Search

Suspension Culture of Mammalian Cells and Macromolecular Growth-Promoting Fractions of Calf Serum

WE suggest a novel biological role for a specific plasma-binding protein in the regulation of oestrogen and androgen activity in man. The biological activity of oestradiol and testosterone, as with other steroids1, is probably exerted by the unbound fraction in plasma; this is only 1–3% of the total concentration. The plasma proteins that bind testosterone and oestradiol include sex-hormone-binding β globulin (SHBG)2, which has high affinity for both steroids; and albumin, which has low affinity but is present in three thousand times the concentration. Further, corticosteroid-binding globulin (CBG) binds testosterone3 though its main affinity is for cortisol and progesterone.

THE mechanism by which antilymphocyte globulins act on immunocompetent cells remains unknown. Recent studies have demonstrated that antilymphocyte sera (ALS) and phytohaemagglutinin (PHA) display a similarity in their action on the lymphocyte1–6. We have studied the action of these substances on the incorporation of amino-acids by human peripheral lymphocytes.

THERE is evidence that at least some yolk proteins of vertebrates originate in the liver and are transported to developing oocytes through the circulation, for example in the fish1, frog and chicken2. Marked pinocytotic activity during oocytic yolk accumulation has been noted in a variety of organisms and is probably related to protein uptake3. In view of the apparent ready uptake of yolk proteins by oocytes, we became interested in the responses of phagocytic cells of the reticuloendothelial system (RES) to frog yolk protein and found that yolk of Rana pipiens, labelled with fluorescein and solubilized with sodium chloride, is readily cleared from the blood of mice4. In addition, however, we noted that yolk injected intravenously also seemed to increase the rate of clearance of other materials. We have therefore investigated the opsonic effect of frog yolk.

THE globulin permeability factors obtainable from the serum of man1, guinea pig2,3, rat4 and rabbit4 appear to be proteases because they are highly active on vascular endothelium—on a molar basis, they are 60–2,000 times as active as histamine, itself a potent permeability factor5; and because they are readily inhibited by prior treatment in vitro with the trypsin inhibitors from soya bean, lima bean and bovine pancreas. The action of these inhibitors, however, is apparently very rapid, a mixture of factor and inhibitor only 1 min. old being inactive when injected into the skin of animals with circulating pontamine blue4,7. Consequently, it is impossible to distinguish inactivation of a protease permeability factor in vitro from inactivation of a protease newly activated in vivo. Moreover, even the best preparations of permeability factors also contain known serum proteases, and so far no enzyme substrate has been found which is hydrolysed by serum fractions in strict proportion to their content of permeability factor.

IN recent work on the serum iron and total iron-binding capacity of groups of healthy and diseased Africans, we found the total iron-binding capacity of pellagra patients to be approximately 60 per cent of healthy controls1. Zone electrophoresis on filter paper in an earlier work2 did not show that the concentration of the β-globulins in pellagra was any lower than healthy controls and this suggested that there may be a relationship between transferrin polymorphism and incidence of this disease, in view of the findings of Gibbett et al.3 that the faster-migrating transferrins take up more iron.

Surgenor, Koechlin and Strong1,2 have described the isolation of the β1-metal-combining globulin (siderophilin, transferrin) from Cohn's plasma fraction IV–4 by alcohol fractionation. The crystallization, described by Koechlin2, could not easily be reproduced by other investigators. Improved conditions for the purification and the crystallization starting from fraction IV–7 have recently been published by Inman3. Already the preparation of fraction IV–7 requires many steps and much time, which may be one of the reasons why only very few clinical experiments with this iron-transmitting globulin have been reported.

CLINICAL and pathological observations on Chinese hamsters (Cricetulus griseus) with spontaneous hereditary diabetes mellitus have been reported previously in several papers1–7. Generally speaking, the disease in Chinese hamsters is primarily pancreatogenic, with degranulation, hydropic degeneration and deficiency of β-cells. Other forms of pathological expression noted in symptomatic animals are glomerulosclerosis, arteriosclerosis, increased periodontal breakdown, and proneness toward the formation of adenocarcinomas of the pancreas.

EARLIER investigations have shown that incubation of isolated rat peritoneal mast cells with purified anti-rat IgG-globulin and fresh serum in vitro results in a cytotoxic immune reaction1. Data demonstrating that this process is inhibited by various agents, especially by alkyl phosphates and synthetic chymotrypsin substrates, indicate that the activation of a chymotrypsin-like esterase is of some importance for the reaction2. The enzyme, which is contained in the heat labile serum portion, shows many striking similarities to an enzyme of the complement complex (C′1-esterase), and was therefore thought to be identical with it3. Further experiments, however, revealed that neither a euglobulin preparation with high C′1-esterase activity nor purified human (C′1a-esterase, irrespective of whether or not antibody is also present, produces detectable morphological or pharmacological changes in isolated rat mast cells (ref. 3 and Lepow, I. H., personal communication). The question thus arose as to what extent the immunological behaviour of mast cells would be influenced by pre-treatment with various enzymes. The data presented here provide some insight into the problem.

