and residual feed intake after divergent genetic selection for high- and low- oxygen consumption in mice.

The aims of the present study were to identify the differences between two mouse lines (high (H)- and low (L)-oxygen consumption) in terms of mitochondrial respiratory activity when GMP (glutamate, malate, and pyruvate) and succinic acid are used as substrates and to examine the relationship between mitochondrial respiration activity and feed efficiency in both lines. The average daily feed intake, feed conversion ratio (FCR), and residual feed intake (RFI) were significantly higher in the H than the L line. The correlation between FCR and RFI was significant (r = 0.60, p < 0.05). RFI was effective as an indicator of feed efficiency. When succinic acid was used as a substrate, mitochondrial respiration states 2-4, ACR, and proton leak were significantly higher in the H than the L line. When GMP was used as a substrate, respiration states 3 and 4 in the H line were significantly higher than those in the L line, and there were significant positive correlations between FCR and RFI and mitochondrial respiration states 2-4. The results indicated that selection for high or low OC changed the basal metabolic rates estimated from liver mitochondrial respiration activity and feed efficiency.

Software-Based Three-Dimensional Deconvolution Microscopy of Cytoskeletal Proteins in Cultured Fibroblast Using Open-Source Software and Open Hardware.

As conventional fluorescence microscopy and confocal laser scanning microscopy generally produce images with blurring at the upper and lower planes along the z-axis due to non-focal plane image information, the observation of biological images requires "deconvolution." Therefore, a microscope system's individual blur function (point spread function) is determined theoretically or by actual measurement of microbeads and processed mathematically to reduce noise and eliminate blurring as much as possible. Here the author describes the use of open-source software and open hardware design to build a deconvolution microscope at low cost, using readily available software and hardware. The advantage of this method is its cost-effectiveness and ability to construct a microscope system using commercially available optical components and open-source software. Although this system does not utilize expensive equipment, such as confocal and total internal reflection fluorescence microscopes, decent images can be obtained even without previous experience in electronics and optics.

Effects of butyrate supplementation in antibiotic-free milk replacer and starter on growth performance in suckling calves.

The aim of this study was to evaluate butyrate supplementation of antibiotic-free milk replacer and starter on growth performance in male Holstein calves. Twenty-nine calves were divided into two groups. Group C (n = 13) was fed antibiotic-free milk replacer without supplementation, and Group B (n = 16) was fed antibiotic-free milk replacer supplemented with butyrate (1.6 % DM of Gustor BP70® ). Starter in Group B contained 0.3 % DM of Gustor BP70® . The intake of milk replacer was lower in group B than in C (p = 0.07 for the treatment x week interaction). Body weight (BW) and heart girth (HG) in group B was higher than in C during the experimental period (p = 0.07 and 0.01 for the treatment × week interaction, respectively). The duration of the weaning period in group B was shorter than in group C (p = 0.02). ß-hydroxybutyrate (BHBA) was higher in group B than in C (p = 0.04). Insulin like growth factor-1 (IGF-1) concentrations tended to be higher in group B than in C (p = 0.07 for treatment × week interaction). Our results show that butyrate supplementation in antibiotic-free milk replacer and starter exerted positive effects on growth performance in suckling calves.

Comparative transcriptome analysis of rumen papillae in suckling and weaned Japanese Black calves using RNA sequencing.

The length and density of rumen papillae starts to increase during weaning and growth of ruminants. This significant development increases the intraruminal surface area and the efficiency of VFA (acetate, propionate, butyrate, etc.) uptake. Thus, it is important to investigate the factors controlling the growth and development of rumen papillae during weaning. This study aimed to compare the transcriptomes of rumen papillae in suckling and weaned calves. Total RNA was extracted from the rumen papillae of 10 male Japanese Black calves (5 suckling calves, 5 wk old; 5 weaned calves, 15 wk old) and used in RNA-sequencing. Transcript abundance was estimated and differentially expressed genes were identified and these data were then used in Ingenuity Pathway Analysis (IPA) to predict the major canonical pathways and upstream regulators. Among the 871 differentially expressed genes screened by IPA, 466 genes were upregulated and 405 were downregulated in the weaned group. Canonical pathway analysis showed that "atherosclerosis" was the most significant pathway, and "tretinoin," a derivative of vitamin A, was predicted as the most active upstream regulator during weaning. Analyses also predicted IgG, lipopolysaccharides, and tumor-necrosis factor-α as regulators of the microbe-epithelium interaction that activates rumen-related immune responses. The functional category and the up-regulators found in this study provide a valuable resource for studying new candidate genes related to the proliferation and development of rumen papillae from suckling to weaning Japanese Black calves.

