Free Calcium Levels Research Articles

Development of a QPatch-Automated Electrophysiology Assay for Identifying TMEM16A Small-Molecule Inhibitors.

The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC50s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents, but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC50s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure–activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.

Curcumin loaded nanoparticles reversed monocrotophos induced motor impairment and memory deficit: Role of oxidative stress and intracellular calcium level

Monocrotophos is one of the extensively used organophosphorus insecticides in agriculture and animal husbandry in India. It induces neurotoxicity by inhibiting acetyl cholinesterase enzyme and elevating oxidative stress. Curcumin, dietary polyphenol, well known for its antioxidant and anti-inflammatory properties which could elicit protective effect against monocrotophos induced neurobehavioural abnormalities. Therefore this study is aimed to investigate ameliorative effect of curcumin loaded Nanoparticles on monocrotophos induced neurotoxicity.Curcumin loaded PLGA nanoformulation was prepared using nanoprecipitation technique and was characterized using zeta sizer, FTIR and NMR spectroscopy. For in-vivo study, 30 male rats were utilized. Exposure of monocrotophos was done at 2 mg/kg (p.o.) for 28 days. Curcumin bulk and cucurmin loaded Nanoparticles were administered with or without monocrotophos. Effect of monocrotophos exposure on behavioural changes was assessed by analysing spontaneous locomotor activity, motor coordination in rotarod test and contextual fear memory in passive shock avoidance paradigm. Effect on biochemical alteration in blood and brain were estimated.The average particle size of Curcumin loaded Nanoparticles was 114.9 nm and in-vitro release study suggested suitable release of Curcumin in simulated intestinal fluid. Results of in-vivo study indicated, monocrotophos exposure caused a significant increase in reactive oxygen species, thiobarbituric acid reactive substances, and intracellular free calcium levels along with altered behavioural abnormalities in locomotor activity, motor coordination, learning and memory. Decline in antioxidant defence level might suggest reduced host defence response. Treatment with curcumin alone did not significantly reversed monocrotophos induced neurotoxicity in rats. Whilst, treatment with curcumin loaded nanoparticles significantly reduced monocrotophos induced behavioural and biochemical alteration in rat brain.The outcome of the present study suggested improved efficacy of curcumin loaded nanoparticles, possibly via its enhanced bioavailability. Further alleviation of oxidative stress and intracellular free Ca2+ level might be speculated as one of the plausible neuroprotective mechanism of curcumin in this study.

Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells.

Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H37Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.

Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.

A Proposed Mechanism for Texture Property of Woody Breast in Broilers

ObjectivesWoody breast is a myopathy observed in chicken breast meat (Pectoralis major) characterized by its tough and rubbery texture. However, the exact causation of woody breast texture is still unknown. We hypothesize that sarcoplasmic reticulum (SR) dysfunctionality early postmortem results in rapid leakage of intracellular calcium may partially contribute to the abnormal meat texture observed in woody breast meat. The objective of this preliminary study was to investigate this hypothesis.Materials and MethodsFourteen Ross line broiler breast fillets (7 severe woody breast and 7 normal) were collected at 3 h postmortem from a commercial processing plant located in the southeast United States. The 7 woody breast samples also exhibited moderate to severe white striping. The 7 normal samples did not exhibit any signs of white striping or woody breast. Each sample was trimmed, weighed, vacuum packaged and frozen at –20°C at approximately 8 h postmortem. One 1.9 cm strip across the cranial end of each fillet was fabricated and pulverized in liquid nitrogen to measure sarcomere length (Laser Scan Confocal Microscope with a 100x/NA 1.4 objective), calpain activity (immunoblotting for µ-calpain autolysis), proteolysis (immunoblotting for troponin-T degradation) and collagen content (hydroxyproline content). Purge was also collected from each sample to evaluate protein (bicinchoninic acid assay) and free calcium concentration (atomic absorption).ResultsWoody breast fillets were heavier than normal chicken breast fillets (522.9 vs. 446.9g; P < 0.05). Woody breast samples tended to have shorter sarcomeres (1.70 vs. 2.02 µm; P = 0.0543) and less intact troponin-T compared to normal breast samples (relative intact troponin-T band density: 49.98 vs. 56.97%; P = 0.0515) at 8 h postmortem. It was interesting to note that no µ-calpain band was detected through immunoblotting for both the woody breast and normal samples at 8 h postmortem. Other studies have found similar results as poultry µ-calpain autolyzed at a much rapid rate than µ-calpain in mammalian species. In addition, the purge from woody breast samples also had higher levels of free calcium compared to normal samples (6.2 vs. 4.2 nmol calcium/mg protein; P < 0.05). Lastly, there was more collagen present in the woody breast samples compared to normal chicken breast samples (3.89 vs. 2.08 mg collagen/g muscle tissue; P < 0.05).ConclusionThe results indicated that the cause of texture abnormality of woody breast may be the combined effects of more calcium being released from the SR early postmortem resulting in shorter sarcomere length and more collagen being deposited in the chicken breast meat. Additional research with the focus on SR integrity and functionality as well as collagen crosslinks are needed to further elucidate the basic mechanism of woody breast texture formation.

