Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XI. Horses Introduced to Australia and European Horses ( Equus caballus )

The diffusional water permeability (P _d) of red blood cells (RBCs) from horses introduced to Australia and from European horses was measured by a Mn²⁺ doping nuclear magnetic resonance (NMR) technique. The values of P _d were ∼3.5 × 10⁻³ cm/s at 25°C, 4.1 × 10⁻³ cm/s at 30°C, 5.6 × 10⁻³ cm/s at 37°C and 6.3 × 10⁻³ cm/s at 42°C with no significant differences between the two strains of horse. Systematic studies on the effect of p-chloromercuribenzene sulphonate (PCMBS) on water diffusion indicated that the maximal inhibition (60%) was reached in 60 min at 22°C with 1 mm PCMBS. The basal permeability to water was estimated at 1.2 × 10⁻³ cm/s at 25°C, 1.7 × 10⁻³ cm/s at 30°C, 2.0 × 10⁻³ cm/s at 37°C and 2.7 × 10⁻³ cm/s at 42°C. The activation energy (E _a,d) of water diffusion was ∼25 kJ/mol and increased to ∼39 kJ/mol after incubation with PCMBS, in conditions of maximal inhibition of water diffusion. The membrane protein electrophoretic pattern of horse RBCs was compared with its human counterpart. The horse RBC membranes had lower amounts of the proteins migrating as bands 4.1 and 4.2 and higher amounts of the protein migrating as band 4.9, and band 6 (glyceraldehyde-3-phosphate dehydrogenase) was undetectable. A noteworthy feature was the appearance of considerable differences in protein migration distances in the region of bands 7 and 9, between horse and human membranes.     

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XII. Dog (<Emphasis Type="BoldItalic">Canis familiaris</Emphasis>) and Cat (<Emphasis Type="BoldItalic">Felis domestica</Emphasis>)

The diffusional water permeability (P _d ) of dog and cat red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulphonate (PCMBS). The values of P _d were in the case of cat RBC ∼3.0 × 10⁻³ cm/s at 15 °C, 3.5 × 10⁻³ cm/s at 20 °C, 4.2 × 10⁻³ cm/s at 25 °C, 4.4 × 10⁻³ cm/s at 30 °C and 5.9 × 10⁻³ cm/s at 38 °C. In case of dog RBC the values of P _d were higher ∼3.8 × 10⁻³ cm/s at 15 °C, 4.6 × 10⁻³ cm/s at 20 °C, 5.0 × 10⁻³ cm/s at 25 °C, 5.9 × 10⁻³ cm/s at 30 °C and 7.9 × 10⁻³ cm/s at 37 °C. Systematic studies of the effect of PCMBS on water diffusion indicated that in the case of dog RBCs the maximal inhibition was reached in 15–30 min with 1 mm PCMBS, whereas in the case of cat RBCs in 60 min with 1 mm PCMBS or in 30 min with 2 mm PCMBS. The values of maximal inhibition in the case of cat RBC were in the range of 55–60% at 15 °C, 60–68% at 20 °C and 25 °C, 50–60% at 30 °C and 50–55% at 37 °C. In the case of dog RBC the corresponding values were higher, 75–80% at 15 °C, 70–80% at 20 °C and 25 °C, 65–70% at 30 °C and 55–60% at 37 °C. The basal permeability to water was estimated to be ∼1 × 10⁻³ cm/s −2 × 10⁻³ cm/s in the range of temperatures of 25–37 °C. The activation energy of water diffusion E _a,d was ∼19 kJ/mol for the dog RBC and ∼23 kJ/mol for the cat RBC. After incubation with PCMBS the values of E _a,d increased, reaching 40 kJ/mol in conditions of maximal inhibition of water exchange. The membrane polypeptide electrophoretic pattern of dog and cat RBCs has been compared with its human counterpart. Dog and cat RBCs contained higher amounts of spectrin (band 1 and 2) and lower amounts of bands 4.4, 4.2, band 5 and band 7 compared to human RBCs. Band 4.9 was decreased only in the cat RBCs, whereas band 6 was decreased only in the dog RBCs. Correspondence and offprint requests to: Gheorghe Benga, Department of Cell and Molecular Biology, ‘Iuliu Haţieganu’ University of Medicine and Pharmacy, 6 Pasteur St, 3400 Cluj-Napoca, Romania. Tel:/Fax: 40–64–194373; e-mail: GBenga@personal.ro; gbenga@umfcluj.ro     