Immunological Identification of a Rivanol ‘purified’ Fraction of Macrocryoglobulinaemia (Waldenstrom) Serum

DISSOCIATION of gamma-globulin molecules by reduction in urea with such thiol reagents as mercapto-ethanol and thioglycolate1,2, or in neutral aqueous solution3, or by S-sulphonation4,5, allows the identification of two types of polypeptide chain, called H, (A) and L (B), which may be separated by gel filtration.

ALTHOUGH haptoglobin is almost always present in normal adult serums, it is demonstrable in only about 10 per cent of cord blood serums by the technique of starch-gel electrophoresis1. The factors accounting for this large percentage of ahaptoglobinaemic new-borns are obscure. The present communication is based on a survey of cord-blood serums undertaken in an attempt to evaluate some of the factors accounting for the high frequency of ahaptoglobinaemia in the new-born. At the same time, work was carried out to substantiate that the new-born baby with detectable haptoglobin produced it himself.

ACCORDING to Bregman and Kirsner1 normal human serum globulins are not usually fixed to tanned red blood cells and this is in contrast to some pathological serum globulins which have a binding capacity for these cells. These conclusions were drawn from the observation that tanned red blood cells coated with normal sera were not usually agglutinated by antiglobulin serum, whereas their coating with some pathological sera resulted in a positive Coombs's test. The difference between normal and pathological sera was even more evident when an anti-non-γ-globulin serum was applied instead of antiglobulin serum. In the anti-non-γ-globulin the anti-γ-constituent of the Coombs's serum is blocked by previous addition of γ-globulin, so that it reacts predominantly with the component of the non-γ-globulin serum2. In the present investigation, in which the same method was applied, evidence was obtained for the existence of an inhibitor in normal concentrated sera which interferes with the binding of the non-γ-globulin component to tanned red blood cells.

RECENT studies have shown that cationic proteins from leucocytes produce tissue injury and responses characteristic of immunological reactions1–4. In an earlier publication we put forward the hypothesis that homograft rejection may be caused by such cationic proteins5. The specificity of the homograft reaction could only occur if the basic protein were transported to the homograft site via specific antibody (globulin) or in cells which had specificity through some chemotactic response of the cell to antigen.

THE chick embryo is capable of developing hypersensitivity of the immediate type (anaphylaxis) to foreign proteins during most of its developmental life1,2. It has also been demonstrated that, from the 12th day of incubation, sensitization of the delayed type to transplantation antigens can be elicited by injection of whole blood3. Due to the fact that chick embryos also are susceptible to immunological suppression and are not capable of humoral antibody production4,5, a new interpretation was needed to account for such distinctive immunological properties of the chick embryo. In proposing such a theory it was postulated that, during development of the embryo, tissue-bound antibodies can be elaborated by cells derived from inactive mesenchymal precursor cells. Such tissue-bound antibodies, in common with classical antibodies, have anaphylactogenic properties1. It was assumed that chicks, sensitized as embryos, would be capable of manifesting hypersensitive reactions for only a short time after hatching. This was based on a common observation that chick embryos and young animals injected with antigen do not manifest anaphylaxis when tested one month or more after the initial injection4,6. The term hapto-antibody was suggested for these tissue-bound embryonic antibodies which manifest this anaphylactic property.

Detection by paper electrophoresis. Using starch gel electrophoresis, six cattle1, fourteen sheep2, three goat (see later) and three human3,4 β-globulin phenotypes have been recognized and indicated to be under the genetic control of three, five, two and three allelomorphs respectively. As one of us had noticed a variation in the number and position of the β-globulin zones in paper electrophoresis of animal sera, the two methods have been compared on sera from cattle, sheep and goats. Starch gel electrophoresis was carried out in phosphate buffer1 pH. 7.6, paper electrophoresis on a wide strip of horizontally suspended Whatman 3MM paper in barbiturate buffer pH 8.6, I 0.05. Typical examples of corresponding electropherograms of sheep and cattle sera are shown in Figs. 1 and 2. The electropherograms of goat sera shown in Fig. 3 are discussed below.