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs

Glycogen synthase kinase 3 (GSK3) was identified as an enzyme regulating sperm protein phosphatase. The GSK3α paralog, but not GSK3β, is essential for sperm function. Sperm lacking GSK3α display altered motility and are unable to undergo hyperactivation, which is essential for fertilization. Male mice lacking sperm-specific calcineurin (PP2B), a calcium regulated phosphatase, in testis and sperm, are also infertile. Loss of PP2B results in impaired epididymal sperm maturation and motility. The phenotypes of GSK3α and PP2B knockout mice are similar, prompting us to examine the interrelationship between these two enzymes in sperm. High calcium levels must exist to permit catalytically active calcineurin to function during epididymal sperm maturation. Total and free calcium levels are high in immotile compared to motile epididymal sperm. Inhibition of calcineurin by FK506 results in an increase in the net phosphorylation and a consequent decrease in catalytic activity of sperm GSK3. The inhibitor FK506 and an isoform-selective inhibitor of GSK3α, BRD0705, also inhibited fertilization of eggs in vitro. Interrelated functions of GSK3α and sperm PP2B are essential during epididymal sperm maturation and during fertilization. Our results should enable the development of male contraceptives targeting one or both enzymes.

RyRs mediate lead-induced neurodegenerative disorders through calcium signaling pathways

Heavy metal lead (Pb) is widely distributed in the environment and can induce neurodegeneration. Accumulating evidence has shown that ryanodine receptors (RyRs) play vital roles in neurodegenerative brain. However, whether aberrant RyRs levels contribute to Pb-induced neurodegeneration has largely remained unknown. In the present study, we report the important role of elevated levels of RyRs in Pb-induced neurodegeneration. Pb was found to upregulate the levels of RyRs in the rat hippocampal tissues and rat pheochromocytoma (PC12) cells. Furthermore, exposure to Pb induced neurodegenerative cognitive impairment in rats, depressed the long-term potentiation (LTP) in the rat brain slices, increased the neuronal intracellular free calcium concentration ([Ca2+]i), inhibited the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine 3′,5′-monophosphate (cAMP) response element binding protein (CREB) as well as the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl2), and activated the phosphorylation of extracellular regulated protein kinases (Erk) protein both in vitro and in vivo. In addition, the knockdown of RyR3 in PC12 cells significantly decreased the [Ca2+]i levels, increased the CaMKIIα and CREB phosphorylation, decrease the phosphorylation of Erk, and elongated the cognitive function-related neurite outgrowth after exposure to Pb. Moreover, treatment with a RyRs agonist showed the involvement of RyRs in Pb-induced depression in LTP in the rat brain slices. In summary, we determined that Pb-mediated upregulation of RyRs led to neurodegeneration via high levels of free calcium, depression of the calcium-dependent CaMKIIα/CREB mnemonic signaling pathway, and activation of the calcium-dependent Erk/Bcl2 apoptotic signaling pathway. These findings on the impact of Pb on the levels of RyRs could further improve our understanding of Pb-induced neurotoxicity and provide a promising molecular target to antagonize Pb-induced neurodegenerative diseases.

Sensing Extracellular Calcium - An Insight into the Structure and Function of the Calcium-Sensing Receptor (CaSR).