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel ( Camelus dromedarius ) and alpaca ( Lama pacos )

The diffusional water permeability (P _d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were, in the case of alpaca RBC≈4.6×10⁻³ cm/s at 25°C, 5.4×10⁻³ cm/s at 30°C, 6.6×10⁻³ cm/s at 37°C and 7.7×10⁻³ cm/s at 42°C. In case of camel RBC the values ofP _d where ≈4.2×10⁻³ cm/s and 9.0×10⁻³ cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10⁻³ cm/s at 25°C, 1.7×10⁻³ cm/s at 30°C and of camel RBC as 1.8×10⁻³ cm/s at 25°C and 3.0×10⁻³ cm/s at 37°C. The values of the activation energy of water diffusion (E _{a, d}) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel (Camelus dromedarius) and alpaca (Lama pacos)

The diffusional water permeability (P _d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were, in the case of alpaca RBC≈4.6×10^?3 cm/s at 25°C, 5.4×10^?3 cm/s at 30°C, 6.6×10^?3 cm/s at 37°C and 7.7×10^?3 cm/s at 42°C. In case of camel RBC the values ofP _d where ≈4.2×10^?3 cm/s and 9.0×10^?3 cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10^?3 cm/s at 25°C, 1.7×10^?3 cm/s at 30°C and of camel RBC as 1.8×10^?3 cm/s at 25°C and 3.0×10^?3 cm/s at 37°C. The values of the activation energy of water diffusion (E _{a, d}) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.     

Haematology of Persian Fallow Deer ( Dama mesopotamica )

The haematology of Persian fallow deer was studied and the means of various parameters were determined for sex and age groups. For total samples, the mean ± standard deviation of haematological parameters were: red blood cells (RBC), 7.42 ± 1.27 × 10¹²/l; haematocrit (PCV), 38.83 ± 7.38%; haemoglobin (Hb), 148.0 ± 17.3 g/l; mean cell volume (MCV), 50.84 ± 7.26 fl; mean corpuscular haemoglobin (MCH), 20.17 ± 1.98 pg; mean corpuscular haemoglobin concentration (MCHC), 38.71 ± 3.88%; white blood cells (WBC) 3.200 ± 1.697 × 10⁹/l; neutrophils, 1.410 ± 0.446 × 10⁹/l, lymphocytes, 1.382 ± 1.116 × 10⁹/l, monocytes, 0.048 ± 0.100 × 10⁹/l, eosinophils, 0.341 ± 0.339 × 10⁹/l, basophils, 0.009 ± 0.005 × 10⁹/l, neutrophil:lymphocyte ratio (N:L), 2.11 ± 2; platelets, 354.8 ± 116.62 × 10⁹/l and fibrinogen, 2.855 ± 1.126 g/l. Three types of haemoglobin were separated by electrophoresis: HbC with higher migration, HbB and HbA with lowest movement. Significant differences were seen for RBC, MCV, MCH, HbC, and HbB concentrations between sexes.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. IX. Australian feral chicken and domestic chicken (Gallus domesticus)

The diffusional water permeability (P _d) of Australian feral chicken and Australian and European domestic chicken red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique. The values of P _d were around 1.7 × 10^?3 cm/s at 15°C, 2.0 × 10^?3 cm/s at 20°C, 2.5 × 10^?3 cm/s at 25°C, 3.7 × 10^?3 cm/s at 30°C, 4.3 × 10^?3 cm/s at 37°C, and 6.1 × 10^?3 cm/s at 42°C, with no significant differences between the three strains of chicken. There was no effect of p-chloromercuribenzene sulphonate on water diffusion. The activation energy of water diffusion was around 37 kJ/mol for all strains of chicken. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of chicken introduced to Australia (and that membrane proteins play little role in the diffusion of water across chicken RBC membrane).     