Two main haemoglobin-binding serum globulin fractions have been demonstrated by using the technique of immuno-electrophoresis. One precipitation line corresponds to the α2 region and is sometimes doubled1; the other to the β region1,2. On the other hand, starch-gel electrophoresis shows numerous haemoglobin-binding globulins which are now well known as the haptoglobins 1 and 2 of Smithies3. The relation between the immuno-electrophoretic and starch-gel haptoglobins is not yet quite clear. Allison's4 hypothesis of polymerization of the α2 haptoglobins is yet to be confirmed.

THE term thyroxine-binding globulin is often used to denote a serum protein fraction with a mobility intermediate between those of α1 - and α2-globulins in paper or moving-boundary electrophoresis in veronal buffer, pH 8.6, ionic strength 0.05–0.10 (ref. 1). However, with the introduction of supporting media such as agar and starch gels and the use of different buffers, ionic strength or pH for paper electrophoresis, binding of thyroid hormones has also been detected in the ‘prealbumin’ zone2. Although the proteins of the ‘prealbumin’ zone obtained under different conditions are not likely to be identical, a strong thyroxine-binding property was detected in human serum prealbumin rich in tryptophan3, as obtained from Prof. Schultze4. From chemical and immunological observations I concluded that the tryptophanrich prealbumin, present as a complex in the α-globulin fraction, might be responsible for much of thyroxine-binding in human serum5. Since then, an α-globulin-type of thyroxine-binding protein has been obtained in this laboratory (unpublished results); the nature of this protein does not support the hypothesis that the tryptophan-rich prealbumin is involved in binding thyroxine. One possible explanation for this discrepancy is that Schultze's prealbumin preparation contained traces (1–2 per cent) of thyroxine-binding globulin which were responsible for the production of antibodies to it present in antisera against this prealbumin. In fact, multiple precipitation lines were observed in immunological studies using rabbit anti-human prealbumin serum5. The experiments to be described support this explanation and throw further light on the nature of the thyroxine-binding globulin in human serum.

USING starch-gel electrophoresis Smithies and Hickman1 recognized five cattle β-globulin phenotypes. In a preliminary communication Ashton2 also described five phenotypes, but after critical examination of some matings he detected a sixth phenotype3. The results indicated that the β-globulin types were determined by three alleles named βA, βDand βE, with no dominance1,3. However, usually it seems to be difficult to distinguish the two phenotypes D/E and E/E.

INDIVIDUAL variations in the serum β-globulin patterns obtained on starch-gel electrophoresis have been shown to be genetically determined in a number of species1–5 and in some the proteins concerned have been shown to be transferrins7,8. These variations have been ascribed to a number of alleles5,6 which have been used in the genetic characterization of animal populations2,3,7. This communication reports the serum β-globulin patterns found in red deer (Cervus elaphus) with some observations and experiments on their stability, shows that they are due to transferrins and makes some comparisons with the β-globulin patterns in other species.

ABNORMALLY high concentrations of macroglobulins are known to occur in a number of disease states1. Detection has depended on certain preliminary tests2,3, at times complicated by either false positive or false negative reactions. Definitive evidence is provided only by analytical ultracentrifugation, a procedure not conveniently available at all medical centres.

POLYMORPHISM, detected by antibody from individuals of the same species (allotypy), has so far been observed only in the γ- (ref. 1) and β-globulins (ref. 2). In man, a class of α-globulin Gc1 and Gc2 is subject to genetic variations and has been revealed by antibodies of heterologous origin3. Polymorphism of other α-globulins, the haptoglobins in several mammalian1 species and recently the trypsin inhibitors in man4,5 have been detected by other means. So far, the polymorphism of serum proteins has been most thoroughly examined in man and rabbit. In the mouse the genetic variation of the transferrins6 and of an allelic pair of γ-globulins (MuA1 and MuA2) (refs. 7–10) have been examined. We wish to report here a new allotypic specificity among the γ-globulins of the mouse.

