Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein

SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation.     

An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

A histological study of the hypophysial-gonadal system during sexual maturation and spawning in the milkfish, Chanos chanos (Forskal)

The pituitary gland of the milkfish, Chanos chanos , was studied at different stages of sexual maturation and spawning. Consecutive median sagittal sections were treated with a range of stains to demonstrate the different cell types and regions. The milkfish pituitary consists of a neural component, the neurohypophysis, and an epithelial component, the adenohypophysis, which in turn consists of three regions: the rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). However, unlike most teleosts, the pituitary gland of the milkfish is encased in a bony chamber, has dorsal and ventral lobes and extends anteriorly from its point of origin at the base of the brain. PAS (+) basophils are found in all regions of the adenohypophysis, but mostly in the proximal pars distalis. These cells undergo hypertrophy and hyperplasia during sexual maturation, shrinkage and degranulation during spawning.     

A comparative study of the tapetum, retina and skull of the ferret, dog and cat

Results of this investigation indicate that the ferret (Mustela putorius) closely resembles the dog (Canis familiaris) and should be a useful research animal alternative. The tapetum lucidum is a common structure present in the eyes of dogs, cats (Felis catus) and other nocturnal animals. Our study showed that this structure was present in ferret eyes. The color or reflection of the ferret and dog tapetum was remarkably reduced by the general fixation with glutaraldehyde. However, this color fading phenomenon was not observed in the cat tapetum. These observations led to this comparative study on several morphological, histochemical and biochemical parameters on mature ferrets, dogs and cats including: (1) the number of center tapetum cell layers, (2) thickness of center tapetum, (3) presence of a microtubule-like structure in each tapetal rod, (4) presence of electron-dense cores in tapetal rods after prolonged fixation in glutaraldehyde, (5) retention of reflection or color of tapetum after prolonged glutaraldehyde fixation, (6) zygomatic bones of eye orbits, (7) zinc content in tapetum, (8) cysteine in the tapetum, (9) cysteine sulfinic acid decarboxylase in liver, (10) thickness of retina from center tapetum, (11) anterior view of skull configuration, and (12) lateral view of skull configuration (jaw and teeth). Among these 12 parameters, ferret and dog were similar in the first nine points; ferret and cat were similar to each other only in the last two points. There was no difference in retinal thickness among these three animals.     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

Effects of exercise on insulin binding and glucose metabolism in muscle

To elucidate the mechanism of enhanced insulin sensitivity by muscle after exercise, we studied insulin binding, 2-deoxy-D-[1-14C]glucose (2-DOG) uptake and [5-3H]glucose utilization in glycolysis and glycogenesis in soleus and extensor digitorum longus (EDL) muscles of mice after 60 min of treadmill exercise. In the soleus, glycogenesis was increased after exercise (P less than 0.05) and remained sensitive to the action of insulin. Postexercise insulin-stimulated glycolysis was also increased in the soleus (P less than 0.05). In the EDL, glycogenesis was increased after exercise (P less than 0.05). However, this was already maximal in the absence of insulin and was not further stimulated by insulin (0.1-4 nM). The disposal of glucose occurred primarily via the glycolytic pathway (greater than 60%) in the soleus and EDL at rest and after exercise. The uptake of 2-DOG uptake was not altered in the soleus after exercise (4 h incubation at 18 degrees C). However, with 1-h incubations at 37 degrees C, a marked increase in 2-DOG uptake after exercise was observed in the soleus (P less than 0.05) in the absence (0 nM) and presence of insulin (0.2-4 nM) (P less than 0.05). A similar postexercise increase in 2-DOG uptake occurred in EDL. Despite the marked increase in glucose uptake and metabolism, no changes in insulin binding were apparent in either EDL or soleus at 37 degrees C or 18 degrees C. This study shows that the postexercise increase of glucose disposal does not appear to be directly attributable to increments in insulin binding to slow-twitch and fast-twitch muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.     

The significance of the aprotic solvent in monomer studies related to the primary subunit environment in the macromolecule

An advantage of aprotic polar solvent systems in the study of monomer interactions relevant to the macromolecular state is demonstrated with the measurement of nucleoside amino proton exchange rates in DMSO/water mixtures. The DMSO/water solvent provides the first unequivocal observation of general acid catalysis of nucleic acid amino proton exchange, which is undetectable in aqueous solution due to the formation of the endocyclic protonated nucleobase. Suppression of nucleobase protonation in the presence of buffer acid is a consequence of anion desolvation in the aprotic solvent. The detected route of general acid catalysis is demonstrated as a consequence of Watson-Crick H-bonding, leading to the implication that amino chemistry is modulated in the helical state to decrease amino proton lifetime in the closed macromolecular context of conformational information obtained by hydrogen exchange methods. This useful property of the aprotic solvent can be extended to monomeric studies pertaining to specific local site interactions affecting the function and conformation of proteins and nucleic acids.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.