The influence of either no fluid or carbohydrate-electrolyte fluid ingestion and the environment (thermoneutral versus hot and humid) on running economy after prolonged, high-intensity exercise

This study investigated the effects on running economy (RE) of ingesting either no fluid or an electrolyte solution with or without 6% carbohydrate (counterbalanced design) during 60-min running bouts at 80% maximal oxygen consumption (V˙O_2max). Tests were undertaken in either a thermoneutral (22–23°C; 56–62% relative humidity, RH) or a hot and humid natural environment (Singapore: 25–35°C; 66–77% RH). The subjects were 15 young adult male Singaporeans [V˙O_2max = 55.5 (4.4 SD) ml kg⁻¹ min⁻¹]. The RE was measured at 3 m s⁻¹ [65 (6)% V˙O_2max] before (RE1) and after each prolonged run (RE2). Fluids were administered every 2 min, at an individual rate determined from prior tests, to maintain body mass (group mean = 17.4 ml min⁻¹). The V˙O₂ during RE2 was higher (P < 0.05) than that during the RE1 test for all treatments, with no differences between treatments (ANOVA). The mean increase in V˙O₂ from RE1 to RE2 ranged from 3.4 to 4.7 ml kg⁻¹ min⁻¹ across treatments. In conclusion, the deterioration in RE at 3 m s⁻¹ (65% V˙O_2max) after 60 min of running at 80% V˙O_2max appears to occur independently of whether fluid is ingested and regardless of whether the fluid contains carbohydrates or electrolytes, in both a thermoneutral and in a hot, humid environment. Accepted: 30 October 1997     

Glucose 6-phosphate dehydrogenase from larval Taenia crassiceps (cysticerci): purification and properties

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified to homogeneity from the soluble fraction of larval Taenia crassiceps (Eucestoda: Cyclophyllidea) by a three-step protocol. Specific activity of the pure enzyme was 33.8 ± 2.1 U mg⁻¹ at 25°C and pH 7.8 with d-glucose 6-phosphate and NADP⁺ as substrates. The activity increases to 67.6 ± 3.9 U mg⁻¹ at 39°C, a more physiological temperature in the intermediary host. Enzyme activity was maximal between pH 6.7 and 7.8. K _m values were 14 ± 1.7 μM and 1.3 ± 0.4 μM for glucose 6-phosphate and NADP⁺, respectively. The enzyme showed absolute specificity for its sugar substrate. NAD⁺ was also a substrate but with a low catalytic efficiency (207 M⁻¹ s⁻¹). No essential requirement for Mg⁺⁺ or Ca⁺⁺ was observed. Relative molecular mass of the native enzyme was 134,000 ± 17,200, while a value of 61,000 ± 1,700 was obtained for the enzyme subunit. Thus, glucose 6-phosphate dehydrogenase from T. crassiceps exists as a dimeric protein. The enzyme’s isoelectric point was 4.5. The enzyme’s activity dependence on temperature was complex, resulting in a biphasic Arrhenius plot. Activation energies of 9.91 ± 0.51 and 7.94 ± 0.45 kcal mol⁻¹ were obtained. Initial velocity patterns complemented with inhibition studies by product and substrate’s analogues support a random bi bi sequential mechanism in rapid equilibrium. The low K _i value of 1.95 μM found for NADPH suggests a potential regulatory role for this nucleotide.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. VIII. Adult and fetal guinea pig (Cavia procellus)

