Normal Rabbit Plasma Research Articles

Degradation of endogenous protein by rabbit pulmonary macrophages.

Pulmonary macrophages were preincubated for 1 or 20 h with L-[U-14C]phenylalanine and the degradation of labeled proteins studied by reincubating these cells in the presence of 4 mM L-phenylalanine and measuring rates of [14C]phenylalanine released from the cells into the medium. We found that proteins prelabeled in 1 or 20 h were degraded 8.0 and 3.0%.h-1, respectively. Decreases in cell viability reduced the rate of protein degradation. Lack of exogenous glucose slowed the rate of degradation of proteins labeled in 1 h, but not of those labeled in 20 h. Varying amino acids in the medium from normal to 5 times normal rabbit plasma levels had no effect on the degradation of either group of proteins. Rates of degradation of both fast and slowly turning over proteins were inhibited during phagocytosis of polystyrene latex particles by about 62 and 33%, respectively. The time course of the changes in protein degradation suggests they are related to intracellular events in the phagocytic process, rather than particle attachment or uptake.

Rabbit blood coagulation factor XI. Purification and properties

Rabbit blood coagulation Factor XI was purified in 30% yield from plasma by ion exchange chromatography. The purified Factor XI zymogen gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of β-mercaptoethanol with an apparent molecular weight of 83,000. On alkaline disc gel electrophoresis Factor XI appeared either as a monomer or a dimer depending upon the conditions prior to electrophoresis. After dialysis against buffer at physiological pH and ionic strength Factor XI appeared as a monomer only. The apparent molecular weight of rabbit Factor XI in rabbit plasma on Sephadex G-200 chromatography was 200,000, probably reflecting a complex formed between high MW kininogen and Factor XI. The concentration of Factor XI in citrated normal rabbit plasma was 3.3 μg/ml.

Pseudocoagulase activity of staphylococci

A total of 245 strains of staphylococci isolated from various pathological specimens derived from cases of human infections was tested for staphylocoagulase activity. Test systems employing normal citrated rabbit plasma and the same substrate supplemented with inhibitors of thrombin and proteolytic enzymes (but not influencing the staphylocoagulase activity) were used for testing suspensions of bacteria and cell-free culture supernatants. A total of 237 strains clotted normal rabbit plasma; however, addition of Trasylol and heparin resulted in positive results in 222 strains, whereas plasma supplemented with Trasylol and hirudin was coagulated definitely by only 173 strains. It is postulated that proteolytic enzymes of staphylococci interfere with staphylocoagulase-induced clotting and may simulate coagulase-positive activity of staphylococci. To avoid such false results, a test system for detection of staphylocoagulase should include proteolytic enzyme inhibitors. Possible mechanisms of these findings are discussed.

Myocardial Depression in Sepsis

Myocardial failure is uniformly fatal when associated with post-traumatic sepsis and multisystem failure. Controversy exists as to whether endotoxin has a direct effect on the myocardium. A nonanoxic isolated arterially perfused rabbit interventricular septum was used in this study to evaluate the effects of endotoxin, live E. coli, and endotoxin/septic shock plasma on myocardial function and ultrastructure. Purified E. coli endotoxin and live E. coli bacteria did not have a significant direct effect on rabbit cardiac muscle function or ultrastructure. Perfusion of the rabbit septum with plasma from rabbits exsanguinated following a 2-hour septic or endotoxin shock insult, however, caused significant (p less than 0.02) myocardial depression when compared with control septa perfused with normal rabbit plasma. Septa perfused with shock plasma demonstrated ultrastructural alterations of mitochondria that were not noted in control preparations.

