Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: IV. Masculinization of the spinal nucleus of the bulbocavernosus with testosterone metabolites

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. SNB motoneurons and their perineal target muscles are present in adult males but reduced or absent in females. This sexual dimorphism is due to the presence of androgen during development; females treated with testosterone (T) perinatally have a masculine SNB system. To assess whether masculinization of the SNB could involve the conversion of testosterone into its active metabolites, dihydrotestosterone (DHT) and estrogen, we examined the development of the SNB in females treated perinatally with estrogen alone or in combination with dihydrotestosterone. Counts of motoneurons in the developing SNB in all groups showed the typical prenatal increase followed by a differential postnatal decline; the incidence of degenerating cells reflected this decline. Motoneuron numbers and the frequency of degenerating cells in females treated with estrogen (E) alone did not differ from those of normal females, with both groups losing large numbers of motoneurons and having a high incidence of degenerating cells. In contrast, females treated with both estrogen and dihydrotestosterone did not show the female-typical decline in motoneuron number and had a low, masculine incidence of degenerating cells. By postnatal day 10, females treated with estrogen and dihydrotestosterone had a fully masculine SNB motoneuron number, suggesting that dihydrotestosterone alone or in conjunction with estrogen may be involved in the development of the sexually dimorphic SNB system.     

A comparative study of bark lectins from three elderberry (Sambucus) species

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

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.