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a key role in calcium homeostasis, by sensing free calcium levels in blood and regulating parathyroid hormone secretion in response. The CaSR is highly expressed in parathyroid gland and kidney where its role is well characterised, but also in other tissues where its function remains to be determined. The CaSR can be activated by a variety of endogenous ligands, as well as by synthetic modulators such as Cinacalcet, used in the clinic to treat secondary hyperparathyroidism in patients with chronic kidney disease. The CaSR couples to multiple G proteins, in a tissue-specific manner, activating several signalling pathways and thus regulating diverse intracellular events. The multifaceted nature of this receptor makes it a valuable therapeutic target for calciotropic and non-calciotropic diseases. It is therefore essential to understand the complexity behind the pharmacology, trafficking, and signalling characteristics of this receptor. This review provides an overview of the latest knowledge about the CaSR and discusses future hot topics in this field.

Upregulation of α2-adrenoceptor synthesis in SHR cardiomyocytes: Recompense without sense – Increased amounts, impaired commands

Aimsto investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. Methodsexperiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2–2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. Resultspercentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. Conclusiondata indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.

Potential anti-neuroblastoma agents from Juniperus oblonga

Neuroblastoma (NB) is a neuroendocrine tumor derived from neural crest cells. Approximately 90% of cases occur in children less than 5 years old. The amplification of MYCN correlates with high-risk neuroblastoma and patients with MYCN amplified showed poorer prognosis than those without MYCN amplification. In this study, three compounds isolated from Juniperus oblonga showed anti-proliferative activity against NB cell lines with and without tetracycline inducible MYCN over-expression which were identified as (−)-deoxypodophyllotoxin (1), (−)-matairesinol (2) and (+)-isocupressic acid (3). The effects of compounds 2 and 3 in NB cells included a decrease in NB cell viability and induction of apoptosis. Compound 1 was more effective in NB cells over-expressing MycN. Compound 1 also showed almost 2-fold induction of intracellular free calcium levels in M2(+) cells, which may indicate a different mechanism of action for this compound. Cytotoxicity studies against the human embryonic kidney cell (HEK-293) showed compounds 1, 2 and 3 were ineffective in the non-cancer cells at concentrations approximating their IC50 against the NB cell lines. These results may lead to safer and more effective treatment options for NB patients especially for those with high-risk NB.

Antidepressant-Like Effect and Mechanism of Action of Honokiol on the Mouse Lipopolysaccharide (LPS) Depression Model

There is growing evidence that neuroinflammation is closely linked to depression. Honokiol, a biologically active substance extracted from Magnolia officinalis, which is widely used in traditional Chinese medicine, has been shown to exert significant anti-inflammatory effects and improve depression-like behavior caused by inflammation. However, the specific mechanism of action of this activity is still unclear. In this study, the lipopolysaccharide (LPS) mouse model was used to study the effect of honokiol on depression-like behavior induced by LPS in mice and its potential mechanism. A single administration of LPS (1 mg/kg, intraperitoneal injection) increased the immobility time in the forced swimming test (FST) and tail suspension test (TST), without affecting autonomous activity. Pretreatment with honokiol (10 mg/kg, oral administration) for 11 consecutive days significantly improved the immobility time of depressed mice in the FST and TST experiments. Moreover, honokiol ameliorated LPS-induced NF-κB activation in the hippocampus and significantly reduced the levels of the pro-inflammatory cytokines; tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interferon γ (IFN-γ). In addition, honokiol inhibited LPS-induced indoleamine 2,3-dioxygenase (IDO) activation and quinolinic acid (a toxic product) increase and reduced the level of free calcium in brain tissue, thereby inhibiting calcium overload. In summary, our results indicate that the anti-depressant-like effects of honokiol are mediated by its anti-inflammatory effects. Honokiol may inhibit the LPS-induced neuroinflammatory response through the NF-κB signaling pathway, reducing the levels of related pro-inflammatory cytokines, and furthermore, this may affect tryptophan metabolism and increase neuroprotective metabolites.

A Mixture of Functional Complex Extracts from Lycium barbarum and Grape Seed Enhances Immunity Synergistically In Vitro and In Vivo