Rabbit Factor VIII: Identification of Size Heterogeneity

Two forms of rabbit factor VIII procoagulant activity, distinguishable by size on gel filtration and ultracentrifugation, have been identified in normal rabbit plasma. These studies have been carried out with citrate-anticoagulated rabbit plasma obtained by cardiac puncture. Two peaks of factor VIII activity were obtained on agarose gel chromatography, using physiologic ionic strength buffers: a high molecular weight peak eluting at the void volume and a second peak eluting with smaller plasma proteins. The presence of high and low molecular weight factor VIII activities was confirmed by sucrose density gradient centrifugation. The two peaks of factor VIII activity remained distinct when proteolytic inhibitors were added to the plasma and eluting buffers. Both the high and low molecular weight factor VIII procoagulant activities were inhibited by antibodies to human and rabbit factor VIII, and both were activated by thrombin. The identification of size heterogeneity of factor VIII in normal rabbit plasma, in the absence of any modification by ionic strength, may permit more satisfactory study of the relationship of factor VIII size to function.

Rabbit factor VIII: identification of size heterogeneity

Two forms of rabbit factor VIII procoagulant activity, distinguishable by size on gel filtration and ultracentrifugation, have been identified in normal rabbit plasma. These studies have been carried out with citrate-anticoagulated rabbit plasma obtained by cardiac puncture. Two peaks of factor VIII activity were obtained on agarose gel chromatography, using physiologic ionic strength buffers: a high molecular weight peak eluting at the void volume and a second peak eluting with smaller plasma proteins. The presence of high and low molecular weight factor VIII activities was confirmed by sucrose density gradient centrifugation. The two peaks of factor VIII activity remained distinct when proteolytic inhibitors were added to the plasma and eluting buffers. Both the high and low molecular weight factor VIII procoagulant activities were inhibited by antibodies to human and rabbit factor VIII, and both were activated by thrombin. The identification of size heterogeneity of factor VIII in normal rabbit plasma, in the absence of any modification by ionic strength, may permit more satisfactory study of the relationship of factor VIII size to function.

Stimulation of cholesterol ester exchange by lipo-protein-free rabbit plasma

A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin: cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.

Interaction Between Rabbit Erythroblast Ferritin and Normal Plasma Proteins

Abstract Erythroblast ferritin (EF), isolated from phenylhydrazine-anemic rabbit marrow, interacts with components of normal plasma and forms a complex separable by starch granule electrophoresis and by dialysis techniques. The binding is facilitated at low ionic strength. The plasma factors responsible for binding EF are present in autologous, as well as homologous, plasma of normal nonimmune rabbits, although binding activities are quite variable in different plasmas. The binding activity for rabbit EF is also present in heterologous plasma of mouse, guinea pig, and man. The active principles in plasma were identified as two heat-stable components which fractionate as immunoglobulins, one as IgG and the other as IgM. The results support the hypothesis that natural antiferritin antibodies of low titer are present in normal plasma.

Plasma clearance of 125I-labelled haemopexin in normal and haem-loaded rabbits.

Summary. Plasma clearance of 125I‐labelled rabbit haemopexin has been studied in six normal control rabbits and in a test group of rabbits haem‐loaded with 25 mg of haem.Following intravenous injection, equilibration of 125I‐haemopexin between intravascular and extravascular compartments appeared to be complete within 24 hr and the decline in plasma activity then became constant in rate. The plasma activity curve was resolved into two exponential functions of time and from the data, fractional catabolic rate, intravascular:extravascular distribution of haemopexin and total haem binding capacity were calculated. Control group. Mean plasma half‐clearance time of haemopexin was 20.3 hr; fractional catabolic rate was 1.8 (SD 0.19) times the intravascular haemopexin pool per day. Extravascular haemopexin was 0.6 (SD 0.11) times the mass of haemopexin in the intravascular pool. Test group. 90–95% of injected 125I‐activity was eliminated from the plasma with a mean half‐clearance time of 1.57 hr and was associated with a significant urinary excretion of labelled iodine. Fractional catabolic rate was 5.84 (SD 0.3) times the intravascular haemopexin pool per day and extravascular haemopexin was 3.38 (SD 1.03) times the mass of haemopexin in the intravascular pool.Haemopexin concentration in normal rabbit plasma was 31–52 mg/100 ml; intravascular plus extravascular haemopexin was 54.5 mg (mean value) and total haem binding capacity of haemopexin was 0.53 mg (mean value).Intravascular haem rapidly complexed with haemopexin and the data suggested that this complex was promptly eliminated from the plasma and the haemopexin catabolized.