IT has been established by several investigators that serum protein components of chick embryo undergo qualitative as well as quantitative changes throughout the developmental period until the time of hatching1–4. The serum protein composition of the newly hatched chick, however, resembles that of the adult bird, as revealed by moving-boundary and paper electro-phoresis. The technique of starch-gel electrophoresis suggested by Smithies5 is promising in that it enables one to detect numerous fractions in the pattern of the serum proteins, particularly in the α- and β-globulin regions, which cannot be detected by the conventional method of Tiselius or paper electrophoresis. By using the starch-gel method, I have been able to demonstrate differences in the patterns of serum proteins of the day-old and week-old chicks when compared with pooled sera of one- and two-year -old roosters, as shown in Fig. 1. The electrophoresis was performed on a vertical tray6 for 18 hr. using borate buffer of pH 8.6 and a field-strength of 6 V./cm. Repeated electrophoretic runs gave patterns similar to those shown in Fig. 1, for the four age groups. Twelve fractions could be distinguished on the patterns of the adult roosters (two pre-albumin components are not seen on the photographs). The same number of fractions could also be seen on the electrophoretic patterns of the young chicks. The mobility and the number of fractions of α-globulins of the newly hatched chicks are distinctly different from those of adult birds. The fraction b of the serum of a day-old chick does not seem to be present in the serum of an adult bird, and of fraction d only traces are observed. Very little γ-globulin is present in day-old and week-old chicks. A distinct narrow band which is immediately behind the albumin (a post-albumin ?) in the adult bird cannot be seen in the young chicks.

VARIOUS β-globulin types have been demonstrated, using starch-gel electrophoresis, in several species of mammals, namely, man1, cattle2, sheep3, goat4 and horse5. The present communication reports the discovery of similar variations in two species of monkey, Macaca mulatta and M. irus. Variations in the monkey β-globulins were initially detected by paper electrophoresis using barbital buffer at pH 8.6. However, the β-globulins were much better resolved when starch-gel was used instead of paper.

THIS communication reports the results of analytical ultracentrifuge analyses on sera of 50 acutely mentally disturbed patients. Sera were taken within 24 hr. of admission to hospital and prior to institution of definitive therapy. Phenothiazine, monoamine oxidase inhibitor or electroshock therapy had not been given to any patient during the three months prior to serum collection. The diagnoses were varied; but all patients had an acute mental disturbance of sufficient severity to warrant hospital admission. Consecutively admitted patients were used excluding only those with known organic brain disease, alcoholism and those known to have had drug or electro-shock treatment during the previous three months. Sera of 48 blood bank donors were used as controls. Sera were stored at −15° C. until analysis, which was usually within 8 weeks of collection. (Analytical ultracentrifuge examinations were made by the Institute of Medical Physics, Belmont, California.)

WE have shown that the adult rat, thymectomized at birth, has a global defect in delayed hypersensitive responses correlated with a depletion of small lymphocytes from blood, lymph nodes and spleen1. The deficiency in antibody mediated responses, in contrast, is selective. The nature of this latter defect has not been clarified. It has been shown that plasma cell and serum γ-globulin levels are normal in thymectomized rats2.

OCCASIONALLY, ultracentrifugation of sera from patients with multiple myeloma yields an abnormal protein fraction with a sedimentation constant of 8–12 S. Jahnke and Scholtan1 referred to such cases as “atypical macroglobulinaemia”, because the sedimentation constant of these pathological proteins is smaller than that of the ‘true’ macroglobulins such as found in Waldenstrom's macroglobulinaemia.

MICE given intraperitoneal injections of antigen–adjuvant mixture1–3 produce large amounts of ascitic fluid which contains specific antibodies. Inbred AKR mice injected intraperitoneally with isologous tumour cell fragments in modified Freund's adjuvant developed large amounts of peritoneal fluid which contained specific cytotoxic and neutralizing antibodies2,4. These observations had immediate practical importance since immunological studies have been limited by the small amount of antiserum obtainable from each mouse. Since little or no information on the biophysical properties of mouse ascitic fluid is available, an investigation was made of its electrophoretic and immunological characteristics.

ON electropapergrams, the alpha-2-globulin protein peak has been shown to coincide exactly with the locus of maximum non-specific alkaline phosphatase activity1. This enzyme may be detected during the run by an adaptation of the Huggins and Talalay sodium phenolphthalein phosphate method, which determines alkaline phosphatase by ascertaining the colour due to free phenolphthalein produced at pH. 9.2 by incubating a serum sample with the colourless substrate (a blank correction is made for non-enzymatic substrate hydrolysis). Because of its broad optimum pH range, alkaline phosphatase has considerable activity at pH. 8.6, which is most often used for paper electrophoresis, and a significant proportion of free phenolphthalein is coloured at this pH. To detect alkaline phosphatase during paper electrophoresis, 0.1–1.0 per cent of sodium phenolphthalein phosphate is included in the buffer; the region of enzymatic activity is seen as a well-localized red band. At pH 9.2 the band is considerably more impressive in colour.

