Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions.

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37 C, 60 min) or glucose-starved (37 C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14-15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under non-starving conditions.     

A model-dependent approach to correlate accelerated with real-time release from biodegradable microspheres

The purpose of this study was to determine the feasibility of applying accelerated in vitro release testing to correlate or predict long-term in vitro release of leuprolide poly(lactideco-glycolide) microspheres. Peptide release was studied using a dialysis technique at 37°C and at elevated temperatures (50°C–60°C) in 0.1 M phosphate buffered saline (PBS) pH 7.4 and 0.1 M acetate buffer pH 4.0. The data were analyzed using a modification, of the Weibull equation. Peptide release was temperature dependent and complete within 30 days at 37°C and 3 to 5 days at the elevated temperatures. In vitro release profiles at the elevated temperatures correlated well with release at 37°C. The shapes of the release profiles at all temperatures were similar. Using the modified Weibull equation, an increase in temperature was characterized by an increase in the model parameter, α, a scaling factor for the apparent rate constant. Complete release at 37°C was shortened from ∼30 days to 5 days at 50°C, 3.5 days at 55°C, 2.25 days at 60°C in PBS pH 7.4, and 3 days at 50°C in acetate buffer pH 4.0. Values for the model parameter β indicated that the shape of the release profiles at 55°C in PBS pH 7.4 (2.740) and 50°C in 0.1 M acetate buffer pH 4.0 (2.711) were similar to that at 37°C (2.577). The E_a for hydration and erosion were determined to be 42.3 and 19.4 kcal/mol, respectively. Polymer degradation was also temperature dependent and had an E_a of 31.6 kcal/mol. Short-term in vitro release studies offer the possibility of correlation with long-term release, thereby reducing the time and expense associated with longterm studies. Accelerated release methodology could be useful in the prediction of long-term release from extended release microsphere dosage forms and may serve as a quality control tool for the release of clinical or commercial batches. Selected for the 2005 AAPS Outstanding Graduate Student Research Award in Pharmaceutical Technologies Sponsored by Solvay Pharmaceuticals.     

Impact of thermohaemolysis-associated membrane alteration on the passive ion permeability and life-span of erythrocytes

Suspending erythrocytes in medium containing sucrose prevented heat-induced lysis at its early stage. This allowed determination of the thermohaemolysis-related ion permeability by measuring the initial rate of the stipulated shrinkage of erythrocytes. Thus, correspondingly, the coefficient P of the ion permeability was calculated for heated human erythrocytes using ouabain-pretreated cells in 37–45°C range and intact cells in 50–58°C range. The values obtained for P obeyed a straight line Arrhenius plot over the entire 37–58°C range suggesting that the ion permeability was activated by a single mechanism earlier identified as relevant to thermohaemolysis. At the 37–58°C range, the activation energy of the P was 250±15 kJ/mol which was markedly different from the value of 56 kJ/mol known for the 10–37°C range. For erythrocytes from five mammals, similar temperature dependencies of the P were obtained over 45–60°C range. For erythrocytes from all species, excluding horse, the P, extrapolated at 37°C, had a value comparable with the known coefficient of the passive, ouabain-insensitive cation permeability at 37°C. For ouabain-treated human erythrocytes at 37°C, the period of thermohaemolysis-related shrinkage in sucrose containing media was found to be about six times shorter than the life-span of intact cells which substantiated the role of the active transport in balancing the thermohaemolysis-related diffusion of ions at 37°C. Consequently, the thermal resistance of erythrocytes, which was earlier related to their sphingomyelin content, was now found also to be in good correlation with their life-span in the circulation of 11 mammals.     

Influence of heat shock on chlorophyll fluorescence of white oak (<Emphasis Type="Italic">Quercus pubescens</Emphasis> Willd.) leaves

Chlorophyll fluorescence parameters of Quercus pubescens Willd. as response to heat shock (HS) by immersing leaves for 5 and 15 min in water of temperatures between 38 and 59 °C were examined. Fluorescence was measured after different periods of recovery (15, 30, 90, 210, and 1 440 min at 24/26 °C night/day temperature and 100 % humidity). The effective quantum yield of photosystem 2 (Y) in control and HS-treated leaves was always measured after previous 15 min irradiation. Under a 5 min HS, Y did not change after using temperatures below 44 °C, was rapidly restored after HS of moderate temperatures (44–48 °C), and progressively decreased and recovered eventually to the initial value after HS of high temperatures (48–52 °C). Y did not recover after HS with temperatures higher than 52 °C. Increase in the duration of HS from 5 to 15 min lead to change of the initial Y at each HS temperature, but the recovery processes were similar to those characteristic after 5 min incubation. The processes of recovery may depend mainly on the specificity of injuries caused by different heat shock temperatures. Thus Q. pubescens is able to preserve and recover the functional potential of its photosynthetic apparatus in response to HS up to 52 °C.     

