Pig Red Blood Cells Research Articles

Identification and Phylogenetic Comparison of Salem Virus, a Novel Paramyxovirus of Horses

A virus that could not be identified as a previously known equine virus was isolated from the mononuclear cells of a horse. Electron microscopy revealed enveloped virions with nucleocapsid structures characteristic of viruses in the Paramyxoviridae family. The virus failed to hemadsorb chicken or guinea pig red blood cells and lacked neuraminidase activity. Two viral genes were isolated from a cDNA expression library. Multiple sequence alignments of one gene indicated an average identity of 45% as compared to Morbillivirus N protein sequences. A weaker relationship was found with Tupaia paramyxovirus (TPMV) and Hendra virus (HeV) N proteins. In the second gene, multiple open reading frames (ORFs) were identified, corresponding to the arrangement of the P, V, and C ORFs in the Morbillivirus and Respirovirus viruses. Short stretches in the C-terminal regions of the P and C proteins showed limited homologies to viruses in the Morbillivirus genus but no obvious relationship to viruses in other genera. The V ORF translation product contained a highly conserved, cysteine-rich domain that is common to most viruses in the Paramyxovirinae subfamily. Sequencing of P gene cDNA clones confirmed the use of a cotranscriptional editing mechanism for the regulation of P/V expression. Based on the location of its origin it has been named Salem virus (SalV).

Antibacterial and hemolytic activity of the skin of the terrestrial salamander,Plethodon cinereus

As resistance increases against fungal antibiotics, antimicrobial peptides are receiving attention as possible replacements. The dermal glands of frogs secrete, among other things, antimicrobial peptides. As part of the innate immune system, stressors may affect the production of antimicrobial peptides by dermal glands. The dermal secretions of some salamanders have been examined for their toxic secretions, but little attention has been given to salamander antimicrobial peptides. This study examines the skin from the tail region for the production of antimicrobial peptides in the terrestrial salamander, Plethodon cinereus. Fractions of tail extracts were isolated using cation-exchange chromatography and reverse-phase HPLC. An HPLC fraction eluting at 15.75 min (HPLC run: 30 min, 30-80% acetonitrile/water gradient, Aquapore RP-300 C18 column) showed activity against Staphylococcus aureus but not against Escherichia coli. The antibacterial activity gradually increased over a 4-hr incubation time up to about 85% inhibition of bacterial growth. Lysis of guinea pig red blood cells also increased gradually over a 1-hr time period. J. Exp. Zool. 287:340-345, 2000.

Characterization of a pig-to-goat orthotopic lung xenotransplantation model to study beyond hyperacute rejection

Background: A pig-to-goat orthotopic lung xenograft model was developed to test whether depletion of goat xenoreactive antibodies against pig red blood cells would prolong pig lung xenograft survival. Methods: Adult goats with anti-pig xenoreactive antibodies underwent left pneumonectomy followed by orthotopic transplantation of pig left lung (group 1) or immunodepletion of their xenoreactive antibodies by extracorporeal right pig lung perfusion before transplantation without (group 2) or with (group 3) complete clampage of the right pulmonary artery. In group 4, goat left lungs were orthotopically transplanted into pigs and served as negative controls (pig serum does not have anti-goat xenoreactive antibodies). Each study group included 5 animals. Immunosuppression in surviving recipients included cyclosporine and azathioprine. Results: Group 1 recipients died 7 ± 3 hours after xenograft reimplantation of severe pulmonary hypertension and dysfunction and vasogenic shock, with little evidence of histologic xenograft injury. Group 2 xenografts had a stable circulatory and respiratory function on reperfusion and survived 9 ± 4 days. Group 3 animals also tolerated complete occlusion of the right pulmonary artery, and xenografts assured the total respiratory support for 4 ± 1 days. After immunodepletion, goat serum showed no detectable titers of xenoreactive antibodies, which began to reappear by postoperative day 2, where xenografts showed histologic stigmata of acute (humoral and cellular-mediated) rejection that evolved to a complete xenograft necrose at death. Group 4 xenografts showed scattered features of acute rejection 5 ± 1 days after the operation. Conclusions: Pig left lung xenografts can provide prolonged and complete respiratory support after depletion of goat xenoreactive antibodies, but they ultimately necrose once recipient xenoreactive antibodies return to pretransplantation values. (J Thorac Cardiovasc Surg 1999;118:805-14)