The Platelet in C-Blood Coagulation Interaction

Abstract Investigation of the coagulation abnormality in the blood of rabbits congenitally deficient in C6 indicates that C can markedly enhance platelet coagulant activity and that failure of this mechanism is responsible for the clotting defect. Addition of inulin to platelet-rich normal rabbit plasma enhances platelet coagulant activity 20-fold more than either collagen, kaolin, or ADP. No effect on coagulant activity of C6-deficient platelet-rich plasma is seen after addition of inulin, nor can an effect be demonstrated on platelet-free normal rabbit plasma. Addition of inulin to C6-deficient platelet-rich plasma does induce partial aggregation, probably due to C3 binding, and partial release (less than 5%) of vasoactive amines. Subsequent addition of C6 is followed by maximal aggregation (due to both ADP-independent and ADP-dependent mechanisms), as well as additional release of vasoactive amines and evolution of clot-promoting activity. Generation of clot-promoting activity and vasoactive amine release are complete before significant leakage of cytoplasmic lactic dehydrogenase.

Studies of the Mechanism of the Jarisch—Herxheimer Reaction in Louse-Borne Relapsing Fever: Evidence for the Presence of Circulating Borrelia Endotoxin

1. Certain aspects of the Jarisch—Herxheimer reaction (JHR) after treatment of louse-borne relapsing fever have been studied in nine patients. Peripheral vasoconstriction immediately preceded the chill phase and profound vasodilation accompanied the flush phase. 2. From four patients 50–150 ml of blood was taken at the onset of the chill and reinjected intravenously into the same patient the next day. One patient experienced a reaction identical with JHR 40–60 min after reinjection, suggesting that the mediator of JHR was present in that blood. 3. From five patients 40 ml of blood were taken serially throughout JHR and 2-ml samples of plasma, free of cells and spirochaetes, were injected into pairs of normal rabbits. All samples taken while the patients had fever were pyrogenic. After incubation with normal rabbit plasma these samples failed to produce fever in endotoxin-refractory rabbits. 4. These results suggest that in the plasma of patients with relapsing fever there is a powerful endotoxin. We were unable to show that the concentration of endotoxin increased during JHR, nor could we demonstrate the presence of leucocyte pyrogen. This failure could be a problem of quantity of plasma used or of an endotoxin-refractory state. 5. The bearing of these observations on the mechanism of JHR and the uselessness of cortisol in modifying the reaction are discussed.

Phospholipid composition of aqueous humor, plasma and lens in normal and alloxan diabetic rabbits

The phospholipid composition of the aqueous humor, plasma and ocular lens of normal and diabetic rabbits is reported. The presence of this class of compounds in the aqueous humor has been demonstrated for the first time, lysophosphatidyl choline, sphingomyelin and phosphatidyl choline constituting the main fractions. The phospholipids of the plasma were lysophosphatidyl choline, sphingomyelin, phosphatidyl choline, phosphatidyl inositol and phosphatidyl ethanolamine. Lens, in addition to the phospholipids present in the plasma, also contained phosphatidyl serine. In the aqueous humor of diabetic animals, the concentration of lysophosphatidyl choline, phosphatidyl choline and some unidentified lipids were found to be reduced while that of sphingomyelin increased. In contrast, the concentrations of nearly all of the plasma phospholipids, including that of phosphatidyl choline, were observed to increase. The phospholipid composition of the lens in normal and diabetic animals was essentially similar, except that in the latter the concentration of phosphatidyl inositol was lower.

Free fatty acids and glycerol in plasma and tissues of normal, hypothermic, and hypotrophic newborn rabbits.

Free fatty acids and glycerol in plasma and tissues of normal, hypothermic, and hypotrophic newborn rabbits.

Detection of thrombopoietic activity in plasma by stimulation of suppressed thrombopoiesis.