A mixture of multiple ingredients is often more effective than the individual ingredients. The functions of Lycium barbarum polysaccharide (LBP) glycoconjugate and grape seed procyanidins (GSP) are widely known. Here, we investigated the synergistic immune-enhancing activity of LBP and GSP. Atomic force microscopy results suggested that the mixture of LBP and GSP exhibited circular structure unlike LBP alone, and the addition of polyphenols may change the spatial conformation of the sugar chain. The changes in the structure were related to the synergistic effect of the two functional agents on immune recovery. In vitro, the proliferation rate of splenocytes was higher in LBP + GSP group (64.16%), rather than the sum of LBP group (13.01%) and GSP group (43.61%) individually used. This synergistical proliferation of splenocytes may be correlated to the increasing intracellular free calcium levels. Furthermore, the mixture significantly enhanced the immunity in vivo, as evident from the recovery of peripheral white blood cell counts in LBP + GSP group (18.535 × 109 /L) to normal group levels (18.115 × 109 /L) and higher B cell proliferation than normal group (P < 0.05). These results highlight the immune-enhancing activity of the combination of LBP and GSP associated with the structural changes, which may facilitate the development of functional foods with fewer resources but enhanced activities. PRACTICAL APPLICATION: The synergistic effects of LBP and GSP on immunomodulatory were better than the sum of the effects of the individual agents both in vitro and in vivo. Our results may provide a research-based support for the development of related functional products and an insight into the production of food resources with a fewer but more effective functional agents for better results.

Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: In search of the key particle property.

One of the main goals of materials science in the 21st century is the development of materials with rationally designed properties as substitutes for traditional pharmacotherapies. At the same time, there is a lack of understanding of the exact material properties that induce therapeutic effects in biological systems, which limits their rational optimization for the related medical applications. This study sets the foundation for a general approach for elucidating nanoparticle properties as determinants of antibacterial activity, with a particular focus on calcium phosphate nanoparticles. To that end, nine physicochemical effects were studied and a number of them were refuted, thus putting an end to frequently erred hypotheses in the literature. Rather than having one key particle property responsible for eliciting the antibacterial effect, a complex synergy of factors is shown to be at work, including (a) nanoscopic size; (b) elevated intracellular free calcium levels due to nanoparticle solubility; (c) diffusivity and favorable electrostatic properties of the nanoparticle surface, primarily low net charge and high charge density; and (d) the dynamics of perpetual exchange of ultrafine clusters across the particle/solution interface. On the positive side, this multifaceted mechanism is less prone to induce bacterial resistance to the therapy and can be a gateway to the sphere of personalized medicine. On a more problematic side, it implies a less intense effect compared to single-target molecular therapies and a difficulty of elucidating the exact mechanisms of action, while also making the rational design of theirs for this type of medical application a challenge.

Diabetic Cardiomyopathy.

Diabetic cardiomyopathy was initially described as a human pathophysiological condition in which heart failure occurred in the absence of coronary artery disease, hypertension, and valvular heart disease. Recent studies in diabetic animal models identify decreased cardiomyocyte function as an important mediating mechanism for heart failure. Decreased cardiomyocyte function is in part mediated by abnormal mitochondrial calcium handling and a decreased level of free matrix calcium levels which could be a good target for new therapeutic interventions.

Ginsenoside Rg5 induces apoptosis in human esophageal cancer cells through the phosphoinositide‑3 kinase/protein kinase B signaling pathway.

The role of ginsenoside in the prevention of cancer has been well established. Ginsenoside Rg5 is one of the main components isolated from red ginseng, which has been demonstrated to have anti-tumor effects by inhibiting cell proliferation and causing DNA damage. However, the role of ginsenoside Rg5 and its molecular mechanisms remain unclear in human esophageal cancer. In the present study, Rg5 was investigated as a novel drug for the chemotherapy of esophageal cancer in in vitro experiments. Esophageal cancer Eca109 cells were exposed to various concentrations of ginsenoside Rg5 (0–32 µΜ) for 24 h. Subsequent cell proliferation assays demonstrated that treatment with ginsenoside Rg5 resulted in the dose-dependent inhibition of proliferation, while a significant increase in apoptotic rate and increased activities of caspase-3, −8 and −9 were observed. In addition, the mitochondrial membrane potential was decreased and the cytoplasmic free calcium level increased following treatment with ginsenoside Rg5. Furthermore, the expression of B-cell lymphoma 2 and phosphorylated-protein kinase B (p-Akt) decreased. The specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002 promoted this effect, while insulin-like growth factor-1, a specific PI3K activator, inhibited this action. Taken together, the results suggested that ginsenoside Rg5 may have a tumor-suppressive effect on esophageal cancer by promoting apoptosis and may be associated with the downregulation of the PI3K/Akt signaling pathway.