Binding of bovine IgG2 a and IgG2 b allotypes to protein A, protein G, and Haemophilus somnus IgBPs

Immunoglobulin binding proteins (IgBPs) are thought to be virulence factors which enable pathogens to evade the host’s immune response. Since bovine IgG2 is important in protection against pyogenic infections, the binding characteristics of Staphylococcus aureus protein A (PrA), streptococcal protein G (PrG), or Haemophilus somnus high molecular weight IgBPs to the two bovine IgG2 allotypes were examined. For PrA or PrG binding of IgG2, guinea pig red blood cells coated with specific IgG2 a or IgG2 b antibodies were used in a competitive binding inhibition assay with unlabeled and horseradish peroxidase-labeled PrA or PrG. To determine which sizes of H. somnus IgBPs bind to the two IgG2 allotypes, immunoblots with H. somnus culture supernatant were probed with anti-DNP IgG2 a and IgG2 b. This detects only Fc binding because anti-DNP does not cross-react with H. somnus antigens. Both IgG2 allotypes bound equally well to PrA and PrG. However, IgG2 b but not IgG2 a bound to H. somnus high molecular weight IgBPs. The lack of differential binding of bovine IgG2 allotypes to PrA and PrG means that these IgBPs can be considered to be unbiased reagents in assays for detection of bovine IgG2 or for immunoaffinity purification. The differential binding of H. somnus IgBPs to the IgG2 allotypes indicates that animals having one allotype may be more resistant to H. somnus infection than animals having the other allotype.

Differential complement activation by bovine IgG2 allotypes

Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2 a and IgG2 b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2 a and IgG2 b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2 b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2 a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2 b had a more rigid hinge than IgG2 a, perhaps partially explaining the greater efficiency of IgG2 b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2 a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.

Myocardial oxygenation in vivo: optical spectroscopy of cytoplasmic myoglobin and mitochondrial cytochromes.

The oxygenation state of myoglobin and the redox state of cytochrome c provide information on the PO(2) in the cytosol and mitochondria, respectively. An optical "window" from approximately 540 to 585 nm was found in the pig heart in vivo that permitted the monitoring of myoglobin and cytochrome c without interference from Hb oxygenation or blood volume. Scanning reflectance spectroscopy was performed on the surgically exposed left ventricle of pigs. Difference spectra between control and a total left anterior descending coronary artery occlusion revealed maxima and minima in this spectral region consistent with myoglobin deoxygenation and cytochrome c and b reduction. Comparison of in vivo data with in vitro fractions of the heart, including Hb-free tissue whole heart and homogenates, mitochondria, myoglobin, and pig red blood cells, reveals minimal contributions of Hb in vivo. This conclusion was confirmed by expanding the blood volume of the myocardium and increasing mean Hb O(2) saturation with an intracoronary infusion of adenosine (20 microgram. kg(-1). min(-1)), which had no significant effect on the 540- to 585-nm region. These results also suggested that myoglobin O(2) saturation was not blood flow limited under these conditions in vivo. Work jump studies with phenylephrine also failed to change cytochrome c redox state or myoglobin oxygenation. Computer simulations using recent physical data are consistent with the notion that myoglobin O(2) saturation is >92% under basal conditions and does not change significantly with moderate workloads. These studies show that reflectance spectroscopy can assess myocardial oxygenation in vivo. Myoglobin O(2) saturation is very high and is not labile to moderate changes in cardiac workload in the open-chest pig model. These findings indicate that myoglobin does not contribute significantly to O(2) transport via facilitated diffusion under these conditions.

Inhibition of complement-mediated immune hemolysis by peptides derived from the constant domain of immunoglobulin.

High-dose administration of intravenous immunoglobulin is reported to be useful for inhibiting complement-dependent immune cytolysis. We have found that, among the proposed C1q-binding sites of the Fc portion of human IgG1, only residues 282-292 inhibited pig red blood cell lysis by human serum. Moreover, a hexadecemeric multiple antigen peptide of residues 282-292 from IgG showed significantly greater activity in suppressing complement-mediated immune cytolysis and can be used in place of high-dose intravenous immunoglobulin, which is extracted from donors and thus is expensive.

Intravenous synthetic alphaGal saccharides delay hyperacute rejection following pig-to-baboon heart transplantation.