The diffusional water permeability (P _d) of adult, pregnant female and fetal guinea-pig red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were around 5.0 × 10^?3 cm/s at 15 °C, 5.3 × 10-3 cm/s at 20 °C, 6.6 × 10^?3 cm/s at 25 °C, 7.5 × 10^?3 cm/s at 30 °C and 8.6 × 10^?3 cm/s at 37 °C with no significant differences between adult, pregnant female and fetal RBCs. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 10 min at 37 °C with 0.1 mm PCMBS. The values of maximal inhibition ranged from 70%–77% at 15–30 °C to 57%–63% at 37 °C in the case of adult and from 64%–67% at 15–30 °C to 51% at 37 °C in the case of fetal RBCs. The basal permeability to water was estimated at 1.1 × 10^?3 cm/s at 15 °C ,1.3 × 10^?3 cm/s at 20 °C, 1.6 × 10^?3 cm/s at 25 °C, 2.2 × 10^?3 cm/s at 30 °C and 3.2 × 10^?3 cm/s at 37 °C for adult and slightly higher values for fetal guinea pig RBCs as 1.6 × 10^?3 cm/s at 15 °C, 2.0 × 10^?3 cm/s at 20 °C, 2.4 × 10^?3 cm/s at 25 °C, 2.6 × 10^?3 cm/s at 30 °C and 4.2 × 10^?3 cm/s at 37 °C. The activation energy of water diffusion was around 22 kJ/ mol, with no significant differences between the adult pregnant female and fetal RBCs, and increased to about 40 kJ/mol in the case of adult and pregnant RBCs and 34 kJ/mol for fetal RBCs after incubation with PCMBS in conditions of maximal inhibition of water diffusion. The membrane polypeptide electrophoretic pattern of adult and fetal guinea-pig RBCs was compared with its human counterpart. The guinea-pig membrane contained higher amounts of spectrin (band 1 and 2), whereas the proteins in bands 4.1, 4.2 and 6 were present in lower amounts. Considerable differences in polypeptides migrating in the region of bands 7 and 8 and in front of them were apparent between the two sources of RBC membranes where some bands were present only in the guinea-pig RBC membranes. The adult guinea-pig membranes contained smaller amounts of proteins migrating in band 4.5 and lacked band 8.     

Effect of cold exposure (−15°C) and Salbutamol treatment on physical performance in elite nonasthmatic cross-country skiers

The effects of whole-body exposure to ambient temperatures of −15°C and 23°C on selected performance-related physiological variables were investigated in elite nonasthmatic cross-country skiers. At an ambient temperature of −15°C we also studied the effects of the selective β₂-adrenergic agonist Salbutamol (0.4 mg × 3) which was administered 10 min before the exercise test. Eight male cross-country skiers with known maximal oxygen uptakes (V˙O_{2 max} ) of more than 70 ml · kg⁻¹ · min⁻¹ participated in the study. Oxygen uptake (V˙O₂), heart rate (f _c), blood lactate concentration ([La⁻]_b) and time to exhaustion were measured during controlled submaximal and maximal running on a treadmill in a climatic chamber. Lung function measured as forced expiratory volume in 1 s (FEV₁) was recorded immediately before the warm-up period and at the conclusion of the exercise protocol. Submaximal V˙O₂ and [La⁻]_b at the two highest submaximal exercise intensities were significantly higher at −15°C than at 23°C. Time to exhaustion was significantly shorter in the cold environment. However, no differences in V˙O_{2 max} or f _c were observed. Our results would suggest that exercise stress is higher at submaximal exercise intensities in a cold environment and support the contention that aerobic capacity is not altered by cold exposure. Furthermore, we found that after Salbutamol inhalation FEV₁ was significantly higher than after placebo administration. However, the inhaled β₂-agonist Salbutamol did not influence submaximal and maximal V˙O₂, f _c, [La⁻]_b or time to exhaustion in the elite, nonasthmatic cross-country skiers we studied. Thus, these results did not demonstrate any ergogenic effect of the β₂-agonist used. Accepted: 18 August 1997     