Lead exposure-induced cognitive impairment through RyR-modulating intracellular calcium signaling in aged rats

Lead is widely distributed in the environment and has become a global public health issue. It is well known that lead exposure induces not only neurodevelopmental toxicity but also neurodegenerative diseases, with learning and memory impairment in the later stage. However, the molecular mechanisms remain elusive. The present study investigated the effects of early life and lifetime lead exposure on cognition and identified the molecular mechanisms involved in aged rats. The results herein demonstrated that the lead concentration in peripheral blood and brain tissues in aged rats was significantly increased in a lead dose-dependent manner. High-dose lead exposure caused cognitive functional impairment in aged rats, concomitant with a longer escape latency and a lower frequency of crossing the platform via Morris water maze testing compared to those in the control and low-dose lead exposure groups. Importantly, neuron functional defects were still observed even in early life lead exposure during the prenatal and weaning periods in aged rats. The neurotoxicity induced by lead exposure was morphologically evidenced by a recessed nuclear membrane, a swollen endoplasmic reticulum, and mitochondria in the neurons. Mechanistically, the exposure of aged rats to lead resulted in increasing free calcium concentration, reactive oxygen species, and apoptosis in the hippocampal neurons. Lead exposure increased RyR3 expression and decreased the levels of p-CaMKIIα/CaMKIIα and p-CREB/CREB in the hippocampus of aged rats. These findings indicated that early life lead exposure-induced cognition disorder was irreversible in aged rats. Lead-induced neurotoxicity might be related to the upregulation of RyR3 expression and high levels of intracellular free calcium with increasing lead concentration in injured neurons.

Quality effects on beef strip steaks from cattle fed high-protein corn distillers grains and other ethanol by-products.

The objective of this study was to evaluate the effects of feeding high-protein corn distillers grains on fresh beef quality. Steers (n = 60) were fed one of five diets for 190 d (six pens with 10 cattle/pen/diet): a corn control (Control), 40% high-protein dry distillers grains plus solubles (HP-DDGS), 40% dry distillers grains plus solubles (DDGS), 40% wet distillers grains plus solubles (WDGS), or 40% bran plus solubles (Bran). Eighteen Choice carcasses (three cattle per pen) were randomly selected within each treatment; however, two carcasses were lost during carcass selection. Strip loins (Longissimus lumborum, IMPS # 180) were collected, divided into three equal portions, and aged for 2, 9, or 23 d. Steaks were fabricated following each aging period and placed under retail display (RD) conditions for 0, 4, or 7 d. Pen was the experimental unit. Hot carcass weight at harvest was 391 kg (SD 31.6 kg). Dietary treatment had no effect on tenderness (Warner-Bratzler shear force) within each aging period (P > 0.05). After 7 d of RD, following all aging periods, steaks from cattle fed HP-DDGS had the greatest visual discoloration except for DDGS and Bran after 23 d (P < 0.05). The steaks from cattle fed HP-DDGS had lower (P < 0.05) redness (a*) values than all other treatments following 7 d of RD. Lipid oxidation increased (P < 0.001) during RD at all aging periods, as measured via thiobarbituric acid reactive substance (TBARS). A day of RD-by-dietary treatment effect (P < 0.001) was observed for lipid oxidation. After 7 d of RD, steaks from cattle fed HP-DDGS had higher TBARS than all other treatments except WDGS (P < 0.01). A trend was found for sarcomere length (P = 0.07), with steaks from cattle fed Bran having the longest sarcomere length compared with all other treatments. An aging effect was found for free calcium content (P < 0.001) where steaks aged 9 and 23 d had significantly higher levels of free calcium than those aged 2 d. There was a tendency (P = 0.07) that steaks from cattle fed WDGS contained more free calcium than steaks from cattle fed DDGS or Bran. Although there were no statistical differences, there was a trend (P = 0.07) that steaks from cattle fed HP-DDGS had higher levels of PUFAs and C18:2. These results suggest that feeding high protein distillers grains has no detriment on tenderness, but may alter the lipid profile of the muscle, resulting in decreased color stability, increased lipid oxidation, and decreased shelf life.