Several oligosaccharides containing the terminal structure Gal(alpha)1-3Gal (alphaGal) and different side chains were tested in vitro for their ability to block natural anti(alpha)Gal antibodies. A di-and a trisaccharide (di(alpha)Gal and tri(alpha)Gal) were selected. A blood group B baboon, having IgG and IgM natural antipig titers of 1:256 and 1:1024 and a hemolytic titer (to pig red blood cells, RBCs) of 1:8, was chosen to measure pharmacokinetic parameters of the saccharides and to assess the extent of in vivo neutralization of the antibodies. Three grams each of the di(alpha)Gal and the tri(alpha)Gal dissolved in saline were administered by bolus intravenous (i.v.) injection. Blood samples were collected at various times and urine was collected at 8 and 24 h. Plasma and urine concentrations of the alphaGal saccharides were estimated by an ELISA specially developed for this study. A fast distribution phase followed by equilibrium and excretion phases were observed, indicating a T1/2 in the order of 1 h. Fifty-eight per cent of the saccharides were recovered in the urine within 24 h. Determination of antipig antibody binding by FACS analysis and of serum cytotoxicity titers for pig endothelial cells demonstrated that a 70% reduction in binding and cytotoxicity could be achieved with plasma saccharide levels of 300-400 microg/ml. Six months later, a pig heart was transplanted heterotopically into the baboon. A 3-g bolus of the saccharide mixture (1.5 g of each saccharide) was given i.v. before allowing blood reperfusion of the transplanted heart, followed by an i.v. infusion of 1 g/hr for 1 hr and 0.5 g/hr for the 3 succeeding hours. Blood concentrations of the saccharides, CH50, hematology and cytotoxicity for PK15 cells were estimated in blood samples taken at various times. Heart function was observed to be satisfactory for 8 h, but was found to have ceased at 18 h. Myocardial biopsies taken at 3 and 5 h showed congestion only, suggestive of minimal vascular rejection, but by 18 h demonstrated severe vascular rejection. In conclusion, alphaGal saccharide therapy given for a period of 4 h delayed, but did not totally prevent, the development of vascular rejection in the pig-to-baboon heart transplant model. alphaGal saccharide therapy may be one of several useful approaches for the prevention of hyperacute rejection in pig-to-primate organ transplantation.

Properties of the Ca 2+ influx reveal the duality of events underlying the activation by vanadate and fluoride of the Gárdos effect in human red blood cells

The properties of the 45Ca 2+ influx by human red blood cells (RBC) induced by NaVO 3 or NaF were compared. The NaVO 3-induced 45Ca 2+ influx was slower and less extensive than that induced by NaF. Both processes were saturable with Ca 2+. Substitution of Na + by K + inhibited the 45Ca 2+ influx induced by NaVO 3 but stimulated that by NaF. The NaVO 3-induced Ca 2+ influx was sensitive to nifedipine (IC 50=50 mol/l), Cu 2+ (IC 50=9 mol/l), DTNB (5,5′-dithiobis-(dinitrobenzoic acid)) (IC 50=12 mol/l) (maximal inhibition 16%, 18%, and 28%, respectively, if NaF was used as inducer). On the other hand, tetrodotoxin (TTX) and cyclosporin A inhibited only the NaF-induced 45Ca 2+ influx (IC 50=21 mol/l and 28 mol/l, respectively). Pig RBC, known not to display the NaVO 3-induced Ca 2+ influx, exhibited Ca 2+ influx induced by NaF. The results show that NaVO 3 activates the Ca 2+ influx via a pathway homologous to the L-type Ca 2+ channel while the NaF-induced Ca 2+ influx is mediated via the TTX-sensitive Na + channel in the presence of NaF with possible participation of calcineurin or cyclophilin. Thus, the Gárdos effect induced by NaVO 3 and NaF represents two phenomena activated by different mechanisms present in the cryptic state in the RBC membrane.

HLA-specific antibodies in highly sensitized patients can cause a positive crossmatch against pig lymphocytes.