Rabbits in which thrombocytosis had been produced by five daily transfusions of platelet concentrates had suppressed endogenous thrombopoiesis, as reflected by decreased incorporation of selenomethionine-(75)Se ((75)SeM) into the circulating platelet mass. Rabbits in which endogenous thrombopoiesis had been suppressed by transfusion-induced thrombocytosis were used to detect thrombopoietic activity in rabbit plasma. Thrombopoietic activity was demonstrated in the plasma of both normal and thrombocytopenic donor rabbits. A dose response relationship was observed between the incorporation of (75)SeM into platelets and the dose of plasma administered. Infusion of 20-150 ml of plasma from thrombocytopenic donors increased the incorporation of (75)SeM into platelets from 52 to 107% above control values. A dose response effect also was seen after infusion of normal plasma, but normal plasma produced less effect than comparable doses of plasma from thrombocytopenic donors. Rabbits with transfusion-induced thrombocytosis appear to be more sensitive assay animals for the detection of thrombopoietic activity than animals with normal platelet counts. Changes in the rate of appearance and levels of (75)SeM may primarily indicate changes in platelet protein or platelet size and are apparently more sensitive indicators of the state of thrombopoiesis than are alterations in the numbers of circulating platelets. The results strongly support the concept of a humoral agent, i.e. thrombopoietin, that acts on megakaryocytes to regulate platelet production.

Mechanisms of Release of Constituents from Rabbit Platelets by Antigen-Antibody Complexes and Complement

Abstract Two mechanisms have been described by which antigen-antibody complexes in the presence of complement induce release of vasoactive amines from rabbit platelets. The reaction of soluble antigen, antibody in excess, normal rabbit plasma and platelets caused adherence of the platelets to the immune complexes (which required the action of C3) and lysis of the platelets (which required the action of C6). The lysis was observed microscopically and was accompanied by release of vasoactive amines, nucleotides and cytoplasmic and lysosomal enzymes. The release did not require active participation of the platelet and was postulated to result from cell lysis by complement components activated by the immune complexes in or on the platelet membrane. A nonlytic, complement-dependent release of vasoactive amines resulted from immune adherence of platelets to erythrocytes or zymosan that had fixed complement components through C3. Vasoactive amines and nucleotides were released by the reaction, but not intracellular enzymes or potassium (measured with rubidium 86). Platelet energy and activated platelet esterases were required for the release, which appeared to result from the C3-induced adherence of the platelets to the particulate antigens.

Leishmania tropica, L. donovani and L. enriettii: Immune rabbit serum inhibitory in vitro

Plasma from rabbits injected with Leishmania tropica showed an inhibitory effect on the growth of Leishmania tropica, enriettii and donovani in vitro. This inhibitory effect was assayed by counting the viable leptomonads under the microscope, using trypan blue as a vital stain. The number of viable leptomonads in cultures containing plasma from injected rabbits decreased steadily, while in cultures of leptomonads with normal rabbit plasma the numbers of leptomonads increased rapidly, reaching a peak on the fifth day of culture. Some effect of the plasma from injected rabbits on the leptomonads could be observed after they were incubated together for 4 hours. The growth inhibitory factor in plasma from injected rabbits is thermostable at 56 °C., its action is independent of the presence of complement, and it could not be adsorbed out by the homologous organism under our experimental conditions. The degree of inhibitory activity of the plasma factor was correlated with the complement fixation titer of the plasma against a soluble antigen of L. tropica.

