Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins

A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that 1) its apparent molecular weight is not changed by reduction and alkylation; 2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; 3) binding of lipoproteins is not inhibited by EDTA; and 4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins. It is unlikely that this protein ever binds lipoproteins in vivo; however, its lipoprotein binding activity has facilitated its purification to homogeneity and suggests that this protein has unusual structural features. The role of the 165-kDa protein in Ca2+ homeostasis in the sarcoplasmic reticulum, if any, remains to be determined.     

Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins

Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.     

Immunohistochemical localization of bursin in epithelial cells of the avian bursa of Fabricius

Antibodies to the avian B-cell-differentiating hormone bursin (lysyl-histidyl-glycine amide) were raised in mice and rabbits by immunizing with bursin conjugates in Freund's adjuvant. Immunohistochemical staining with these bursin-specific antibodies was restricted to follicular and dendritic reticular epithelial cells of the bursa of Fabricius, and was not found in control avian tissues.     

Molecular basis for a polymorphism involving Fc receptor II on human monocytes

IgG Fc receptor II (Fc gamma RII) on human monocytes is polymorphic with respect to its appearance on gels after isoelectric focusing and with respect to its ability to mediate T lymphocyte proliferation induced by murine anti-CD3 mAb of the IgG1 isotype (i.e., its ability to bind murine IgG1). To determine the molecular basis for this polymorphism, we isolated total cellular RNA from PBMC of responders and nonresponders (defined by Leu-4-induced [3H] thymidine incorporation) and synthesized corresponding cDNA. Sequences encoding the extracellular domain of Fc gamma RII were then amplified using the Taq polymerase chain reaction. Amplified DNA fragments were cloned into pUC vectors, and sequenced. Analysis of clones from two nonresponders revealed a single base change (G for A) at position 519, which would result in the substitution of a histidine for an arginine at residue 133 in the mature Fc gamma RII protein. These findings suggest that the polymorphism involving human monocyte Fc gamma RII results from allelic variation of a single gene.     

Prolonged changes in plasma concentrations of catecholamine metabolites following a single infusion of an MPTP analog

The magnitude and duration of effects of a single intravenous injection of 4'-amino MPTP, an analogue of the dopamine neurotoxin, MPTP, on plasma levels of catechols and normetanephrine were examined in conscious dogs. Plasma samples were collected prior to treatment with intravenous saline or 4'-amino MPTP.2HCl (22.5 mg/kg) and at weekly intervals for six weeks following treatment. Saline treatment had no effect on plasma levels of any of the measured compounds. Following 4'-amino MPTP, plasma DHPG fell to 14% of the pre-injection value and remained decreased for the full 6-week test period, with partial recovery by week 6 to 42% of the pre-injection value. Plasma DOPAC levels fell to 28% of pre-injection values 1 week after treatment with 4'-amino MPTP and showed no evidence of recovery during the 6-week test period. Plasma DOPA fell to 58% of the pre-injection level, while concentrations of the catecholamines NE, EPI, and DA were generally unaffected. The plasma concentration of the O-methylated NE metabolite, normetanephrine, was also unchanged by 4'-amino MPTP treatment. There were no differences in the concentrations of DA, NE or EPI within the adrenal medulla between saline and 4'-amino MPTP treated groups. This pattern of changes in plasma levels of catechols, which is consistent with presynaptic inhibition of MAO within sympathetic terminals, may be a useful indicator of exposure to MPTP-like neurotoxins.     

The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.     

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.     

Probabilistic reconstruction of ancestral protein sequences

Using a maximum-likelihood formalism, we have developed a method with which to reconstruct the sequences of ancestral proteins. Our approach allows the calculation of not only the most probable ancestral sequence but also of the probability of any amino acid at any given node in the evolutionary tree. Because we consider evolution on the amino acid level, we are better able to include effects of evolutionary pressure and take advantage of structural information about the protein through the use of mutation matrices that depend on secondary structure and surface accessibility. The computational complexity of this method scales linearly with the number of homologous proteins used to reconstruct the ancestral sequence.     

Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.