In Vitro Distribution of Gold in Serum Proteins after Incubation of Sodium Aurothiomalate and Auranofin with Human Blood and its Pharmacological Significance

This study presents a comparative drug–protein, in vitro, binding profile of sodium aurothiomalate and auranofin. It was found that about 40% of total protein-bound gold is attached to albumin after incubation of aurothiomalate with whole blood for 24 h and about 29% of it was with α₁-globulin and the least amount was found with γ-globulin (6.1%). On the other hand, approximately 84% of the protein-bound auranofin gold attached to globulins of which 51% was found with β-globulin band. It was almost equally distributed among albumin, α₂-globulin and γ-globulin, and showed least affinity for α₁-globulin. The gold analyses were performed by standardless instrumental neutron activation method duly validated by use of an established atomic absorption method. The results of this study explain to some extent the difference in, in vivo, pharmacokinetics and pharmacodynamics of the two drugs.     

Heat Denaturation and Emulsifying Properties of Plasma Protein

The effects of pH and NaCl on the denaturation of plasma protein during heat treatment were investigated, as well as the relationship between protein structure and emulsifying properties. When the plasma protein solution (1% w/v) was heated at 80°C, precipitation was accelerated by the presence of NaCl. The measurement of SH groups, surface hydrophobicity and CD spectrum revealed that denaturation occurs easily by heat treatment in the neutral pH region and in the presence of NaCl. The emulsifying activity index (EAI) did not change much after heat treatment at pH 3 irrespective of the presence of NaCl, but it decreased about 60% after heat treatment at pH 7 in the absence of NaCl. Gel filtration patterns indicated that a high molecular weight peak arose upon heat treatment at neutral pH. We concluded that the decrease in EAI was owing to the polymerization of serum albumin and γ-globulin, which are the main components of plasma protein, and disulfide bonds participated in this process.     

Carotenes from Mutants of the Dinoflagellate, Crypthecodinium cohnii

SYNOPSIS.
The carotenoid compositions of 15 nitrosoguanidine-induced mutants of Crypthecodinium cohnii , a heterotrophic dinoflagellate, were determined by chromatographic and mass spectral analyses. Wild-type C. cohnii grown with irradiation of 250 W/cm² visible light at 27 C synthesizes β-carotene (33%) and γ-carotene (67%) amounting to 0.083 mg/g dry wt. There are 4 types of carotenoid-deficient mutants: (I) albinos which synthesize no C₄₀-carotonoids: (II) albinos blocked at the level of phytoene desaturation; (III) cream-colored cells which accumulate mainly §–carotene, with phytoene and/or β-zeacarotene also present; and (IV) light-orange strains which synthesize reduced amounts of β-carotene and γ-carotene.
Dark-grown wild-type cells produced 35% as much carotenoids as light-grown cells. Inhibition studies revealed that diphenylamine (3 γ) caused phytoene accumulation; nicotine at 0.9 mM blocked the final cyclization, to cause γ-carotene to accumulate in wild-type cells. Inhibition by adenine and guanine (1.5 mM) of carotenogenesis was demonstrated for the first time in any system. The effect of these purines was similar to that of diphenylamine addition: phytoene desaturation was largely inhibited.
The carotenogenic system in this dinoflagellate is similar to that of green algae and higher plants, and is under nuclear genetic control.     

Some Effects of Protein Deficiency in Young Growing Pigs

No significant differences between the serum protein concentrations of the totally starved pigs and control animals given a milk protein supplemented diet were found. Serum lipid levels rose in the totally starved group. During the first 42 days of the experimental period sharp falls in serum protein concentrations were noted in the protein deprived pigs. The losses were greatest in the albumin and two β-globulin fractions, γ-globulin levels rose but at a much slower rate than in the control group. In the pigs which were refed a protein containing diet during the second 42 day period, the concentrations of all the serum protein fractions except for γ-globulin had reached control levels by the end of the investigation. The pigs which continued with the protein free diet showed further losses of albumin, a reduction in some α-globulin fractions and a cessation of net γ-globulin synthesis. The trends observed by measurements of protein bound carbohydrate were similar to those obtained from polypeptide determinations indicating quantitative rather than qualitative changes in the serum proteins. The reduction in the serum lipid concentrations of the pigs undergoing protein starvation was largely accounted for by losses of α- and β-lipoproteins. The protein deprived pigs maintained their initial body weight, while a continuous fall in weight was found in the totally starved group and a continuous increase in the control pigs.     