Changes in erythrocyte membranes after fractioned hyperthermia

The structural changes in erythrocytes membranes were examined before and after the second heat shock of erythrocytes. Electrophoretic separation of protein erythrocyte membranes for cells incubated at 48.5°C was different from control i.e. from erythrocytes incubated at 37°C. No quantitative or qualitative changes were spotted in comparison with protein membranes isolated from the erythrocytes following single or double heat shock. Fluidity of erythrocytes membranes was determined by using spin labels, 5-doxylstearic acid and 16-doxylstearic acid. The membranes were more rigid in their hydrophobic regions after incubation of cells at 44°C. It can be suggested that erythrocyte membranes play some role in thermotolerance and heat damage of enuclate cells.     

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

Effects of heat stress in the leaf mitotic cell cycle and chromosomes of four wine-producing grapevine varieties

Grapevine varieties respond differentially to heat stress (HS). HS ultimately reduces the photosynthesis and respiratory performance. However, the HS effects in the leaf nuclei and mitotic cells of grapevine are barely known. This work intends to evaluate the HS effects in the leaf mitotic cell cycle and chromosomes of four wine-producing varieties: Touriga Franca (TF), Touriga Nacional (TN), Rabigato, and Viosinho. In vitro plants with 11 months were used in a stepwise acclimation and recovery (SAR) experimental setup comprising different phases: heat acclimation period (3 h—32 °C), extreme HS (1 h—42 °C), and two recovery periods (3 h—32 °C and 24 h—25 °C), and compared to control plants (maintained in vitro at 25 °C). At the end of each SAR phase, leaves were collected, fixed, and used for cell suspensions and chromosome preparations. Normal and abnormal interphase and mitotic cells were observed, scored, and statistically analyzed in all varieties and treatments (control and SAR phases). Different types of chromosomal anomalies in all mitotic phases, treatments, and varieties were found. In all varieties, the percentage of dividing cells with anomalies (%DCA) after extreme HS increased relative to control. TF and Viosinho were considered the most tolerant to HS. TF showed a gradual MI reduction from heat acclimation to HS and the lowest %DCA after HS and 24 h of recovery. Only Viosinho reached the control values after the long recovery period. Extrapolating these data to the field, we hypothesize that during consecutive hot summer days, the grapevine plants will not have time or capacity to recover from the mitotic anomalies caused by high temperatures.     

Erythrocyte membrane defects in hemolytic anemias found through derivative thermal analysis of electric impedance

Hereditary hemolytic anemias originate mainly from defects in hemoglobin and plasma membrane proteins. Here, we propose a new method, thermal analysis of impedance, sensitive to membrane defects. It detects three processes in erythrocyte membrane; fall in membrane capacity at 49.5 °C and activation of passive PO₄²⁺ permeability at 37 °C and inorganic ions at 61.5 °C. The denaturation of spectrin is involved in the first process whilst the anion channel is involved in latter processes. Using this method three persons with xerocytosis were found whereby the fall in membrane capacity and spherization of erythrocytes were both postponed (53 °C) compared to control (49.5 °C). In contrast to control cells, strong activation of passive permeability for Cl⁻ at 37 °C and sucrose at 61 °C were detected that were both eliminated by pre-inhibition of the anion channel with 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS). In addition, erythrocytes from 15 patients with various forms of anemia were studied in intact state and after refreshment. The results were compared with the data of clinical laboratory and osmotic fragility test. The final conclusion is that this method detects membrane defects with altered spectrin and anion channel syndrome (hereditary xerocytosis, spherocytosis, poikilocytosis and pyropoikilocytosis, elliptocytosis and stomatocytosis) and, after refreshment, helps differentiate them from the anemia with hemoglobinopathy.     

Erythrocytes <Emphasis Type="SmallCaps">l</Emphasis>-Arginine y+ Transporter Inhibition by N-Ethylmaleimide in Ice-bath

Erythrocytes l-arginine uptake is conveyed by y+ and y+L membrane transport systems. Pre-incubation with N-ethylmaleimide for 10 min at 37°C inhibits the y+ system. The aim of this study was to determine the ideal pre-incubation temperature in evaluating y+ and y+L systems. Cells were pre-incubated with or without N-ethylmaleimide for 10 min at 4°C and 37°C. l-Arginine uptake was quantified by radioisotope and standard erythrocytes membrane flux methodology. Results demonstrate that erythrocytes l-arginine content is depleted by pre-incubation at 37°C for 10 min, thus changing the V _max measurement. The inhibitory effect of N-ethylmaleimide pre-incubation was temperature independent and already complete after 1 min of incubation. No significant difference in kinetic parameters was detected between cells pre-incubated at 37°C or 4°C, under zero-trans conditions. In conclusion, we suggest that measurement of erythrocytes l-arginine uptake by y+ and y+L systems could be carried out without N-ethylmaleimide pre-incubation at 37°C.     