Downregulated angiopoietin-like protein 8 production at calving related to changes in lipid metabolism in dairy cows.

Acute physiological adaptation of lipid metabolism during the postpartum transition period of cows facilitates peripheral metabolic regulation. Hepatokines, which are hormones secreted from hepatocytes, are presumed to play a critical role in systemic metabolic regulation. Angiopoietin-like protein 8 (ANGPTL8) has been identified as a novel hepatokine associated with circulating triglyceride concentrations in mice and humans. However, regulation of ANGPTL8 and its physiological effects is still unknown in cattle. The present study aimed to reveal changes in ANGPTL8 expression and secretion during the periparturient period, and to investigate its regulatory effect on adipocytes and mammary epithelial cells. In the peripartum period, liver ANGPTL8 mRNA expression was lesser on the day of parturition and 1 wk postpartum than it was 1 wk before parturition (P < 0.05). Moreover, plasma ANGPTL8 concentrations decreased on the day of parturition as compared with that 1 wk before parturition (P < 0.05). In addition, ANGPTL8 expression in cultured bovine hepatocytes was downregulated after oleate and palmitate treatment but upregulated after insulin treatment (P < 0.05). ANGPTL8 decreased hormone-sensitive lipase (HSL) expression in differentiated adipocytes and cluster of differentiation 36 (CD36), fatty acid synthase (FAS), acetyl-coa carboxylase (ACC), and stearoyl-coa desaturase (SCD) in cultured bovine mammary epithelial cells (P < 0.05). These data suggest that hepatic ANGPTL8 production was downregulated postpartum when the cows experienced a negative energy balance. This downregulation was associated with increased concentrations of NEFA and decreased concentrations of insulin in lactating cows, and it facilitated lipid mobilization from adipose tissue to the mammary glands. We speculate that ANGPTL8 might have beneficial effects in reverting or improving the physiological adaptation and pathological processes of lipid metabolism during the peripartum period.

Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts.

Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, the author examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK-/-) fibroblasts. FAK-/- cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK-/- cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK-/- cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.

Post-prandial decrease in plasma growth hormone levels is not related to the increase in plasma insulin levels in goats.

OBJECTIVE: In the present study, we examined whether the post-prandial reduction in plasma growth hormone (GH) levels is related to the increase in plasma insulin levels in ruminants. METHODS: We performed two experiments: intravenous bolus injection of insulin (0.2 IU/kg body weight) or glucose (1.0 mmol/kg body weight) was administered to increase the plasma insulin levels in male Shiba goats. RESULTS: In the insulin injection experiment, significant (p<0.05) increase in GH concentrations was observed, 15 to 20 min after the injection; it was accompanied with a significant (p<0.01) increase in cortisol concentrations at 45 to 90 min, when compared to the concentrations in the saline-injected controls. The glucose injection significantly (p<0.05) increased the plasma GH concentration at 20 to 45 min; this was not accompanied by significantly higher cortisol concentrations than were observed for the saline-injected control. Hypoglycemia induced by the insulin injection, which causes the excitation of the adrenal cortex, might be involved in the increase in insulin levels. CONCLUSION: Based on these results, we conclude that post-prandial increases in plasma insulin or glucose levels do not induce a decrease in GH concentration after feeding in the ruminants.

Search for an index for the taste of Japanese Black cattle beef by panel testing and chemical composition analysis.

To search for an index for chemical composition related to superior taste in Japanese Black beef, we conducted panel tests and analyzed the chemical composition of seven beef brands. Thirty-five sirloin beefs from five heifers were used in this study, sold under seven beef brands graded as more than A4 on the Japanese Meat Grade scale. The chemical composition analyses assessed both raw and roasted meat, the latter of which was roasted under the same conditions as those used for the panel test. Results of the panel test and chemical composition analyses revealed that fatty acid composition, sugar content, adenosine triphosphage (ATP)-related compounds, amino acid composition and odor composition in the sirloin meat differed among beef brands. Furthermore, the correlations of chemical compositions between roasted and raw meat were significantly high. Sugar content and ATP-related compounds in roasted meat were significantly correlated with the item 'overall evaluation' of the panel test. ATP-related compounds, such as inosinic acid, carnosine and taurine, in roasted and raw meat were correlated significantly with the item 'umami intensity' of the panel test. These results suggest that the composition of these components is important for an index related to the overall evaluation of beef.