HCO 3 − -dependent volume regulation in α-cells of the rat endocrine pancreas

Ion transport activity in pancreatic α-cells was assessed by studying cell volume regulation in response to anisotonic solutions. Cell volume was measured by a video imaging method, and cells were superfused with either 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-buffered or HCO₃⁻-buffered solutions. α-Cells did not exhibit a regulatory volume increase (RVI) in response to cell shrinkage caused by hypertonic solutions. A RVI was observed, however, in cells that had first undergone a regulatory volume decrease (RVD), but only in HCO₃⁻-buffered solutions. RVI was also observed in response to a HCO₃⁻-buffered hypertonic solution in which the glucose concentration was increased from 4 to 20 mM. The post-RVD RVI and the glucose-induced RVI were both inhibited by 10 μM 5-(N-methyl-N-isobutyl) amiloride or 100 μM 2,2′-(1,2-ethenediyl) bis (5-isothio-cyanatobenzenesulfonic acid), but not by 10 μM benzamil nor 10 μM bumetanide. These data suggest that Na⁺–H⁺ exchangers and Cl⁻–HCO₃⁻ exchangers contribute to volume regulation in α-cells.     

Measurement and prediction of peak shivering intensity in humans

Prediction equations of shivering metabolism are critical to the development of models of thermoregulation during cold exposure. Although the intensity of maximal shivering has not yet been predicted, a peak shivering metabolic rate (Shiv_peak) of five times the resting metabolic rate has been reported. A group of 15 subjects (including 4 women) [mean age 24.7 (SD 6) years, mean body mass 72.1 (SD 12) kg, mean height 1.76 (SD 0.1) m, mean body fat 22.3 (SD 7)% and mean maximal oxygen uptake (V˙O_2max) 53.2 (SD 9) ml O₂ · kg⁻¹ · min⁻¹] participated in the present study to measure and predict Shiv_peak. The subjects were initially immersed in water at 8°C for up to 70 min. Water temperature was then gradually increased at 0.8 °C · min⁻¹ to a value of 20 °C, which it was expected would increase shivering heat production based on the knowledge that peripheral cold receptors fire maximally at approximately this temperature. This, in combination with the relatively low core temperature at the time this water temperature was reached, was hypothesized would stimulate Shiv_peak. Prior to warming the water from 8 to 20 °C, the oxygen consumption was 15.1 (SD 5.5) ml · kg⁻¹ · min⁻¹ at core temperatures of approximately 35 °C. After the water temperature had risen to 20 °C, the observed Shiv_peak was 22.1 (SD 4.2) ml O₂ · kg⁻¹ · min⁻¹ at core and mean skin temperatures of 35.2 (SD 0.9) and 22.1 (SD 2.2) °C, respectively. The Shiv_peak corresponded to 4.9 (SD 0.8) times the resting metabolism and 41.7 (SD 5.1)% of V˙O_2max. The best fit equation predicting Shiv_peak was Shiv_peak (ml O₂ · kg⁻¹ · min⁻¹)=30.5 + 0.348 ×V˙O_2max (ml O₂ · kg⁻¹ · min⁻¹) − 0.909 × body mass index (kg · m⁻²) − 0.233 × age (years); (P=0.0001; r ²=0.872). Accepted: 7 September 2000     

Development of a fluorinated polyimide hollow fiber for medical devices

 We fabricated an asymmetric polyimide hollow fiber for medical devices. A dry/wet phase inversion process was applied to a spinning process to prepare the hollow fiber. The outer diameter was 330 μm with a wall thickness of 70 μm. Transfer rates of O₂ and CO₂ in the asymmetric polyimide fiber were 6.9 × 10⁻³ and 5.5 × 10⁻³ cm³ (STP)/(cm² s cmHg), respectively, which are approximately 10 times higher than those measured in the Menox and Si-polypropylene fibers of the presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber was evaluated in vivo, indicating that polyimide had excellent blood compatibility when compared with silicone-coated fiber. Additionally, we fabricated a novel porous membrane with three-dimensional fine structure from cylindrical microscale pores and examined possibility of a porous membrane for use in hemodialysis. Received: October 3, 2002 / Accepted: February 20, 2003 Acknowledgments This work was supported by a JSAO Grant. The authors wish to thank Mr. K. Kuwana of Senko Medical Instrument Mfg. Co., Ltd., for providing the Si-PP hollow fibers and Mr. K. Sakai of Dainippon Ink and Chemicals, Inc., for providing the Menox hollow fibers. Also the authors thank Dr. S. Nagaoka, Mr. M. Niwa, and Ms. Y. Taketani of Tokyo Metropolitan University for their useful comments.     

