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).     

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.     

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.     

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.     

Cytokines and chemokines in postovulatory follicle regression of domestic chicken (Gallus gallus domesticus)

The mechanism of postovulatory follicle (POF) regression in birds is still poorly understood. In the current study, expression of IL-1beta, IL-6, GM-CSF, IFN-gamma, IL-2, IL-4, IL-13, chCXCLi2, chCCLi2, chCCLi4, chCCLi7, IL-10 and TGF-beta2 mRNAs was estimated in regressing POF by semi-quantitative RT-PCR. In addition, the changes in immune cell population, histological and apoptotic changes were also studied in regressing POF. The expression of cytokines (IL-1beta, IL-6, IL-10 and TGF-beta2) and chemokines (chCXCLi2, chCCLi2, chCCLi4 and chCCLi7) was upregulated in POFs, suggesting a role for these molecules in tissue regression. The histological findings suggested a significant infiltration of immune cells, especially heterophils, lymphocytes and macrophages, into the regressing POF. The flow cytometry analysis of lymphocyte subpopulations revealed that CD3(+), CD4(+), CD8(+) and Bu-1(+) lymphocytes were significantly increased during this regression. The significant up-regulation of chemokines might have attracted the immune cells during POF regression. The percentage of apoptotic cells was significantly increased during the regression of POF. The up-regulation of IL-1beta, IL-6, IL-10 and TGF-beta2 and down-regulation of GM-CSF might have induced apoptosis during the POF regression. However, expression of IFN-gamma, IL-2, IL-4 and IL-13 was not significantly altered during POF regression. In conclusion, cytokines appear to play an important role in the regression of POF in chicken. Furthermore, the regression of chicken POF seems to be an inflammatory event similar to luteolysis of the mammalian corpus luteum.     

Comparative distribution of NADPH-diaphorase activity and tyrosine hydroxylase immunoreactivity in the diencephalon and mesencephalon of the domestic chicken (Gallus domesticus)

We described the distribution of NADPH-diaphorase-containing neurons in relation to tyrosine hydroxylase immunoreactivity in the diencephalon and mesencephalon of the chicken. In the diencephalon, both markers were found in the lateral hypothalamus, dorsal hypothalamic area, hypothalamic periventricular nucleus, paraventricular nucleus and mamillary area. A close examination showed that the fine distribution of these markers differed slightly, so that they were never observed in the same neurons. In the mesencephalon, NADPH-diaphorase and tyrosine hydroxylase immunoreactivity were found in the ventral pedunculopontine area (nucleus tegmenti pedunculopontinus pars compacta, adjacent areas surrounding the quintofrontal tract and the nucleus mesencephalicus profundus ventralis), the coeruleus complex (locus coeruleus, ventral and dorsal subcoeruleus nuclei), the ventral tegmental area and the central gray. The majority of these neurons contained either diaphorase or tyrosine hydroxylase. Nevertheless, in a few cases both markers appeared to colocalize in the same neuron, typically in large perikarya of the ventral pedunculopontine area.     

Histomorphometric study of the left and right brachiocephalic arteries in different ages of chicken (Gallus domesticus)

Brachiocephalic arteries (left and right) in chicken are large arteries and information about their quantitative microanatomy remains scarce. The purpose of this study was to compare the width diameter of the brachiocephalic arteries layers in chicken at different ages (Gallus gallus domesticus). Three healthy chickens at four different ages (days 5, 18, 26, and 40) were euthanized. The left and right brachiocephalic arteries were separately collected and fixed in 10 % buffered formalin. After preparation of histological sections, the samples were stained with hematoxylin–eosin and Masson's trichrome stain. The thickness of the intima, media, and adventitia layers and the diameter of the arteries were microscopically measured. Brachiocephalic arteries in chicken were elastic type. The thickness of the tunica media of the right artery was more than that of the left one from days 18 to 40 (P?<?0.05). At day 40, the diameter of the left brachiocephalic artery was higher than the right one, but it was not significant (P?=?0.29).     

The rate of absorption into the embryonic chicken (Gallus domesticus) of 3H-testosterone.

The maximum absorption of tritiated testosterone by 5-day old embryos occurred one hour after its application to the eggshell. This decreased to relatively low levels during a 77 hour period. When the hormone was applied at the eleventh day of incubation there was a relatively large initial uptake by the bursa, spleen and thymus in that order and a subsequent diminution. Activity absorbed by the spleen remained essentially unchanged throughout the experimental period. The bursa appeared to have the greatest affinity for the hormone.     