Thermotolerance of IVM‐derived bovine oocytes and embryos after short‐term heat shock

A series of experiments were designed to study the effect of elevated temperatures on developmental competence of bovine oocytes and embryos produced in vitro. In experiment 1, the effect of heat shock (HS) by a mild elevated temperature (40.5°C) for 0, 30, or 60 min on the viability of in vitro matured (IVM) oocytes was tested following in vitro fertilization (IVF) and culture. No significant difference was observed between the control (39°C) and the heat‐treated groups in cleavage, blastocyst formation, or hatching (P > 0.05). In experiment 2, when the HS temperature was increased to 41.5°C, neither the cleavage rate nor blastocyst development was affected by treatment. However, the rate of blastocyst hatching appeared lower in the HS groups (13% in control group vs. 3.9% and 5.6% in 30 min and 60 min, respectively; P < 0.05). When IVM oocytes were treated at 43°C prior to IVF (experiment 3), no difference was detected in blastocyst and expanded blastocyst development following heat treatment for 0, 15, or 30 min, but heat treatment of oocytes for 45 or 60 min significantly reduced blastocyst and expanded blastocyst formation (P < 0.05). In experiment 4, the thermotolerance of day 3 and day 4 bovine IVF embryos were compared. When embryos were pre‐treated with a mild elevated temperature (40.5°C) for 1 hr, and then with a higher temperature (43°C) for 1 hr, no improvement in thermotolerance of the embryos was observed as compared to those treated at 43°C alone. However, a higher thermotolerance was observed in day 4 than day 3 embryos. In conclusion, treatment at 43°C, but not 40.5°C or 41.5°C significantly reduced oocyte developmental competence. An increase in thermotolerance was observed from day 3 to day 4 of in vitro embryonic development, which corresponds to the maternal to zygotic transition of gene expression in bovine embryos. Mol. Reprod. Dev. 53:336–340, 1999. © 1999 Wiley‐Liss, Inc.     

The role of sialic acid in the determination of survival of rabbit erythrocytes in the circulation

Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating erythrocytes of rabbit. Neuraminidase is shown to remove only sialic acid from these erythrocytes. Neuraminidase-treated and intact rabbit erythrocytes have similar in vitro properties, except those of cellular charge and cellular adhesion in their sera. These properties include similar shape, osmotic fragility curve, autohemolysis at 37°, K⁺ retention and pyruvate kinase activity. The D-glucose 6-phosphate dehydrogenase and the cholinesterase activities are higher on the neuraminidase-treated erythrocytes than on the intact ones. After injection into rabbits, the sialic acid-less erythrocytes tested, were promptly removed from the circulation; intact erythrocytes, previously incubated under the same conditions but without neuraminidase, were removed from the circulation after a significantly longer period.     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

Indium-111 oxine labeled erythrocytes: Cellular distribution and efflux kinetics of the label

Indium-111 oxine labeled erythrocytes are useful in scintigraphic studies of splenic function because of the high yield of γ-photons [172(90%) and 247(94%) keV] of indium-111. However, the effects of indium-111 oxine on the structural and functional integrity of erythrocytes which might influence their reticulo-endothelial (RE) sequestration are unknown. We examined the morphology of human and rat indium-111 labeled erythrocytes by SEM, the distribution of the label within the cell by analysis of the membrane and cytosol (hemoglobin solution) and the kinetics of efflux of indium-111 from erythrocytes incubated at 37 °C in plasma or physiological buffer. Indium-111 oxine labeled red cells retain their discocytic morphology and the cell indices, and density characteristics on phthalate ester are similar to those of the control cells. The efficiency of labeling may be as high as 97%. Human or rat erythrocyte membranes retain 33 and 41% of indium-111, and the cytosol contains 67 and 59%, respectively. About 98% of the indium-111 is bound to the membrane proteins and 1% to the lipid bilayer. Efflux of indium-111 from cells in autologous plasma showed a multiphasic release resulting in about 4–5% release of the label in 2 h and 11.5% in 20 h. Cells in PBS showed 1–5% release of the label during the incubation period. These findings suggest that indium-111 oxine labeling of erythrocytes does not grossly alter the structural and deformability integrity of the cells to induce selective RE sequestration, unless the cells have been damaged prior to or during the labeling procedure, or the spleen is hyperactive.     