The presence of IgG HLA-specific antibodies in the serum of patients awaiting transplantation indicates T- and B-cell priming and would result in acute rejection of a poorly matched human allograft. Recent advances in xenotransplantation, with the amelioration of hyperacute rejection using transgenic pig kidneys, may benefit such patients. However, accelerated cellular rejection might result from the primed T-cell recognition of antigenic epitopes shared between pig and human MHC molecules. We have compared the reactivity of IgG antibodies from 8 nonsensitized (NS) and 13 highly sensitized (HS) patients with human and pig lymphocytes by flow cytometry. Xenoreactive natural antibodies (XNA) were absorbed with pig red blood cells, and HLA class I-specific antibodies were further absorbed with pooled human platelets. Before XNA absorption, 20 of the 21 patients had a positive IgG crossmatch with pig lymphocytes, and there was no difference between NS and HS patients. In contrast, after XNA absorption, none of the 8 NS patients were positive, compared with 9 of the 13 HS patients (mean of the median channel fluorescence values of 7.7 and 86.5, respectively; P=<0.001). For XNA-absorbed HS patient sera, 20 of 30 (67%) pig lymphocyte crossmatch combinations were positive, with a mean median channel fluorescence value of 125 (range 31 to 294) compared with 9.5 (range 7 to 13) for the 10 crossmatch-negative combinations. Platelet absorption resulted in a concomitant reduction in antibody binding to pig lymphocytes in three of six HS patient sera, indicating that HLA class I-specific antibodies are responsible, at least in part, for the positive crossmatch. These results suggest that some IgG HLA-specific antibodies can bind to pig lymphocytes, analogous to a positive crossmatch with allogeneic donors.

Isolation of influenza A(H3N2) virus with “O”→“D” phase variation

We report the isolation of two influenza A(H3N2) virus strains which were unable, in the first passages in MDCK cell culture, to agglutinate chicken erythrocytes, though reacting with guinea pig and turkey red blood cells. This observation demonstrates that the occurrence of this phenomenon is not exclusive to influenza A(H1N1) viruses, as previously reported. In order to investigate the molecular basis of this phenomenon, we analysed the nucleotide sequence of the HA-1 region, presumed to be involved in the switch of haemagglutination properties, from the virus present in the original samples and in the corresponding strains isolated and cultivated in MDCK cells and in embryonated eggs. The substitution of amino acid 138 (Ala-->Ser) in MDCK cells could be related to the change in haemagglutination characteristics.

Erythrocytes modulate cell cycle progression but not the baseline frequency of sister chromatid exchanges in pig lymphocytes

The effect of co-culturing varying concentrations of pig and human red blood cells (RBCs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression in pig plasma (PLCs) and whole blood leukocyte cultures (WBCs) was studied. No variation in SCE frequency was observed between pig control WBC and PLC. Addition of pig and human RBCs to pig PLCs did not modify the baseline frequency of SCEs. On the other hand, cell proliferation was slower in PLCs than in WBCs. The addition of pig or human RBCs to PLCs accelerated the cell-cycle progression of pig lymphocytes. When RBCs were added to PLCs the concentration and time sequence of RBC incorporation affected the cell-cycle progression of swine lymphocytes. When doses of pig or human RBCs equivalent to those present in WBCs were added immediately after PLC stimulation, the cell-cycle kinetics were similar to those of WBCs. Shorter co-incubation periods or a reduction in the dose of RBCs made cell-cycle progression intermediate between PLC and WBC values. Thus, pig and human RBCs modulated the in vitro cell-cycle progression of pig lymphocytes in a time- and dose-dependent manner, and the low baseline frequency of SCEs of pig lymphocytes is independent of the presence or absence of erythrocytes in culture

Structural requirements for the edema-inducing and hemolytic activities of mastoparan B isolated from the hornet ( Vespa basalis) venom

Mastoparan B (MP-B) is a cationic tetradecapeptide isolated from the black-bellied hornet ( Vespa basalis) venom. It has a primary structure (LKLKSIVSWAKKVL-CONH 2) distinct from other vespine mastoparans. The peptide caused a dose-dependent swelling in rat hind paw and showed a potent hemolytic activity in guinea pig red blood cells. Studies on the structure- activity relationship of the peptide showed that replacing lysine at position 2 (Lys 2) by asparagine (Asn) in the MP-B sequence caused about 40% decrease in its edema-inducing activity at 50 μg/paw and 90% decrease in hemolytic activity at 30 μM of the peptide, while the same substitution at Lys 4 did not cause a significant change in either activity. Replacing either Lys 11 or Lys 12 by leucine (Leu) caused little or no decrease in the edema-inducing and hemolytic activities. Decreases in both activities were observed when both Lys 11 and Lys 12 were replaced by Leu. On the other hand, replacing tryptophan at position 9 (Trp 9) by tyrosine or phenylalanine in MP-B sequence almost abolished its hemolytic activity, while the edema-inducing activity was only partially inhibited. Circular dichroism spectra of the peptides measured in 20% trifluoro- ethanol revealed that substitution of Lys and Trp did not cause a significant change in the conformation of MP-B. It appears that Lys 2 is crucial for both hemolytic and edema-inducing activities of MP-B, while Trp 9 is of special importance to the hemolytic activity of MP-B. Lys 11 and Lys 12 in MP-B probably play a lesser role in both activities.