MORE than one component of human caeruloplasmin has been reported by several investigators1. Morell and Scheinberg1 have observed 4, possibly 5, components of caeruloplasmin by fractionating a pool composed of 9,109 individual sera. In such a large pool, infrequent unusual components would be easy to lose. Furthermore, the complex fractionation procedure provides opportunity for artefactual alteration of specific proteins. The resolving power of vertical starch-gel electrophoresis2 combined with direct chemical and enzymic analysis on the gel provides a simple and elegant means of examining the problem in individual sera. Using this technique we have found 5 electrophoretically distinct proteins having one or more characteristic properties of caeruloplasmin.

Heim and Kerrigan1 have demonstrated by starch gel electrophoresis a slow-moving α2-globulin in the serum of rats after the administration of carbon tetrachloride similar to that previously described, using immuno-electrophoresis, by D'Arcy2,3, in pregnant, neonatal and tumour-bearing rats, and by starch gel electrophoresis, by Beaton et al.4 and Heim5.

ELECTROPHORESIS on paper of serum proteins has enabled us to separate small quantities into albumin, and α-, β- and γ-globulin. By using a comparatively thick filter paper (MunktellNr. 20, Grycksbo, Sweden), 58 cm. × 7 cm., we can separate up to 30 mgm. of protein within 24 hr., with 5.5 V./cm. and 16–18 m.amp. applied in a closed atmosphere. As buffer system veronal/veronal sodium is used, of 0.06 ionic strength and pH 8.9. From the two paper strips used simultaneously, only one is stained afterwards with bromphenol blue; this strip serves as a guide for cutting off the fractions from the still wet, unstained strip.

THE recent publication of a communication on the preparation of caeruloplasmin1, the main cuproprotein of plasma, prompts this brief account of a method of its preparation from the precipitate G2, the mixture of α- and β-globulins obtained in the plasma fractionation scheme of Kekwick and Mackay2. With G2 from batches of plasma of 40–150 l., the following stages were used: (1) Extraction of G2 by 0.07 M sodium chloride at pH7 and denaturation of extracted lipoprotein with ether below − 25° C. by the McFarlane technique3; (2) precipitation of caeruloplasmin from the ether-saturated extract at pH 4.8; (3) solution of the precipitate in ether-saturated 0.1 M sodium chloride, removal of impurities by precipitation at pH 5.35 and a second McFarlane treatment; (4) reprecipitation of caeruloplasmin at pH 4.8 as in (2) and solution in 0.1 M sodium chloride; (5) precipitation of impurities at pH 4.8 by diluting to 0.02 M sodium chloride and precipitation of caeruloplasmin by further dilution to 0.0045 M sodium chloride. The product was dissolved in 0.l M sodium chloride. E 1 cm. (605 mµ)/E 1 cm. (280 mµ), which gives a measure of degree of purification, was 0.019.

zone electrophoresis of serum proteins in starch gels was first described by Smithies1, who employed a borate buffer system and silver electrodes. Poulik2 described a discontinuous system using tris (tri-hydroxymethylamino-methane) and borate buffers for the separation of proteins. Two-dimensional electrophoresis on paper, and then on starch gel as used by Smithies and Poulik3,4, revealed at least six molecular size-groups in the α2-globulin group. A modification of this method is described, which utilizes the combination of haptoglobins with haemoglobin to give a faintly coloured band as visual marker of the α2-region and thus avoids staining. Commercially available starch is used as supporting medium, and provides a wide separation of the individual fractions.

IT is now well established that congenital agammaglobulinaemia is associated with multiple serum protein deficiencies ; the sera of these patients lack 7S γ-globulins, 19S γ-globulins or β2M globulin, and β2A globulin1. Good et al.2,3 have recently reported that the sera of patients with agammaglobulinaemia contain three proteins not found in normal human sera and they have termed them β2K, β2E and γ1A globulins; their work was performed with antiserum from a horse immunized against human serum by Dr. P. Grabar. A review of our own immunoelectrophoretic patterns as well as those of Grabar et al. has failed to reveal any protein constituent other than C-reactive proteins in the sera of agammaglobulinaemic patients which is not ordinarily detectable in normal sera. Consequently the work reported here was undertaken to re-investigate this matter.

CERULOPLASMIN is one of the minor components of plasma proteins. Most of the numerous publications which have appeared in the past several years are concerned with the evaluation and clinical significance of hypo- and hyper-ceruloplasminaemia. Nevertheless, the biological role of ceruloplasmin is still unknown.

MOST investigators who preserve sera for future analysis take it for granted that the serum proteins are stable at temperatures around −20° C. Some evidence supports this assumption: the titres of certain antibodies or of rheumatoid factor, for example, remain unaltered after even several years in a deep freeze. This communication shows that most human sera, from both healthy and sick people, contain an α-2 globulin which loses its antigenic (precipitin) characteristics after prolonged storage at −20° C.