Developmental Changes of N-Acetyl-L-Aspartic Acid, N-Acetyl-α-Aspartylglutamic Acid and β-Citryl-L-Glutamic Acid in Different Brain Regions and Spinal Cords of Rat and Guinea Pig

Abstract The developmental changes of N -acetylaspartic acid (NA-Asp), N -acetyl-α-aspartylglutamic acid (NA-Asp-Glu), and β -citryl-L-glutamic acid ( β -CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β -CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β -CG content decreased to an adult level at birth.     

Actin and Myosin Synthesis During Differentiation of Neuroblastoma Cells

Abstract: The levels and rates of synthesis of actin and myosin in murine neuroblastoma S20 cells have been examined during neurite formation in the absence of serum. Two forms of actin (β, -γ) have been identified in these cells; they have isoelectric points of about 5.43 and 5.45, respectively, which differ from that of murine skeletal muscle actin (5.41). Although the neuroblastoma actins have the same molecular weight as skeletal muscle actin, they can be clearly distinguished from this protein by peptide maps. Actin comprises about 7% of the total protein in these cells and neither the level nor the proportion of the two isoelectric forms change during neurite extension (β: γ:60: 40). Myosin ATPase activity is also similar in morphologically differentiated and undifferentiated cells. The rates of synthesis of actin and myosin relative to total protein synthesis undergo small changes during differentiation. Actin synthesis increases about 30% in the 1st day of induction and falls off to the undifferentiated level by day 4. Myosin synthesis increases by about 30% within 2 days of induction and remains at that level for at least 2 days. In contrast, no change in the relative synthesis of tubulin occurs over 4 days of neurite induction. Thus, neurite extension by these cells is not accompanied by large changes in either the level or rates of synthesis of actin and/or myosin.     

Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.     

Immunological state of adult germfree miniature minnesota pigs

Fundamental hematological and immunological data were obtained on sexually mature germfree miniature pigs fed, after the milk diet period, with cereal-type diet sterilized by γ-radiation, and were compared with data of control conventional animals. Germfree adult pigs had a lower count of peripheral blood leukocytes with a lower percentage of neutrophil granulocytes and without any younger forms, a lower total serum protein level with a negligible amount of γ and α₂ globulin fractions and a higher serum albumin and β globulin level. In the mesenteric lymph nodes and in spleen, surface IgA-bearing cells predominated over surface IgG-bearing cells. Also a large amount of IgA-containing cells was found in the intestinal lamina propria, where the IgG cells were present in a negligible amount. IgM cells were the most frequent surface isotype in peripheral blood. The count of blood T lymphocytes was more than doubled.     

Gamma-COP appendage domain - structure and function

COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the β-, γ-, δ- and ζ-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the α-, β'- and ε-COP subunits. Here, we present the structure of the appendage domain of γ-COP and show that it has a similar overall fold as the α-appendage of AP2. Again, like the α-appendage the γ-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian γ-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of γ-COP, Sec21p, a second binding site is proposed to exist on the γ-COP appendage that interacts with the α,β',ε COPI subcomplex.     

Changes in serum protein profile of healthy adult Nigerians after three decades.

Serum protein profile was recently determined in 55 healthy adult Nigerians and compared with values obtained 3 decades ago. The total serum protein levels were similar, but an increase in titre was recorded in albumin, alpha 1-globulin and beta-globulin. The gamma-globulin level fell markedly in the present study, suggesting improvement in health care and quality of life in the last 3 decades. Changes seen in the serum protein pattern of healthy adult Nigerians were within the normal range compared with values recorded in other Africans and Black Americans. These changes should be considered in the interpretation of laboratory results.     

Longitudinal studies on serum proteins in man during two years of stay at an altitude of 4,000 m

The sequential changes in serum total protein concentration and in various electrophoretic fractions among lowlanders during two years of stay at high altitude (4,000 m) were determined and compared with that of high altitude natives (Ladakhis). The albumin to globulin ratio decreased during the early period of exposure to altitude among lowlanders and continued to remain at that level with minor fluctuations during the entire period of stay at altitude. Among the high altitude natives, a decreased albumin to globulin ratio with low serum protein concentration was observed. The composition of globulins separated electro-phoretically was of similar pattern in altitude natives and in lowlanders after a stay of two years at altitude. Among these subjects the-globulin was significantly higher as compared to values at sea level.     