ROLE OF Fc FRAGMENTS IN INHIBITION OF XENOGENIC HYPERACUTE REJECTION BY INTRAVENOUS IMMUNOGLOBULIN

It has been shown that a high dose administration of human intravenous immunoglobulin G (IVIG) prolonged the survival time of xenografted guinea pig heart in the rat. In this study, we compared the effectiveness of intact IVIG (I-IVIG), Fc fragments of IVIG (Fc-IVIG), and F(ab') fragments of IVIG (Fab-IVIG) in preventing xenogenic hyperacute rejection in the guinea pig-to-rat heart transplantation model. Relatively high dose administration of IVIG (0.6 g/kg body weight) induced xenograft survival times of 32.0 +/- 13.0 min and 33.2 +/- 16.8 min with I-IVIG and Fab-IVIG, respectively, which were significantly longer than times for animals treated with Fc-IVIG or for untreated control animals (survival times of 12.6 +/- 7.4 min and 10.4 +/- 7.6 min, respectively). The in vitro inhibitory effect of guinea pig red blood cell hemolysis by fresh rat serum correlated with the results of in vivo study. On the other hand, the in vitro inhibitory effect of IVIG on pig red blood cell hemolysis by fresh human serum showed that Fc-IVIG, which had little effect in the guinea pit-to-rat combination, had a higher inhibitory effect than Fab-IVIG, or I-IVIG. Fc-IVIG and I-IVIG achieved a complete inhibition of in vitro hemolysis in the pig-to-human combination. It is suspected that an interference with the complement classical pathway by the Fc portion of IVIG could be an attractive tool for inhibition of hyperacute rejection in the pig-to-human combination.

Deformability of red blood cells from different species studied by resistive pulse shape analysis technique

The Cell Transit Analyzer (CTA) is now being used widely in clinical hemorheology. Most of the data obtained by CTA are limited to human blood, although the CTA has an important potential to be used in experimental studies on animal models. However, behavior of red blood cells (RBC) from various species might be different in CTA. Eight parameters reflecting different aspects of cell passage through pores with 5 μm diameter and 15 μm length were determined or human, guinea pig, dog, rabbit, rat, mouse and sheep RBC, together with instrument precision and biological variation. These parameters have a wide range when measured in different species and correlate with cell volume. Sensitivity of these parameters to the glutaraldehyde-induced alterations in RBC deformability was not same for different laboratory mammals. The main reason for this difference seems to be related to the cell size and thus sensitivity might be significantly limited if 5 μm pore-size filters are used to test the smaller RBC. The results of this study may help in designing experimental studies on laboratory mammals using the CTA.

Role of inosine in prevention of methaemoglobinaemia in the pig: in vitro studies.

Methaemoglobin reductase activity was studied in pig, human and cattle erythrocytes incubated in a medium containing glucose or inosine as NADH generator. With glucose, methaemoglobin reduction was very fast in human, less so in cattle and slowest in pig erythrocytes. With inosine, pig red cells reduced methaemoglobin more rapidly than human red cells and in bovine erythrocytes the enzyme activity was undetectable. In intact red cells the ability to reduce methaemoglobin depends on the amount of NADH the cell can produce with glucose or inosine utilization. The results show that pig red blood cells utilize inosine as the NADH generator for enzymatic reduction of methaemoglobin. The greater efficiency of pig red cells could be due to several factors: better inosine transport, more active metabolic pathway for inosine utilization (so that more NADH is produced), or greater MR activity when NADH is in excess. In any case, the high rate of methaemoglobin reduction could explain the lower prevalence of methaemoglobinaemia in pigs than in cattle.

