Effect of gamma globulin on reactivity of the organism. V. Types of biological activity of gamma globulin preparations]

Biological activity of 110 series of commercial gamma-globulin preparations was studied; they were found to contain placental antigens, group-specific blood substances, gonadotropic hormones and antibodies to them. Placental antigens were found in 12% of placental and abortive gamma-globulin batches in titres of 1 : 2--1 : 16; no placental protein was revealed in donor gamma-globulin. There were group-specific blood substances in all the batches of placental and abortive gamma-globulin studied (in titres of 1 : 138--A, 1 : 112 B in the placental gamma-globulin and in titres of 1 : 48.9--A, 1 : 32--B in the abortive gamma-globulin). In the preparations from the venous blood group-specific substances were either absent or present in lowe titres only (1 : 2). The value of gonadotropic hormones in the placental gamma-globulin batches constituted 873+/-157, and in the abortive--991.4+/-147 IU/l; no gonadotropins were revealed in donor gamma-globulin. The mean titres of antibodies to gonadotropin hormone in the gamma-globulin preparations made of placental blood constituted 1 : 236+/-32, of abortive--1 : 131+/-16.6, and of the venous blood--1 : 46+/-24.7. The presence of biologically-active substances in the gamma-globulin preparations pointed to the necessity of increased requirement of their quality; additional requirements to its standardization proved to be also necessary.     

Chemical, biological and immunological properties of pituitary gonadotropins from the donkey (Equus asinus): comparison with the horse (Equus caballus)

Donkey gonadotropins (donkey luteinizing hormone, dLH; donkey follicle-stimulating hormone, dFSH) have been isolated in purified form from 191 donkey pituitaries using essentially the same procedures previously employed for the purification of equine gonadotropins. Chemically, dLH and dFSH were observed to be similar to equine LH (eLH) and FSH (eFSH) in fractionation behavior and glycoprotein nature. Two forms of the dFSH molecule were observed, as is the case for eFSH. Donkey LH had significantly less total carbohydrate (13.5%) and sialic acid (1.9%) than eLH (26.7% and 5.8%, respectively). Carbohydrate (17-21%) and sialic acid (2.4%) content of the two dFSH preparations closely resembled that of eFSH. A slightly higher tyrosine content in the donkey gonadotropins was noted in a comparison of amino acid compositions. Immunologically, in a heterologous FSH radioimmunoassay (RIA), dFSH preparations were equal to or twice as active as eFSH preparations. However, in homologous RIAs for equine chorionic gonadotropin (eCG), eFSH and eLH, both the dLH and dFSH preparations were considerably less active than the equine gonadotropins, and their inhibition curves were all nonparallel. Biologically, in the Steelman-Pohley assay both dFSH preparations were equipotent and as potent as eFSH (approximately 40 times NIH-FSH-S12). In the Sertoli cell assay for cAMP (FSH assay) and the Leydig cell assay for testosterone (LH assay), both dFSH and dLH were 2- or 6-fold more active than eFSH and eLH, respectively. In rat and equine testis FSH homologous radioreceptor assays, dFSH preparations were as active and up to 6-fold more active than eFSH. In contrast, dLH was 10-fold less active than eLH in the equine LH homologous radioreceptor assay. Unlike eLH, dLH was found to possess little intrinsic FSH activity or FSH inhibitory activity, and the small amount of FSH activity observed was most likely due to FSH contamination. Therefore, eLH behaves much like eCG (pregnant mare's serum gonadotropin, PMSG) which also possesses both LH and FSH activity. In contrast, dLH behaves more like donkey chorionic gonadotropin (dCG) which possesses only a low degree of FSH activity.     

Trophoblastic beta-globulin in immunoglobulin preparations

The content of trophoblastic beta-globulin in 142 lots of commercial immunoglobulin preparations from 20 manufacturers, produced from placental, abortion and donor blood sera, has been studied. 83% of lots from abortion blood serum and 94% of lots from placental blood serum have been found to contain the admixture of this beta-globulin, its concentration in the lots from placental blood serum being significantly higher. The method for the detection of trophoblastic beta-globulin may be used for evaluating the quality of immunoglobulin preparations as it indicates the degree of their purification from placental proteins.     

A comparison of menotropin, highly-purified menotropin and follitropin alfa in cycles of intracytoplasmic sperm injection

Background  

Over the last several decades, as a result of an evolution in manufacturing processes, a marked development has been made in the field of gonadotropins for ovarian stimulation. Initially, therapeutic gonadotropins were produced from a simple process of urine extraction and purification; now they are produced via a complex system involving recombinant technology, which yields gonadotropins with high levels of purity, quality, and consistency.     

Fragmentation of gamma globulin preparations manufactured in the USSR]

Gamma-globulin preparations prepared by the alcoholic method from the placental and abortion sera were split during storage under the effect of proteases contained in them; fragments with a molecular weight of 50 000 and 60 000 were formed. The most intensive splitting was seen in the preparations obtained from the precipitates of B placental and abortive sera. Some delay in the gamma-globulin fragmentation could be reached by addition to protease inhibitors--epsilon-aminocapronic acid or cyclocapron.     

