The matrix metalloprotease matrilysin (PUMP) is expressed in developing human mononuclear phagocytes. Matrilysin (PUMP, MMP-7) is a member of the metalloprotease gene family, whose constituents are responsible for the remodeling of extracellular matrix. The matrilysin protein is a 28-kDa zymogen possessing catalytic activities against a broad range of extracellular matrix substrates including proteoglycans, gelatin, fibronectin, laminin, and elastin. To gain insights into the biological expression of matrilysin in human cell types, we generated a monospecific, polyclonal antibody against a 16-amino acid sequence derived from its catalytic domain, a region which lacked significant homology with other matrix metalloenzymes. We found this antibody capable of precipitating a 28-kDa protein from the conditioned media of human bone marrow-derived promonocytes and human peripheral blood monocytes cultivated in vitro. Promonocyte matrilysin was rapidly converted to a 19-kDa form by organomercurial activation. While matrilysin was constitutively synthesized by bone marrow-derived promonocytes, its secretion was markedly up-regulated by the mononuclear phagocyte activator, lipopolysaccharide. Furthermore, despite its expression in monocyte precursors, blood monocytes, and monocyte-derived macrophages, matrilysin was not synthesized by human alveolar macrophages under any tested condition. In situ hybridization studies with matrilysin cRNA confirmed the presence of specific mRNA in both human promonocytes and monocytes. Moreover, a marked increase in hybridizable mRNA was observed with lipopolysaccharide treatment suggesting that matrilysin synthesis is pretranslationally regulated. In summary, this represents the first report documenting constitutive and regulated synthesis of matrilysin by a normal human cell type and suggests that matrilysin is expressed as a significant secreted product of mononuclear phagocytes at an intermediate stage of cellular differentiation.


The human leukocyte platelet-activating factor receptor. cDNA cloning, cell surface expression, and construction of a novel epitope-bearing analog. A human myeloid transcript of approximately 4 kilobases was cloned as a cDNA from an expression library based on homology with the guinea pig cDNA recently described by Honda et al. (Honda, Z-I., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Tok, H., Ito, K., Miyamato, T., and Shimizu, T. (1991) Nature 349, 342-346) as a receptor for platelet-activating factor (PAF). The cloned DNA confers high affinity binding sites for platelet-activating factor when transfected into COS-7 cells and has binding and desensitization properties similar to the human leukocyte receptor. Southern analysis using this cDNA indicates that the PAF receptor gene is present as a single copy in the human genome. The deduced protein sequence predicts seven hydrophobic regions for the PAF receptor, characteristic of the rhodopsin gene family, and is 83% identical to the deduced protein sequence of the corresponding guinea pig molecule. A modified human PAF receptor cDNA was constructed by inserting an additional 30 nucleotides after the 5'-ATG, encoding the amino acid sequence MDYKDDDDKEF, which is specifically recognized by a monoclonal antibody. The modified cDNA encodes a functional PAF receptor and is detected by antibody on the membrane of transfected COS-7 cells. The use of this construct supports the structural model for the rhodopsin-like superfamily of receptors which places the NH2-terminal sequence on the extracellular side of the membrane, and should additionally be useful for affinity purification of the receptor protein.


Human cysteine-rich protein. A member of the LIM/double-finger family displaying coordinate serum induction with c-myc. We previously reported the structure of the placentally derived human cysteine-rich (h crp) cDNA and demonstrated that it encodes a highly conserved and widely distributed zinc finger-like protein. We now report that the expression of both the mouse and human crp genes is induced as a primary response to serum in quiescent Balb/c 3T3 cells and in human fibroblasts. The profile of this primary response is remarkably parallel to that of c-myc in the Balb/c 3T3 cell line. The structure of the 23.2-kilobase h crp gene demonstrates that it is a member of a gene superfamily encoding proteins sharing a highly characteristic 52-amino acid "LIM/double-finger" motif. The evolutionarily conserved structure of cysteine-rich protein, its structural similarity to a number of developmentally critical proteins, its distinctive tissue distribution, and its primary response to early events in the cell cycle suggest that crp plays an important role in cell function.


The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits. Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.


Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue. A cDNA for human adipsin was isolated and shown to encode a protein sharing 98% amino acid sequence similarity with the protein sequence previously determined for purified natural human complement factor D. Like mouse adipsin, recombinant human adipsin displays the enzymatic activity of human complement factor D, cleaving complement factor B only when B is complexed with activated complement component C3. We conclude that human adipsin is equivalent to complement factor D and that adipsin is the homologue of factor D in rodents. Adipose tissue is a major site of synthesis of human adipsin/complement factor D mRNA, but unlike the case in rodents, human adipsin mRNA is also expressed in monocytes/macrophages. The data presented here, demonstrating the equivalence of human adipsin to complement factor D and its high level of expression in fat, suggest a previously unsuspected role for adipose tissue in immune system biology.