Carbon-13 nuclear magnetic resonance studies of poly(N-vinylcarbazole)

The ¹³C NMR spectra of poly(N-vinylcarbazole), [poly(1-(N-carbazolyl)ethylene)], prepared under various polymerization conditions were measured at 25,03 MHz in 1,4-dioxane solution. The spectra were assigned by the chemical shifts and T₁ measurements of the polymers and N-ethylcarbazole as a model compound. The absorption of methylene and methine carbons varied by the polymerization methods. It was tentatively presumed that the polymers obtained by radical polymerization had syndiotactic-rich structures and the polymers prepared with BF₃O(C₂H₅)₂ isotactic-rich structures. The hindered internal rotation of the bulky carbazolyl group at the main chain of poly(N-vinylcarbazole) was also observed. The ¹³C NMR spectra indicated that the polymerization of N-vinylcarbazole with nitroethane proceeded by a radical mechanism while that with chloranil, (2,3,5,6-tetrachloro-1,4-benzoquinone), proceeded by a cationic mechanism.     

Comparative NMR studies of diffusional water permeability of erythrocytes from some animals introduced to Australia: Rat rabbit and sheep

The diffusional water permeability (P _d) of the red blood cell (RBC) membrane of rat, rabbit and sheep, representing some animal species introduced to Australia, has been monitored, by a Mn²⁺-doping ¹H nuclear magnetic resonance (NR) technique using a high-field spectometer operating at 400 MHz. In order to make comparisons with previous measurements on the same species (performed at 25 MHz) an analysis of the influence of instrumental parameters and of the state of blood oxygenation was performed on samples of rat and rabbit blood. It was found that by using a short interpulse delay (around 100 µs) in the Carr-Purcell-Meiboom-Gill sequence, and by performing the measurements of the transverse relaxation time of the water in the cell interior (T _2i) on packed cells (haematocrit >95%), the values of the water exchange time (T _e) obtained with a high-field spectrometer are comparable with those obtained by using a low-field NMR spectrometer. The values of the diffusional water permeability (P _d), calculated from the T _e values, were, at 24.6°C around 10 × 10^–3 cm/s in case of rat and rabbit RBC and around 5 × 10^–3 cm/s for sheep RBC; at 37°C the P _d values were around 16 × 10^–3 cm/s for rat RBC, 14 × 10^–3 cm/s for rabbit RBC and 7 × 10^–3 cm/s for sheep RBC. These values are very close to the P _d values previously reported for the European counterparts of these species. There were no significant differences in the P _d values between laboratory rabbits and wild rabbits, or between castrated males, pregnant or nonpregnant females. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of animal species introduced to Australia or by sex hormone status. If the NMR instrument-parameters are carefully chosen then comparative studies of RBC water permeability in different laboratories, at separate locations and using different instruments are meaningful.     

The transmission of passive immunity to Escherichia coli from mother to young in the domestic fowl (Gallus domesticus)

The serum antibody responses of laying hens that had received a single intravenous injection of dead E. coli bacteria, have been compared with antibody levels in normal birds. The passage of immune antibodies into the yolk and into the sera of the hatched chicks has been observed and no substantial difference was found between progeny of a number of inoculated and control birds.

Some evidence is presented which suggests that a selective concentration of 7Sγ₂-globulin occurs during the process of yolk formation.

     

Salt and water permeability of the epithelium of the coprodeum and large intestine in the normal and dehydrated fowl (Gallus domesticus). In vivo perfusion studies

1. The transmural net flow of salt and water in the coprodeum and large intestine of normal and dehydrated hens was investigated by means of an intraluminal in vivo perfusion technique.2. The lumen was perfused with a raffinose-electrolyte solution having a low sodium concentration (Na(+) = 1 m-equiv/l.). The osmolality of the solution was adjusted in the range 66-585 m-osmolal by adding raffinose. Polyethylene glycol 4000 (PEG) served as a water marker. The experiments permitted estimation of the passive transport parameters: the reflexion coefficient (sigma) of the penetrating solutes (predominantly NaCl), the Na(+) mobility (omega(s)), and the osmotic water permeability coefficient (P(osm)).3. When the luminal fluid had the same osmolality as plasma the net water flow (J(v)) was zero, indicating a sigma of unity. The net flow of Na(+) was zero, (at J(v) = 0) and a transmural electric potential difference close to zero was present both in normal and in dehydrated birds. This indicates an omega(s) of zero. When lumen osmolality was higher than that of plasma, no ;solvent drag' effect on Na(+) was demonstrated in the serosa to mucosa (s-m) direction.4. The P(osm) appeared to be independent of the luminal osmolality in the range of +/-200 m-osmolal from plasma osmolality. In normal birds the P(osms-m) was 3.2 mul./kg.hr.m-osmolal, the P(osmm-s) 5.8 mul./kg.hr.m-osmolal. In dehydrated birds these values were 3.6 and 10.0 respectively. Thus there seems to be rectification of water flow, and it varies with the state of hydration.5. A net K(+) flow of 15-50 mu-equiv/kg.hr in the m-s direction and a net Cl(-) flow of 10-50 mu-equiv/kg.hr in the s-m direction were observed. No relationship was observed between the flow of these solutes and the net water flow.     