THE OCCURRENCE OF HISTOCHEMICAL ACTIVITY OF 3β-HYDROXYSTEROID DEHYDROGENASE IN THE DEVELOPING TESTES OF POECILIA RETICULATA

In the mature testes of the guppy, Poecilia reticulata , some groups of cells, distributed sparsely in the interspace between the peripheral germ cell layer and the hilar duct system, show evident histochemical response for Δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). In the testis of newly delivered guppies, somatic cells are present in the testicular hilus as a compact mass without revealing any structural differentiation. In the testis of juvenile fish of the 8mm stage about 7 days after birth, interstitial cells resembling histologically those of adult testes become differentiated from the somatic cell mass and, though only in some specimens, coincidentally begin to display weak but obvious histochemical response for 3β-HSD. Thereafter the occurrence of enzyme activity becomes increasingly regular in the developing testes, and attains the adult pattern of distribution in testes of all specimens after the 11 13mm stage or 17 ∽ 20 days of age.
The appearance and enhancement of 3β-HSD activity in the testis is concurrent with the differentiation and development of the testicular duct system. Treatments of newly delivered fish with methyltestosterone (30 ∽ 50 μg/g diet) distinctly stimulate the development of the duct system, which suggests a possible role of androgen secretion occurring in the early phase of the testicular development in the control of testicular organogenesis in the guppy.     

Isolation and characterization of a gene expressed mainly in the gastric epithelium, a novel member of the ep37 family that belongs to the βγ-crystallin superfamily

EP37 family proteins are non-lens members of the βγ-crystallin superfamily, of which expression is observed in integumental tissues of the Japanese newt, Cynops pyrrhogaster . In the present study, a gene was isolated that has high homology with ep 37 and is transcribed mainly in the gastric epithelial cells and hence designated gep. The predicted amino acid sequence of the gep cDNA contains four βγ-crystallin motifs in the N-terminal half, as is the case in the integumental EP37 proteins. Immunohistochemical analysis showed that GEP protein was mainly localized on the luminal content of the surface mucous cells of the gastric epithelium in both premetamorphic larvae and adults. In addition, GEP protein was also expressed in fundic glands after metamorphosis. Considering the fact that β- and γ-crystallins are evolutionarily related to stress-induced proteins, this localization suggests that GEP protein may have an evolutionarily conserved role in protection against physicochemical stresses, such as physical abrasion and autodigestion, during assimilation.     

A Radioimmunoassay for Ependymins β and γ: Two Goldfish Brain Proteins Involved in Behavioral Plasticity

Abstract: A radioimmunoassay (RIA) using ¹²⁵I-labeled antigen was developed for the quantitative determination of two goldfish brain proteins (ependymins β and γ). The proteins were isolated from the cerebrospinal fluid (CSF) and cells of the ependymal zone surrounding goldfish brain ventricles. The turnover rates of β and γ were previously shown to be specifically enhanced after the animals successfully acquired a new pattern of swimming behavior. Femtomole quantities of ependymin β were measurable by the RIA. In applications of the assay, β and γ ependymins were found to have common immunological properties, since ¹²⁵I-β-antigen bound to antibody could be displaced by unlabeled ependymin γ as well as ependymin β but not by a variety of other proteins including several purified glycoproteins isolated from goldfish brain. The ependymins were shown to constitute 14% of the total protein content of the brain extracellular fluid and also to be present as a minor component of the serum proteins (0.3%). Ependymins β and γ have an immunological reactivity in these fractions that can be increased by a factor of 30 on heating. The data suggest that the antigenicity of the molecules is highly masked, and that it may require some unraveling of the quaternary structure of the proteins before maximal interaction with the antisera becomes possible.     

The Metabolism of Serum Proteins in Neonatal Rabbits

1. Incorporation of S³⁵-labeled amino acids into serum proteins has been studied in neonatal and developing rabbits. It was found that, per unit weight, neonatal rabbits synthesized only about 1/36 of the gamma globulin, 1/7 of the beta globulin, ½ of the alpha globulin, and ⅛ of the albumin that an adult synthesized. The growing rabbit developed the ability to synthesize various serum proteins at different times. 2. Plasma volumes and serum protein concentrations were determined at different times during the growth period of the rabbit. Plasma volumes were found to be 1 and ½ times larger in newborn animals than in adults, with a gradual decline to the adult level. The total serum protein concentration at birth was about 60 to 65 per cent of the adult value and gradually increased with growth as the plasma volume decreased. 3. Half-lives of homologous albumin and gamma globulin were studied. The half-life of albumin in neonates was nearly twice as long as the half-life in adults, the latter value being reached at 1 month of age. The half-life of gamma globulin in neonates was more than twice as long as the half-life in adults and reached adult values at 2 to 3 months. 4. Attempts were made to alter serum protein metabolism. Gamma globulin synthesis early in life was augmented with antigen injections.     

