Destruction of Enteric Bacteria in Liquid Egg with β-Propiolactone

Liquid whole egg or egg white, inoculated with Escherichia coli 1485, Salmonella senftenberg ATCC 8400, or Salmonella typhimurium 84-I, was treated with concentrations of β-propiolactone ranging from 0.05 to 0.3%. Egg white containing 1 × 10³ to 1 × 10⁶ cells of E. coli 1485 per ml was sterilized in 1 hr at 27 C by lactone concentrations of 0.2 and 0.3%. Egg white containing 1 × 10⁵ cells of S. senftenberg ATCC 8400 per ml was sterilized in 12 hr at 10 C by 0.1% lactone and in 2 to 3 hr by 0.3% lactone at the same temperature.

Liquid whole egg inoculated with 1 × 10⁵ cells of either species of Salmonella was sterilized in 4 to 5 hr at 10 C with 0.2% lactone or in 2 to 3 hr by 0.3% lactone at this temperature. A mild heat treatment of either 15 min at 37 C or 1 min at 55 C markedly shortened the exposure times required for sterilization by β-propiolactone at 10 C.

After disinfection was complete, the lactone-treated liquid whole egg was reinoculated with low cell numbers of either species of Salmonella to determine the presence of residual lactone or toxic products. Liquid whole egg treated with 0.2% lactone would support the growth of salmonellae after 13 to 14 hr at 10 C. A heat treatment of 45 min at 37 C or 10 min at 55 C immediately after addition of 0.2% lactone allowed growth of the salmonellae in the lactone-treated liquid whole egg. No evidence of residual toxicity from the lactone treatment was found.

The amount of lactone needed to prevent the outgrowth of low cell numbers of either strain of Salmonella in liquid whole egg was quantitated. Liquid whole egg containing 0.06 to 0.07% lactone would not support salmonellae growth from inocula of 1 to 10 cells per ml of egg. Lactone concentrations above 0.08% prevented outgrowth of salmonellae inocula of 10 to 200 cells per ml of liquid whole egg.

     

Circadian and Short-Term Regulation of Blood Pressure and Heart Rate in Transgenic Mice with Cardiac Overexpression of The β1-Adrenoceptor

Congestive heart failure is associated with a loss of circadian and short-term variability in blood pressure and heart rate. In order to assess the contribution of elevated cardiac sympathetic activity to the disturbed cardiovascular regulation, we monitored blood pressure and heart rate in mice with cardiac overexpression of the β₁-adrenoceptor prior to the development of overt heart failure. Telemetry transmitters for continuous monitoring of blood pressure and heart rate were implanted in 8 to 9-week-old wildtype and transgenic mice, derived from crosses of heterozygous transgenic (line β₁TG4) and wildtype mice. Cardiovascular circadian patterns were analyzed under baseline conditions and during treatment with propranolol (500 mg/L in drinking water). Short-term variability was assessed by spectral analysis of beat-to-beat data sampled for 30 min at four circadian times. Transgenic β₁TG4 mice showed an increase in 24 h heart rate, while blood pressure was not different from wildtype controls. Circadian patterns in blood pressure and heart were preserved in β₁TG4 mice. Addition of propranolol to the animals’ drinking water led to a reduction in heart rate and its 24 h variation in both strains of mice. Short-term variability in blood pressure was not different between wildtype and β₁TG4 mice, but heart rate variability in the transgenic animals showed a rightward shift of the high-frequency component in the nocturnal activity period, suggesting an increase in respiratory frequency. In conclusion, the present study shows that both the circadian and the short-term regulation of blood pressure and heart rate are largely preserved in young, nonfailing β₁-transgenic mice. This finding suggests that the loss of blood pressure and heart rate variability observed in human congestive heart failure cannot be attributed solely to sympathetic overactivity but reflects the loss of adrenergic responsiveness to changes in the activity of the autonomic nervous system.     

RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice

Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the Reck ^+/- mice compared to wild type-derived fibroblasts. We observed that Reck ^+/- mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1—RECK- β1-integrin.     