Blocking of acid-fixed nuclear binding of Epstein-Barr virus nuclear antigen (EBNA) by different DNA species.

Treatment of Epstein-Barr virus-determined nuclear antigen (EBNA) with DNA resulted in blocking of its ability to convert acid-fixed EBNA-negative cell nuclei to an EBNA-positive form. Epstein-Barr virus (EBV) DNA, herpes simplex virus type 2 (HSV-2) DNA and DNA isolated from three lymphoblastoid cell lines differed in their potency to block this reaction. EBV DNA was found to be about three times more effective than cellular DNAs in abolishing the ability of DNA-cellulose-purified EBNA to convert acid-fixed nuclei to the EBNA-positive form; the effect of HSV-2 DNA was of intermediate character. No difference was found between the blocking potency of DNAs isolated from EBV-genome-negative Ramos cells and EBV-genome-positive Raji and P3HR-1 cells.     

Comparative studies of mast cells from normal (non-immunized) and actively sensitized dogs

Morphologically and functionally intact mast cells were isolated from the lung and mesentery of normal or actively sensitized dogs using the pronase or collagenase tissue dissociation methods. The latter method yielded about 6 times as many metachromatically staining cells. Electron microscopic examination revealed the presence of both mast cells and basophils in all samples, independent of the enzyme used for tissue dissociation. The average histamine content of the cells obtained with the pronase method was significantly higher (21.8±3.1 pg) than after collagenase treatment (16.2±4.2 pg). There was no appreciable difference in the reactivity to secretory stimulation of the cells obtained by the two methods.The cells isolated from actively sensitized dog tissues showed a significantly higher spontaneous histamine release (12.9±1.2%) than those from non-sensitized animals (7.8±1.3%) and responded equally well to challenge with both the antigens ovalbumin and horse serum. In contrast to those from normal animals, the mast cells from actively sensitized dogs released histamine on stimulation with acetylcholine, metacholine or atropine. In addition, the response to the threshold concentration of compound 48/80 (10^–6) was significantly stronger in the sensitized cells. Small molecular polyvinylpyrrolidone (K25) was effective in mast cells from normal and actively sensitized dogs.     

Proton nuclear magnetic resonance measurement of p-boronophenylalanine (BPA): a therapeutic agent for boron neutron capture therapy.

Noninvasive in vivo quantitation of boron is necessary for obtaining pharmacokinetic data on candidate boronated delivery agents developed for boron neutron capture therapy (BNCT). Such data, in turn, would facilitate the optimization of the temporal sequence of boronated drug infusion and neutron irradiation. Current approaches to obtaining such pharmacokinetic data include: positron emission tomography employing F-18 labeled boronated delivery agents (e.g., p-boronophenylalanine), ex vivo neutron activation analysis of blood (and very occasionally tissue) samples, and nuclear magnetic resonance (NMR) techniques. In general, NMR approaches have been hindered by very poor signal to noise achieved due to the large quadrupole moments of B-10 and B-11 and (in the case of B-10) very low gyromagnetic ratio, combined with low physiological concentrations of these isotopes under clinical conditions. This preliminary study examines the feasibility of proton NMR spectroscopy for such applications. We have utilized proton NMR spectroscopy to investigate the detectability of p-boronophenylalanine fructose (BPA-f) at typical physiological concentrations encountered in BNCT. BPA-f is one of the two boron delivery agents currently undergoing clinical phase-I/II trials in the U.S., Japan, and Europe. This study includes high-resolution 1H spectroscopic characterization of BPA-f to identify useful spectral features for purposes of detection and quantification. The study examines potential interferences, demonstrates a linear NMR signal response with concentration, and presents BPA NMR spectra in ex vivo blood samples and in vivo brain tissues.