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10⁻² ng/μl (278-bp, S. enterica), 11.2 × 10⁻² ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10⁻² ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10⁴ copies/μl, 3.58 × 10⁴ copies/μl, and 1.79 × 10⁴ copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.     

Erythropoiesis in the Diploid and Tetraploid Odontophrynus americanus : An Evolutionary Approach in These Cryptic Species (Amphibia, Anura, Leptodactylidae)

Polyploidy is one of Nature’s strategies to create diversity among fauna and flora, resulting in new species. We have used light, scanning and transmission electron microscopy to perform morphometric analyses in maturing erythroid cells of the cryptic species diploid and tetraploid Odontophrynus americanus frogs. Normal blood of both specimens contained 97%–99% erythrocytes and 1%–3% reticulocytes, besides thrombocytes and leucocytes. Mature erythrocytes were flattened, ellipsoidal, nucleated, with cytoplasm rich in haemoglobin. Five days after being made anaemic, 15% and 33% of diploid and tetraploid red blood cells respectively, were in an immature stage, basically proerythroblasts. These cells were also seen at the 10th day, in addition to basophilic and polychromatophilic erythroblasts. By day 15 a higher number, 75% and 89% of reticulocytes in earlier maturation stage was found, respectively. At day 20 of recovery from anaemia, there were 63% and 85% of reticulocytes, respectively, most in an advanced stage of maturation. The number of immature cells then gradually decreased at days 30 and 50. Cytoplasmic inclusions similar to Heinz bodies were found in these cells associated with RNA or RNP. Morphometric analysis showed that the tetraploid erythroid cells synthesise 30% more ribosomes than the diploid erythroid cells. The density of ribosomes/μm² allowed these cells to be classified into seven classes: proerythroblasts, basophilic erythroblasts I and II, polychromatophilic erythroblasts I and II, reticulocytes and erythrocytes. Such morphometric strategy suggested that gene activity was more intense in the tetraploid maturing erythroid cells, despite the marked tendency of these tetraploid cells towards diploidisation of the genome expression.     

Attenuated β-adrenoceptor-mediated cardiac contractile responses following androgenic steroid administration to sedentary rats

Androgenic steroids administered in doses at pharmacological levels to sedentary animals have been shown to result in a reduced β-adrenoceptor-mediated increase in systolic cardiac performance when assessed in vivo. Whether the attenuated adrenergic response occurs as a consequence of alterations in either cardiac loads, heart rate, modifications in left ventricular (LV) geometry, or a decrease in myocardial contractile performance has not been determined. In this study the effect of chronic administration (over 3 months) of an androgenic steroid (nandrolone decanoate, 5 mg · kg⁻¹ biweekly) on the response of load-insensitive indices of myocardial contractile function [the slope of the LV systolic stress-strain relationship (LV-Eⁿ _max, where Eⁿ _max is systolic myocardial elastance)] to an adrenergic-inotropic stimulus was examined ex vivo in paced rat hearts. Systolic cardiac performance was assessed at 300 beats · min⁻¹ in isolated constant flow perfused heart preparations both before and during 10^−8.5 mol · l⁻¹ isoproterenol (ISO) infusion (approximate concentration of ISO eliciting 50% maximal inotropic response to ISO). Steroid administration resulted in left-shifted LV systolic and diastolic pressure-volume (P-V ) relationships. The leftshifted P-V relationships were attributed, in part, to increased slopes of these relationships. However, the steroid-mediated increment in the slope of the systolic P-V relationship (systolic chamber elastance, E_max) was not associated with a similar change in LV Eⁿ _max [control 19.2 (SEM 2.1) g · cm⁻², steroid 18.3 (SEM 2.4) g . cm⁻²] as determined in the absence of ISO. Isoproterenol infusion resulted in an increase in both E_max and Eⁿ _max in the control rats, without altering systolic performance in the steroid treated rats. Consequently, in the presence of ISO, the steroid treated rats exhibited a similar E_max, but a reduction in Eⁿ _max compared to the control rats [control 25.6 (SEM 1.9) g · cm⁻², steroid 18.5 (SEM 1.5) g · cm⁻²; P < 0.05]. In conclusion, these results would suggest that chronic high dose androgenic steroid administration produces a decrease in myocardial contractile reserve to β-adrenoceptor stimulation. Accepted: 3 September 1999     