Studies on Plasma Angiotensinases in Liver Disease

1. Plasma angiotensinase activity was significantly increased in patients with various kinds of liver diseases. The patterns of angiotensinase activity in plasma from normal subjects and patients with liver diseases were compared by horizontal starch block electrophoresis. Angiotensinase activity in normal plasma was found only in the albumin zone, while in plasma from patients with liver diseases it was found not only in the albumin zone but also in the γ-globulin zone. On recovery of the patient, angiotensinase activity in the γ-globulin zone was not demonstrated. This angiotensinase, which was demonstrated electrophoretically in the γ-globulin zone, was named angiotensinase G. 2. On experimental liver damage in rabbits, the findings obtained in human beings were confirmed. 3. Angiotensinase G inactivated both α-asp1-val5-angiotensin II and (α-asp1(NH2)-val5-angiotensin II, but did not inactivate β-asp1 val5-angiotensin II. The optimal pH of the enzyme was between 6.8 and 7.1. The activity of the enzyme was completely inactivated by ethylenediaminetetraacetate treatment and the full activity was restored by addition of manganese. Its activity was inhibited by trasylol as well as diisopropylfluorophosphate. 4. Angiotensinase G could not be detected either in liver extract from normal rabbits or from rabbits with experimental liver damage. When normal rabbit plasma was incubated together with liver extract, angiotensinase G was demonstrated electrophoretically in the incubation mixture. In vitro experiments indicated that γ- or β-globulin fraction of plasma and a heat unstable and non-dialyzable substance in microsome fraction of liver cells were essential for the formation of angiotensinase G. 5. The appearance of angiotensinase G seemed to be one of the factors of an increase of plasma angiotensinase activity in liver diseases. The possible role of an increase of plasma angiotensinase activity on the contribution to the blood pressure reduction in liver diseases was discussed.

腹腔内接種によりマウスに強い毒力を示す特殊ブドウ球菌に関する研究

Serological properties were investigated in the unique staphylococcal strains, E 46 and E 97, which are coagulase-negative but deoxyribonuclease-positive, and which had been described in the preceding parts of the present series of papers. They were compared between these strains and typical strains of each of Staphylococcus aureus (E 111 and B 40) and S. epidermidis (E 241 and B 217). The four strains were also described in the same preceding papers. As antigen were used thimerosal-treated cells of each strain for the cross agglutination and the agglutinin absorption tests, and the condensed culture filtrate of each strain or the polysaccharide fraction from the E 46 strain for the precipitin reaction, and the same polysaccharide fraction for the skin test. Antisera were obtained from rabbits immunized with the killed cells mentioned above. The results obtained are as follows.1) The immunologic tests revealed that both unique staphylococcal strains possessed the same antigenic structure, and that they had a common antigen with both strains of S. aureus, but not with either of the two strains of S. epidermidis.2) As described in the first report of this series, the unique staphylococcal strains gave rise to diffuse colonies in the serum soft agar using normal rabbit serum or plasma, as well as the E. epidermidis strains did, wheres the S. aureus strains formed compact colonies. In the present similar experiments using such anti-rabbit sera as described previously, all the unique staphylococcal and S. epidermidis strains were converted to such extent as to form compact colonies in the case of their respective homologous antisera, but not in the case of their heterologous antisera, whereas the S. aureus strains were not affected at all either by the homologous or by the heterologous antisera.Thus, it may be possible to confirm that the unique staphylococcal strains possess identical morphological, biological, and serological attributes and also intermediate properties between the typical strains of S. aureus and S. epidermidis, although they are much closer to the former species than the latter.

An assay method for leukocyte pyrogen.

A method for assaying leukocyte pyrogen is described which is shown to remain valid despite the presence of very large amounts of bacterial endotoxin. It uses a combination of two procedures to inactivate endotoxin: incubation in normal rabbit plasma for 24 hr at 37 degrees C, and injection into rabbits rendered refractory to endotoxin. The validity of the assay was confirmed by formal statistical methods. A simplified assay method is also described which lacks full statistical validity, but is more suitable for routine use.

Plasma angiotensinase activity in liver disease

The electrophoretic detection of the appearance of plasma angiotensinase activity in the γ-globulin zone, named angiotensinase G, in patients with liver damage was studied using rabbits. Angiotensinase G could not be detected either in liver extract from normal rabbits or from rabbits with experimental liver damage. When normal rabbit plasma was incubated together with liver extract, angiotensinase G activity was demonstrated electrophoretically in the incubation mixture. In vitro experiments indicated that γ- or β- globulin fractions of plasma and a heat unstable substance in microsome fractions of liver cells were essential for the formation of angiotensinase G.