MEASUREMENT OF THE RATE OF ACETYLCHOLINE DIFFUSION THROUGH A BRAIN SLICE AND ITS SIGNIFICANCE IN STUDIES OF THE CELLULAR DISTRIBUTION OF ACETYLCHOLINESTERASE

The rate of ACh diffusion through a 0·8 mm thick slice from the surface of the rat cerebral cortex, under aerobic conditions at 37°C, was determined by bathing the intact surface of the slice (compartment A) with ACh containing buffer and determining the concentration of ACh in buffer bathing the cut surface of the slice (compartment B). With 1 or 5 mM-ACh in compartment A no ACh was detectable in compartment B within 3 h unless at least 95 per cent of the AChE, as assessed on homogenates, was inhibited. With a given level of AChE inhibition, the rate of ACh diffusion was dependent on its concentration in compartment A. With 1 mM-ACh in compartment A the difference between the rates of hydrolysis of ACh during diffusion through slices with an AChE inhibition of 98·3 and 99·4 per cent, as assessed by AChE assays of homogenates made from the slices, was only 6 per cent of the difference between the rates of hydrolysis of 1 m-ACh by the homogenates of that part of the slices through which diffusion took place. For 5 mM-ACh and levels of 95 and 99·2 per cent inhibition the corresponding value was 10-3 per cent. Since the concentration of ACh must fall across the slice it is not possible to calculate from these figures the number of enzyme sites involved in the hydrolysis during diffusion, i.e. the concentration of extracellular AChE. The implications of these observations are discussed, particularly in relation to studies of the release of ACh from the cerebral cortex in vivo     

Temperature sensitivity of Antarctic soil-humic substance degradation by cold-adapted bacteria

Heteropolymer humic substances (HS) are the largest constituents of soil organic matter and are key components that affect plant and microbial growth in maritime Antarctic tundra. We investigated HS decomposition in Antarctic tundra soils from distinct sites by incubating samples at 5°C or 8°C (within a natural soil thawing temperature range of −3.8°C to 9.6°C) for 90 days (average Antarctic summer period). This continuous 3-month artificial incubation maintained a higher total soil temperature than that in natural conditions. The long-term warming effects rapidly decreased HS content during the initial incubation, with no significant difference between 5°C and 8°C. In the presence of Antarctic tundra soil heterogeneity, the relative abundance of Proteobacteria (one of the major bacterial phyla in cold soil environments) increased during HS decomposition, which was more significant at 8°C than at 5°C. Contrasting this, the relative abundance of Actinobacteria (another major group) did not exhibit any significant variation. This microcosm study indicates that higher temperatures or prolonged thawing periods affect the relative abundance of cold-adapted bacterial communities, thereby promoting the rate of microbial HS decomposition. The resulting increase in HS-derived small metabolites will possibly accelerate warming-induced changes in the Antarctic tundra ecosystem.     

Heat shock induces simultaneous rearrangements of all known cytoskeletal filaments in normal interphase fibroblasts

Hyperthermia (heat shock (HS)) induces changes in morphology of nucleoli, cytoplasmic organelles, and cytoskeleton. Responses to hyperthermia are, as a rule, similar in all types of eukaryote cells. However, there is no information on the uniformity of the cytoskeleton heat shock response (CHSR) in different cell types. This has led to the conclusion that the eukaryote CHSR depends on the cell type. We studied CHSR only in one cell type-in normal embryonic mouse fibroblasts (NEMFs) and in normal embryonic rat fibroblasts (NERFs), as well as in normal postnatal rat fibroblasts (NPRFs), by using the method of fluorescence microscopy. Incubation of the cells at 43°C led to a rearrangement of cytoskeleton. Responses of cytoskeleton to HS in NEMF, NERF, and NPRF were similar. Heat shock resulted in disassembly of bundles of actin filaments (AFs), marked changes in microtubule (MT) morphology, and collapse of intermediate filaments (IFs) around the nucleus. Rearrangements of different cytoskeleton filament types occurred simultaneously and were seen as soon as after 2–4 min. After 30–120 min of incubation at 43°C, the cells were still capable of rebuilding the actin cytoskeleton after the temperature had returned to normal (37°C). We believe that the cytoskeleton rearrangement under the action of HS can be of vital importance in cell protection against temperature stress. Data are discussed on possible coupling of the CHSR process with rearrangement of the protein synthesizing system, which leads to initiation and/or stimulation of synthesis of HS proteins.     

Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism

The levels of pyruvate kinase (PYK), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast-to-hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25°C to 37°C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25°C to 37°C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late-exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the Increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape.     

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C₁₂E₈) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C₁₂E₈ DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C₁₂E₈ DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C₁₂E₈ treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C₁₂E₈ to the study of DRMs.     

Changes in the Levels and Forms of Cholinesterases in the Blood Plasma of Normal and Dystrophic Chickens

Abstract: Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were analysed in the blood plasma of developing chickens, both normal and those with inherited muscular dystrophy. The amounts and the molecular forms of each were examined. °ChE concentration rises in the plasma of normal and dystrophic chicks at the end of embryonic development and is maintained after hatching at a constant, relatively high level, accounting for 90-95% of total cholinesterase activity in normal plasma. This level is maintained in normal and dystrophic chickens. In embryonic plasma of both normal and dystrophic chicks, on the other hand, the levels of AChE are higher than those of °ChE. Immediately after hatching the AChE level decreases rapidly in normal plasma, reaching a very low level by 2-3 weeks ex ovo. The AChE level in plasma from dystrophic birds, although less than normal from day 19 in ovo to 2 weeks ex ovo, subsequently increases to peak around 4 months at levels 15-20-fold of those in normal birds. There is virtually no enzyme of either type in the erythrocytes of normal or dystrophic chickens. The changes of AChE in plasma were correlated with the alterations of AChE in dystrophic fast-twitch muscles, suggesting that the latter pool is a precursor of the plasma AChE. Both the AChE and the °ChE in plasma exist in multiple molecular forms, which are similar to certain of those found previously in the muscles of these birds. The major form (60-80%) of both enzymes in the plasma is the M form (sedimentation coefficient ≥11 S) in all cases, but it is accompanied by certain other forms. In no case is there any of the heaviest form (H₂, 19-20 S) of AChE or of °ChE found in normal and dystrophic muscle, which is attached at the synapses in normal muscle. The pattern of forms of plasma °ChE is constant at all ages, and in normal and dystrophic chickens. The pattern of forms of AChE in the plasma, in contrast, varies with age and with dystrophy in a characteristic manner. The sedimentation coefficients and the amounts of the enzymes in fast-twitch muscle of dystrophic animals are compared with those of the plasma forms, and an interpretation is given of the characteristic patterns of AChE and of χE in their blood.     

Effects of supplemental zinc amino acid complex on gut integrity in heat-stressed growing pigs

Heat stress (HS) jeopardizes livestock health and productivity and both may in part be mediated by reduced intestinal integrity. Dietary zinc improves a variety of bowel diseases, which are characterized by increased intestinal permeability. Study objectives were to evaluate the effects of supplemental zinc amino acid complex (ZnAA) on intestinal integrity in heat-stressed growing pigs. Crossbred gilts (43±6 kg BW) were ad libitum fed one of three diets: (1) control (ZnC; 120 ppm Zn as ZnSO₄; n=13), (2) control+100 ppm Zn as ZnAA (Zn220; containing a total of 220 ppm Zn; n=14), and (3) control+200 ppm Zn as ZnAA (Zn320; containing a total of 320 ppm Zn; n=16). After 25 days on their respective diets, all pigs were exposed to constant HS conditions (36°C, ∼50% humidity) for either 1 or 7 days. At the end of the environmental exposure, pigs were euthanized and blood and intestinal tissues were harvested immediately after sacrifice. As expected, HS increased rectal temperature (P⩽0.01; 40.23°C v. 38.93°C) and respiratory rate (P⩽0.01; 113 v. 36 bpm). Pigs receiving ZnAA tended to have increased rectal temperature (P=0.07; +0.27°C) compared with ZnC-fed pigs. HS markedly reduced feed intake (FI; P⩽0.01; 59%) and caused BW loss (2.10 kg), but neither variable was affected by dietary treatment. Fresh intestinal segments were assessed ex vivo for intestinal integrity. As HS progressed from days 1 to 7, both ileal and colonic transepithelial electrical resistance (TER) decreased (P⩽0.05; 34% and 22%, respectively). This was mirrored by an increase in ileal and colonic permeability to the macromolecule dextran (P⩽0.01; 13- and 56-fold, respectively), and increased colonic lipopolysaccharide permeability (P⩽0.05; threefold) with time. There was a quadratic response (P⩽0.05) to increasing ZnAA on ileal TER, as it was improved (P⩽0.05; 56%) in Zn220-fed pigs compared with ZnC. This study demonstrates that HS progressively compromises the intestinal barrier and supplementing ZnAA at the appropriate dose can improve aspects of small intestinal integrity during severe HS.