Regulation of Cell Volume by β2-adrenergic Stimulation in Rat Fetal Distal Lung Epithelial Cells

Cell-volume changes induced by terbutaline (a specific β₂-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively differed with the concentration of terbutaline. Terbutaline of 10⁻¹⁰–10⁻⁸ m induced transient cell swelling. Terbutaline of 10⁻⁷ m induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10⁻⁶–10⁻⁵ m induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10⁻³ m induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10⁻⁶ m suppressed the cell swelling induced by 10⁻¹⁰–10⁻⁸ m terbutaline and quinine of 10⁻³ m inhibited the cell shrinkage induced by 10⁻⁶–10⁻⁵ m terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux. Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10⁻⁹–10⁻³ m): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10⁻⁴ m), like terbutaline (10⁻⁵ m), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 × 10⁻¹⁰ m ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca²⁺ abolished the cell swelling caused by terbutaline of 10⁻¹⁰–10⁻⁸ m. With removal of extracellular Ca²⁺, the initial, rapid cell shrinkage induced by 10⁻⁵ m terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular Ca²⁺. Overall, at low concentrations (10⁻¹⁰–10⁻⁸ m), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca²⁺-dependent; high concentrations (≥⁻⁶) induced quinine-sensitive rapid cell shrinkage, which was Ca²⁺-dependent; high concentrations (≥⁻⁷) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in FDLE cells by cytosolic cAMP and Ca²⁺ through activation of Na⁺ and K⁺ channels. Received: 13 March 1995/Revised: 17 January 1996     

Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K⁺ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (V_m), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in V_m. Cell volume and V_m changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by V_m depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K⁺ or Cl⁻ channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in V_m– define the success of RVD.     

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.     

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C₂C₁₂ myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C₂C₁₂ myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C₂C₁₂ myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm², P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice.     

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn''s activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn''s dynamics at the PM and its relation to its inhibition of exocytosis.     

Protective Effects of Transforming Growth Factor β2 in Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life.     

Optimization of β-Cyclodextrin-Assisted Extraction of Apigenin and Luteolin from Chrysanthemum indicum L. Using Response Surface Methodology Combined with Different Optimization Algorithms and Evaluation of Its Antioxidant Capacity

Cyclodextrins and their derivatives have shown successful applications in extracting active compounds from medicinal plants. However, the use of β-cyclodextrin derivatives for extracting apigenin and luteolin from Chrysanthemum indicum L. remains unexplored. Additionally, the application of nature-inspired optimization algorithms in optimizing extraction conditions has been limited. Therefore, this study was performed with the aims of optimizing the extraction of apigenin and luteolin from C. indicum with the assistance of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) using response surface methodology combined with various optimization algorithms, including desirability function approach, genetic algorithm, particle swarm optimization, and firefly algorithm. The results showed that the optimal conditions obtained by the four algorithms were consistent, with an extraction time of 60 min, HP-β-CD concentration of 30 mg/mL, and a solvent-to-solid ratio of 24 mg/mL. At these conditions, the apigenin and luteolin contents were 1.362±0.008 and 8.724±0.117 mg/g, respectively. The results also showed that HP-β-CD-assisted extraction exhibited significantly higher apigenin and luteolin contents compared to conventional solvent. Comparable results were also yielded from the antioxidant assay. Our study suggested that the nature-inspired optimization algorithms might be potential options in enhancing the effectiveness of the traditional response surface methodology for the optimization of extraction of natural products.     

Different Allele Frequencies of the BAIBLocus Determining Excretion of β-Aminoisobutyric Acid in Healthy Donors and Patients with Coronary Atherosclerosis from the Buryat and Lithuanian Populations

Phenotype and allele frequencies of the genetically dimorphic system determining urinary excretion of -aminoisobutyric acid (BAIB) were studied in population samples of Buryats from the Aginskii Buryat Autonomous District and Lithuanians from Vilnius and in patients with coronary atherosclerosis (CA) from both populations. Frequency of allele BAIB*b, which determines high BAIB excretion, proved to be more than twice higher in Buryats compared with the population sample of Lithuanians (0.620 versus 0.289, respectively). The proportion of individuals with high BAIB excretion in CA patients of either ethnic sample was twice higher than in the corresponding control sample. Frequency of allele BAIB*bin CA patients and healthy individuals was 0.348 and 0.242, respectively, in the Lithuanian population and 0.775 and 0.557, respectively, in the Buryat population. Thus, assessment of urinary excretion of BAIB proved to be prognostically valuable. The method used to detect a variation in BAIB excretion is relatively inexpensive, simple, and suitable for mass screening of patients and healthy individuals (population control). After additional testing with representative samples, the method can be used as an accessory diagnostic test in patients with cardiovascular disorders.