Electrocardiographic, endocrine and biochemical stress indices of electroejaculated Egyptian Baladi goat bucks

This study aimed to evaluate the stress response to electroejaculation (EE) through electrocardiogram (ECG) indices alongside hormonal and biochemical indices. Lead II base-apex ECG was recorded at rest (control, T0) and immediately after EE (T1), repeated in four consecutive weeks. The collected ejaculates were evaluated for standard semen characteristics (ejaculate volume, mass motility, individual motility, sperm cell concentration and total sperm count per ejaculate). Blood samples were collected at rest (T0), 10 (T2) and 120 min (T3) after EE for determination of biochemical and hormonal stress indices. EE induced sinus tachycardia with simultaneous marked increase in P waves amplitude and decrease in all waves and intervals durations (except for QRS complex and PR interval). Similarly, significant variation between the first and the last EE sessions were observed in heart rate (HR) indices (mean RR, mean, maximum and minimum HR), some wave amplitudes and segment durations. While the stress index significantly increased in the 3rd week only. This was associated with significant short term increases in serum level of cortisol and progesterone (P4) with significant decrease of glucose and globulins at T2. Furthermore, at T3 there was significant increase in cardiac troponin I (CTnI) concentration, while free triiodothyronine (FT3), potassium (K+), calcium (Ca2+) and glucose levels decreased significantly. There were significant increases between the first and the last EE sessions in cortisol, FT3 and significant decrease in Lactate dehydrogenase (LDH) concentrations. On contrary, there were no significant changes in time related to EE and weekly repetitions in the corrected QT interval (QTc), root mean square of sum of squares of normal to normal difference (RMSSD), standard deviation of the normal to normal (SDNN) vasovagal tonus index (VVTI) and semen characteristics. In conclusion, although repeated EE causes acute stress response which increases with repetition, the response appeared transient and not deleterious to the cardiac activity and semen characteristics evidenced by non-persistent significant increases in CTnI and absence of significant changes in the standard semen characteristics of the bucks. Additionally, ECG might be beneficial in assessing the welfare of electroejaculated bucks. Further assessment of heart rate variability (HRV) indices of bucks after EE using frequency and non-linear indices in a larger animal population is recommended.

Modulation of Structural and Functional Properties of Human Lymphocytes by Reactive Oxygen Species.

The effects of ROS on functional properties (cytotoxic activity, antibody-producing activity, TNFα synthesis, and free cytosol calcium level), membrane structure (by expression of some surface markers), and apoptosis of lymphocytic cells were estimated in the peripheral blood of healthy volunteers. 1O2, [Formula: see text], OH•, and H2O2 mostly suppressed cytotoxic activity of lymphocytes against Ehrlich ascites carcinoma cells and inhibited IgG synthesis and expression of receptors and surface markers (Fc receptors, CD3, CD19, and CD56). The exposure of lymphocytes to H2O2 (10-6 M), 1O2, and OH• was followed by an increase in the level of a secondary messenger, intracellular calcium, in comparison with non-exposed cells. The presence of exogenous calcium in the medium for lymphocyte suspending induced an increase in the number of cells at early and late stages of apoptosis 6 h after exposure to H2O2 and 1O2 in comparison with lymphocytes incubated in Ca2+-free medium.

Serum Biochemical, Hormonal and Fatty Acid Profiles During the Late Gestation of Pregnancy Ketosis in Boer Cross Goats

Pregnancy ketosis has been recognized as one of the common metabolic disease affecting goat’s meat and milk production. For the present study, sixteen (n=16) individuals of pregnant does at day 80 of pregnancy had been used. A total of 8 does were categorized as control group (healthy pregnant goats), were fed on Napier grass and goat concentrate with water ad libitum, and another 8 does were considered as treatment group which categorized as ketosis based on the clinical signs and presence of ketone body in urine. Blood sample were collected from all goats for biochemical profiles analysis which were glucose, Beta-hydroxybutyrate (BHBA), free fatty acid (FFA), calcium, electrolytes (sodium, potassium, chloride), liver enzyme and hormonal levels (cortisol and insulin). Three does from each group were slaughtered and liver samples were collected for fatty acid profiles study. In this study, the BHBA, FFA, calcium, amino aspartate transferase (AST), gamma glutamyl transferase (GGT) and cortisol hormone were significantly higher in pregnancy ketosis goats as compared to control group. Meanwhile, the concentration of glucose, sodium, potassium, chloride and insulin hormones were lower in pregnancy ketosis goats as compared to control. Furthermore, the fatty acid composition in blood plasma of pregnant goat with ketosis showed higher level of palmitic, stearic and oleic acid, while in liver, palmitic, oleic and linoleic acid was found higher.