New aspects of isolation and purification of thyroxine-binding globulin from human biological fluids

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.     

Biological and immunological properties of zebra pituitary gonadotropins: comparison with horse and donkey gonadotropins

Previous studies from this laboratory have described the properties of purified luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from horse and donkey anterior pituitary glands. The present study afforded the opportunity to further characterize these previously purified hormone preparations and to compare them with enriched gonadotropin fractions from zebra pituitary glands. Although a single LH and FSH fraction was usually obtained for each pool of pituitaries, two separate zebra LH and two donkey FSH preparations were generated. Purified hormone preparations from the horse were designated eLH and eFSH. Preparations zLH-A, zLH-B, and zFSH were obtained from zebra pituitaries, and fractions dLH, dFSH-A, and dFSH-B were prepared from donkey pituitary glands. These preparations were analyzed by LH and FSH radioimmunoassays (RIAs), radioreceptor assays (RRAs), LH bioassay, and chromatofocusing. Clear immunological differences were observed between equid gonadotropins. Homologous RIAs for eLH and eFSH did not cross-react similarly, or in a parallel fashion, with gonadotropins from the donkey and zebra. In contrast, RIAs capable of assessing LH or FSH in a wide number of species showed all equid gonadotropin preparations to have considerable activity and to produce parallel dilution curves. Relative to eLH (1.00), zLH-A was found to have higher LH bioactivity:LH RIA (2.50), LH RRA:LH RIA (1.42), and LH bioactivity: LH RRA (2.21) activity ratios. The dLH and zLH-B fractions only differed from eLH in LH RRA:LH RIA activity (0.69 and 0.62, respectively). Only LH from the horse possessed clear intrinsic FSH-receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity purification of fibrinogen using an Affimer column

BackgroundFibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen.MethodsFibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis.ResultsAffimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII.ConclusionsThe Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations.General significanceOur methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.     

Direct PCR on fruitflies and blood flukes without prior DNA isolation.

DNA is the essential substrate for the polymerase chain reaction (PCR). Standard protocols include a DNA purification step, but this is laborious if a large number of DNA preparations have to be performed, although a variety of simple methods exist for the isolation of crude DNA for PCR. For microorganisms, PCR protocols exist that allow the amplification of sequences directly from the organism without DNA purification. The results introduced in this paper demonstrate that a direct PCR approach also works with fruitflies and blood flukes as examples for intact multicellular organisms.     

Characteristics of biologically active mannan substances, isolated from fodder yeast

The component composition and antiviral properties of the mannan-containing preparations were studied. These preparations were extracted from some laboratory and commercial specimens of the fodder yeast cultivated on different substrates. It was shown, that the main component of pure preparation was the mannan which had molecular mass near 13 kDa. The monosaccaride composition of mannan component was varied depending on the source, degree of purification, methods of purification of the preparations. However, the crude and pure mannan preparations activity relatively to VTV was approximately equal.     

Cell types of the pars distalis: seasonal changes in secretory activity and effect of steroids on spermatogenesis in Rana hexadactyla

Cytomorphological features and tinctorial affinities of the adenohypophyseal gonadotrophs have been studied in stained preparations of the pituitary of adult male frogs Rana hexadactyla Lesson, at monthly intervals during a calendar year. All the five morphologically distinct cell types showed a progressive increase in size from August of February, followed by a decrease from March to July. Extrusion of the synthetized product occurred in the summer and during this period production was low. Injection of steroids viz. stilboestrol, testosterone, oestradiol, doca and cortisol, interfered with the elaboration and secretion of gonadotropins, the adenohypophyseal feedback mechanism, and the sensitivity of the germinal epithelium. Changes in gonadotropins secretion and their impact on the testis are discussed.     

Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.     

Comparison of the effects of gonadotropic preparations of the carp and stellate sturgeon pituitaries on in vivo and in vitro oocyte maturation in the Siberian sturgeon Acipenser baeri brandt

Injections of 2.5 mg/kg of stellate sturgeon (Acipenser stellatus Pall.) pituitary extract and 5 mg/kg of carp (Cyprinus carpio L.) pituitary extract in Siberian sturgeon (Acipenser baeri Brandt) females did not reveal significant differences in the effects of these preparations. There were no differences in the percentage of females that responded by ovulation, duration of the period from injection to ovulation, rate of ovulation, or quality of mature eggs as estimated by the percentage of fertilization or percentage of normal embryos at the small yolk plug stage. Thus, an insufficient efficiency in the artificial reproduction of the Siberian sturgeon grown in captivity is not related to the use of the carp pituitary preparation as a stimulus. Estimation of the ratio of specific activities of the pituitary extracts and purified gonadotropins of the stellate sturgeon and carp by in vitro oocyte maturation has shown that it varies within wide limits as a function of the medium composition and physiological state of follicles. Hence, the ratio of activities of the gonadotropins of different species as determined by in vitro maturation of sturgeon oocytes may markedly differ from that upon injection of these preparations in breeders.     

Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus

The effect in vitro of oocyte maturation inhibitors and the ability of preparations of luteinizing hormone to relieve the arrest induced by these substances was studied in preparations of cumulus-free (naked) rat oocytes, and compared to previously obtained results from oocytes enclosed by their cumulus cells. The development of both the cumulus-oocyte complex and naked oocyte is arrested in vitro by cyclic AMP derivatives or cyclic nucleotide phosphodiesterase inhibitors. While gonadotropins can overcome the effect of these substances in the cumulus oocyte complex, they have no effect on naked oocytes. Cholera enterotoxin, an irreversible activator of adenylate cyclase, maintains developmental arrest in cultured cumulus oocyte complexes but not in naked oocytes. Preparations of luteinizing hormone can partially overcome the effect of cholera enterotoxin in the complexes. Furthermore, the acceleration of oocyte maturation in vitro observed in the presence of gonadotropins, which is seen in cumulus oocyte complexes, can be mimicked by stripping the oocyte of its associated cumulus cells. The results of these and other studies suggest that: (1) the cytoplasmic levels of cyclic AMP in the isolated oocyte are high enough to maintain meiotic arrest if a phosphodiesterase inhibitor is present; (2) the oocyte contains an active phosphodiesterase; (3) the oolemma may lack the adenylate cyclase system and; (4) gonadotropins seem to affect the oocyte indirectly, via the cumulus cells, possibly by interrupting communication between the two cell types.     

A purine N1-C6 hydrolase activity in the chicken egg yolk: a vestigial enzyme?

All attempts to prove the presence of xanthine oxidase and uricase in yolk preparations failed. We were able instead to show that yolk preparations could hydrolyze the N1-C6 bond of certain purine bases. In the case of xanthine, 4-ureido-imidazole-5-carboxylic acid and 4-ureido-imidazole are formed. Activity only becomes apparent during purification. An analogous enzyme was described earlier in Clostridium cyclindrosporum. The liver and the blood plasma of actively laying hens do not contain the enzyme. A scheme for the degradation of egg ribonucleic acids is presented.     

Immunochemical analysis of molecular forms of mammalian DNA ligases I and II

Using specific antibodies against calf thymus DNA ligases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130 000 and 90 000 were detected by SDS-polyacrylamide gel electrophoresis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.     

Seasonal dynamics in the function of avian pituitary gonadotropic cells]

The investigations showed that differentiation and functional activity of gonadotropocytes in ducks at different phases of a sexual cycle change in complete conformity with the quantitative changes of gonadotropic hormones in the hypophysis and peripheral blood. Intensification of the function of gonadotropocytes and an increase in the content of gonadotropins in blood and hypophysis correspond to activation of gonads. The refracteriness of gonads is accompanied by involution of gonadotropocytes and inhibition of synthesis and secretion of gonadotropins in them.     

An evaluation of the quality of tetanus anatoxins by gel electrophoresis]

The study of tetanus toxoids obtained from different manufacturers in the USSR has shown that these preparations exhibit molecular heterogeneity. The method of gel filtration has made it possible to find out that tetanus toxoids from different manufacturers differ in the degree of their purification. The preparations produced by the manufacturing enterprises in Perm and Ufa have been found to contain considerably less ballast substances than the preparations produced in Moscow.     

Comparison of the ability of seven gonadotropin preparations from different mammalian sources to interact with the adenylyl cyclase system in corpora lutea from rabbits and rats

The effects of guanine nucleotides and magnesium (Mg2+) on the interaction of seven different gonadotropin preparations with their rabbit and rat luteal receptors were studied and compared to the ability of these gonadotropins to stimulate luteal adenylyl cyclase activity. In both the rabbit and rat, human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) were less efficacious than the other gonadotropin preparations in stimulating luteal adenylyl cyclase activity and thus behaved as partial agonists. Addition of 2 mM MgCl2 increased the affinity of the rat luteal receptors for all seven gonadotropins tested, while in the rabbit Mg2+ increased the affinities for porcine, bovine, ovine, rat and rabbit LH but did not significantly alter the affinities for hCG or hLH. In no instance did the addition of 100 microM GTP alter the affinity of the receptor from that observed in the absence or presence of Mg2+. A positive correlation existed for both species between the Kd values calculated from binding experiments and the Kact values obtained in adenylyl cyclase assays suggesting that the specific gonadotropin-binding sites present in rabbit and rat luteal membranes represent receptors which mediate the stimulatory effect of LH. The magnitude of the Mg2+-induced increase in affinity of a given gonadotropin preparation for its receptor was correlated with the efficacy with which that gonadotropin stimulated luteal adenylyl cyclase activity in both the rabbit and rat.     

Species specificity of radioreceptor assay and radioimmunoassay for rat FSH

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.