Involvement of alpha5beta1 integrins in interleukin 8 production induced by oral viridans streptococcal protein I/IIf in cultured endothelial cells

Using human endothelial cells, we define a mechanism that accounts for the induction of interleukin 8 (IL-8) by protein I/IIf, an adhesin from Streptococcus mutans serotype f. We report that protein I/IIf interactions with endothelial cells increased the tyrosine phosphorylation of three cellular components with relative mass of 145 000, 125 000 and 70 000 in endothelial cells. These proteins were identified as phospholipase Cγ (PLCγ), focal adhesion kinase (FAK) and paxillin after immunoprecipitation with monoclonal antibodies (mAbs) and immunoblotting with antiphosphotyrosine mAbs. These results suggested that β1 integrins could be one of the components implicated in the modulin activity of protein I/IIf. By incubating protein I/IIf with either purified α5β1 integrins or with α5β1 integrins overexpressing CHO cells, we demonstrated that α5β1 integrins act as cell receptors for protein I/IIf. We also showed that protein I/IIf interactions with α5β1 integrins lead to IL-8 secretion. Using specific inhibitors, we demonstrated that protein I/IIf-induced IL-8 release involves mitogen-activated protein kinases (MAPKs), and that PLCγ and PKC also seem to contribute to protein I/IIf stimulation. However, PI-3K activation is not involved in IL-8 release. Altogether, these results indicate that, after binding to α5β1 integrins, protein I/IIf induces IL-8 release by activating the MAPKs signalling pathways.     

The immune response of the brown trout Salmo trutta to lipopolysaccharide

Brown trout produced high molecular weight, thermostable, dithiothreitol sensitive, non-precipitating, complement-fixing antibodies and agglutinins to lipopolysaccharides after intramuscular injection with adjuvant. Antibodies were first detected on Day 14 and reached maximum titres after 56 to 63 days when a single injection was given. When either a second or a third injection was administered maximum titres occurred 34 to 40 days after the injection. After each injection the titres increased significantly, and the protein concentration of the sera was significantly decreased. In cellulose acetate electrophoresis experiments those bands which migrated in the β- to γ-globulin regions were increased.
Antibody-secreting and antigen-binding cells were detected on Days 8 and 4 respectively and maxima were reached between Day 16 and Day 18. The number of cells per 10⁶ lymphoid cells was higher in the spleen than in the kidney.     

Chemical classification of cattle. 1. Breed groups

From approximately 1000 papers with data on protein polymorphism in some 216 breeds of cattle, 10 polymorphic proteins were compared in means and variances of gene frequencies (arcsin p½) for ten well-recognized breed groups for 196 of the breeds. The polymorphic proteins were α-lactalbumin, β-lactoglobulin, caseins (α_sl, β and x), serum albumin, transferrin, haemoglobin, amylase I and carbonic anhydrase II. The breed groups were North European, Pied Lowland, European Red brachyceros, Channel Island brachyceros, Upland brachyceros, primigenius-brachyceros mixed, primigenius, Indian Zebu, African Humped (with Zebu admixture), and African Humped (Sanga).
The coherence within groups and the differences between groups are often impressive. Only carbonic anhydrase II fails to differentiate at least some of the major breed groups.
In some cases paradoxical distributions of rare genetic variants can be explained by a more detailed inspection of breed history.
The chemical data support the morphological and geographical divisions of cattle into major breed groups. There are three distinct but related brachyceros groups; for some polymorphisms the two Channel Island breeds, the Jersey and the Guernsey, are quite divergent. Although some authorities have considered the Pied Lowland as primigenius, it is a very distinct breed group.     

PRESENCE OF THREE ACTIN TYPES IN SKELETAL MUSCLE OF CHICK EMBRYOS

The types of actin present during development of skeletal muscle in chick embryos were investigated by various electrophoretic methods. The proportions of the actin types in the tissue were estimated by a novel method involving separation of actin bands by electrophoresis, staining of the bands with Coomassie Brilliant Blue R–250, extraction of dye and measurement of its absorbance.
Results showed that α-, β- and γ-actins were all present in embryonic skeletal muscle and were assembled into myofibrils. However, β- and γ-actins disappeared from myofibrils as muscle development proceeded. In embryonic muscle, the proportions of the three actin types in the soluble and myofibril fractions were different: their amounts were in the order β>γ>α in the soluble fraction, and α>β>γ in the myofibril fraction.