Two new Myxidium species (Myxosporea: Myxidiidae) infecting the gallbladder of African flying fish, Cheilopogon nigricans and Suez fusilier, Caesio suevicus from the Red Sea, Egypt: a morphological and morphometric study

Myxidium maamouni sp. n. and Myxidium aydai sp. n. were described from the gallbladder of the African flying fish Cheilopogon nigricans and Suez fusilier Caesio suevicus, respectively. Fishes were collected from the Red Sea at Al-Quseir, Egypt. M. maamouni have irregular to mostly rounded polysporous plasmodia with diameter of 27 μm. Spores were sigmoid or S-shaped and sometimes spindle-shaped in the frontal view with smooth valves. They measured 13.5 × 8.0 × 8.2 μm in size. Their polar capsules were equal pyriform and measured 7.0 × 3.2 μm in size with nine to 12 coils. Spores of M. aydai were spindle-shaped in the frontal view with thin smooth valves. They measured 23.0 × 5.6 × 5.5 μm in size. Their polar capsules were pyriform and measured 7.2 × 3.4 μm in size with eight to nine coils.     

Effects of Duration of Storage and Storage Temperature on Cell Counts of Bovine Blood Samples as Determined by an Automated Haematology Analyser

Analysis of blood cells is an important part of many scientific investigations in the field of cattle herd health. Over the last 30 years, automated blood analysis has all but replaced manual counting of blood cells using counting chambers. The present study investigated the effects of prolonged storage and storage temperatures on cell counts as determined by a haematology analyser. Blood samples from 20 clinically healthy cows were repeatedly analysed with a Cell-Dyn 3500 (Abbott Diagnostika, Delkenheim), within 24 hours after collection and after storage at either 4° C or 20° C. The counts of most blood cells were more stable in samples stored at 20° C than those stored at 4° C. For at least 8 h, the counts of all analysed cell types, with the exception of lymphocytes, remained within ±3 standard deviations that were calculated for fresh samples, provided that the blood was stored at 20° C. Correspondence and offprint requests to: Dr Ulrich Bleul, Klinik für Fortpflanzungskunde, Universit?t Zürich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. IX. Australian feral chicken and domestic chicken (Gallus domesticus)

The diffusional water permeability (P _d) of Australian feral chicken and Australian and European domestic chicken red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique. The values of P _d were around 1.7 × 10^–3 cm/s at 15°C, 2.0 × 10^–3 cm/s at 20°C, 2.5 × 10^–3 cm/s at 25°C, 3.7 × 10^–3 cm/s at 30°C, 4.3 × 10^–3 cm/s at 37°C, and 6.1 × 10^–3 cm/s at 42°C, with no significant differences between the three strains of chicken. There was no effect of p-chloromercuribenzene sulphonate on water diffusion. The activation energy of water diffusion was around 37 kJ/mol for all strains of chicken. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of chicken introduced to Australia (and that membrane proteins play little role in the diffusion of water across chicken RBC membrane).