Alteration of Cerebral Microcirculation by Hemodilution with Hemosome in Awake Rats

Our study showed that hemodilution with modified fluid gelatin resulted in an increase in local cerebral blood flow (LCBF), but no change at all in local cerebral oxygen delivery (LCOD) in rats. Hemosome, a lecithin encapsulated hemoglobin having the oxygen-carrying capacity, was developed to improve LCOD by hemodilution. Therefore, we have hypothesized that LCBF & LCOD would be increased by hemodilution with hemosome. To test this hypothesis, adult male Sprague-Dawley rats weighing approximately 350g were used and divided into the hemodilution and the control groups. Hemosome was made from pig red blood cells and lecithin. It's mean diameter was approximately 0.3 um and hemoglobin concentration was approximately 4g/dl. Isovolemic hemodilution, which lowered the systemic hematocrit from approximately 50% to approximately 30%, was achieved by rapidly replacing blood with the same volume of hemosome. Ten min later, LCBF in 14 brain structures were measured using the 14C-iodoantipyrine technique. Our results showed that LCBF of the control group ranged from 115 +/- 11 ml/100g/min in the medulla to 260 +/- 31 ml/100g/min in the occipital cortex. LCBFs were generally higher (p < 0.05, MANOVA) by 16% in the hemodilution group than in the control group. However LCODs were generally decreased (p < 0.05, MANOVA) by 18% in the hemodilution group than in the control. In conclusion, hemodilution with hemosome indeed improves LCBF but lowers LCOD in awake rats.

Elevating intracellular free Mg2+ preserves sensitivity of Na(+)-K+ pump to ATP at reduced temperatures in guinea pig red blood cells.

Red cells of hibernating species have a higher relative rate of Na(+)-K+ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na(+)-K+ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37 degrees C by measuring ouabain-sensitive K+ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg2+ to 2 mmol.1-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20 degrees C, rather than to decrease, as occurs in cells not loaded with Mg2+. In ground squirrel cells raising intracellular free Mg2+ had little effect on apparent affinity of the pump for ATP at 20 degrees C. ATP affinity rose slightly with cooling both in Mg(2+)-enriched and in control ground squirrel cells. Increased intracellular free Mg2+ in guinea pig cells stimulated Na(+)-K+ pump activity so that at 20 degrees C the pump rate was the same in the Mg(2+)-enriched guinea pig and control ground squirrel cells. Pump activity in Mg(2+)-enriched guinea pig cells at 5 degrees C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na(+)-K+ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg2+. Conversely, in ground squirrel red cells natural rise of free Mg2+ may in part account for the preservation of the ATP affinity of their Na(+)-K+ pump with cooling.

Studies on the haemolytic complement of the dromedary camel ( Camelus dromedarius). II. Alternate complement pathway haemolytic activity in serum

Fresh camel serum caused lysis of unsensitised red blood cells (RBC) of chicken, rabbit and guinea pig. Homologous RBC were resistant to lysis. There was only minimal lysis of goat, sheep, rat and cattle RBC. Lysis of heterologous RBC was attributed to the presence of alternate complement activity (ACP) in the serum as adsorption with respective RBC and addition of 10 mM ethylene glycol-bis-tetraacetate (EGTA) in the SVBS diluent did not abrogate the haemolytic activity. Guinea pig RBC were the most sensitive to lysis, giving a mean ACP activity of 41.5±1.8 CH 50 units ml −1. Clotting, followed by storing of blood between 0 and 37°C for 1 h did not significantly affect ACP activity. However, considerable activity was lost when blood was clotted and stored at 44°C for 1 h, or when serum was kept at 4°C for 24 h. Treatment with zymosan, or incubation at 56°C for 30 min inhibited ACP activity. Maximum ACP activity occurred in the presence of 8 mM Mg 2+ in the SVBS-EGTA diluent, at pH 7.3 and incubation time of 2 h at 37°C. Levels of ACP activity were determined in 79 healthy camels of different age groups, ranging from 3 months to 15 years. Calves between 3 months and 1 year of age had higher ACP activity than camels in the age group of 5 years and above. Highest mean ACP activity of 89±7.9 CH 50 units ml −1 were recorded in 1–5 year old camels ( P<0.0001). The lowest ACP activity was observed in the 10–15 year old camels (52±2.9 CH 50 units ml −1). There was no significant difference in the ACP activity between the sexes.

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 (Pd) 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 ofPd 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.