Beta 1,4-galactosyltransferase: a short NH2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention. Beta 1,4-galactosyltransferase (beta 1,4-GT) is a Golgi-resident, type II membrane-bound glycoprotein that functions in the coordinate biosynthesis of complex oligosaccharides. Additionally, beta 1,4-GT has been localized to the cell surface of a variety of cell types and tissues where it is proposed to function in intercellular recognition and/or adhesion. Thus beta 1,4-GT is an appropriate molecule to be used in analyzing the molecular basis for retention of a membrane-bound enzyme in the Golgi complex and its subsequent or alternative transport to the cell surface. Previously we have shown that the gene for bovine and murine beta 1,4-GT is unusual in that it specifies a short (SGT) and long (LGT) form of the enzyme (Russo, R. N., Shaper, N. L., and Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331). The only difference between the two related forms is in the primary structure of the cytoplasmic domains, where LGT has an NH2-terminal extension of 13 amino acids. In this study, we have tested the hypothesis that LGT and SGT are differentially retained in the Golgi or directed to the cell surface. LGT, SGT or chimeric proteins, containing the NH2-terminal cytoplasmic and transmembrane domain of SGT and LGT fused to the cytoplasmic protein pyruvate kinase, were each stably expressed in Chinese hamster ovary cells. Proteins expressed from each construct were localized by immunofluorescence staining exclusively to a perinuclear region, identified as the Golgi by co-localization with wheat germ agglutinin. Furthermore, the subcellular distribution of both SGT and LGT was restricted to the trans-Golgi compartment as assessed by EM immunoelectron microscopy. These data suggest that both forms of beta 1,4-GT are resident trans-Golgi proteins and that an NH2-terminal segment containing the cytoplasmic and transmembrane domains of SGT (39 amino acids) or LGT (52 amino acids) is sufficient for Golgi retention.


A cardiac troponin T epitope conserved across phyla. Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.


Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme. cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.


Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3). We previously cloned, sequenced, and expressed two distinct mammalian Na+/H+ exchanger isoforms (NHE-1 and NHE-2). We report here the cloning of a composite cDNA which encodes a third mammalian isoform (NHE-3), which is expressed specifically in intestine and kidney. The protein deduced from the longest open reading frame of this composite sequence has 832 amino acids with a calculated Mr of 92,747. The hydrophobicity plot of NHE-3 is very similar to that of NHE-1 and NHE-2. NHE-3 is also predicted to have 10-12 membrane-spanning domains and a long cytoplasmic domain which contains putative protein kinase phosphorylation motifs. NHE-3 exhibits overall 41% amino acid identity with NHE-1. NHE-3 is likely a glycoprotein as it has one potential N-linked glycosylation site, which is conserved in all NHEs identified. Northern blot analysis of poly(A+) RNA isolated from rabbit ileum using NHE-3 cDNA as a probe hybridized to a single 5.4-kilobase transcript. More detailed tissue distribution of message was performed by ribonuclease protection assay. It was found that NHE-3 message is only expressed in intestine and kidney, with the kidney cortex having the most abundant message, followed by intestine and kidney medulla. In intestine, ileum and ascending colon have the same amount of message, with much lesser amounts in jejunum. The message is absent from duodenum and descending colon, which lack the neutral NaCl absorptive process. Thus, NHE-3 might be involved in Na+ absorption in intestinal and renal epithelial cells.


3-Deazaadenosine inhibits thrombin-stimulated platelet-derived growth factor production and endothelial-leukocyte adhesion molecule-1-mediated monocytic cell adhesion in human aortic endothelial cells. Injury to the vascular endothelium and the subsequent inflammatory response are considered prerequisites for the development of atherosclerosis. Platelet-derived growth factor (PDGF) production by and monocyte adhesion to aortic endothelial cells (EC) may participate in this inflammatory process and therefore are two potential targets for control by anti-inflammatory agents. Our previous studies have demonstrated that monocyte adhesion and PDGF production are stimulated by thrombin in EC. Here, we provide evidence that treatment of EC with the anti-inflammatory agent 3-deazaadenosine (c3Ado) effectively abolished thrombin-stimulated PDGF production and monocyte adhesion. c3Ado had no significant effect on either basal monocyte adhesion or constitutive PDGF production. c3Ado was also effective in negating monocyte adhesion induced by other agonists, such as interleukin-1, phorbol 12-myristate 13-acetate (PMA), and lipopolysaccharide. Northern analysis demonstrated that c3Ado significantly reduced thrombin- and PMA-stimulated steady-state levels of PDGF-A chain, PDGF-B chain, and endothelial-leukocyte adhesion molecule-1 (ELAM-1) mRNAs. Nuclear run-on studies demonstrated that a marked transcriptional activation of these genes by thrombin and PMA was abrogated by c3Ado treatment. The transcriptional rate of the alpha-tubulin gene was unaffected by the drug. Antibody binding studies with an anti-ELAM-1 monoclonal antibody 7A9 revealed that thrombin-stimulated EC expression of ELAM-1 was abolished by c3Ado, indicating that the suppression of ELAM-1 expression on EC surface may be a mechanism by which c3Ado interferes with monocyte adhesion. Experiments with the nucleoside transport inhibitor nitrobenzylthioinosine suggested that the transport of c3Ado into EC was required for its inhibitory activity. In addition, L-homocysteine thiolactone was found to potentiate the inhibitory activity of c3Ado, suggesting that the accumulation of intracellular c3Ado homocysteine may be the underlying mechanism by which c3Ado inhibits thrombin-induced EC function. Taken together, these results indicate that c3Ado may prove effective against vascular injury and inflammation through its ability to inhibit induction of both monocyte adhesion and PDGF production.


Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells. To determine if selective activation of individual isozymes of protein kinase C (PKC) might explain the apparently divergent effects of PKC stimulation on platelets, we purified and characterized the isozymes from both platelets and human erythroleukemia (HEL) cells, a cell line that has many features of megakaryocytes. Two peaks of platelet PKC activity were resolved by hydroxylapatite chromatography; immunoblot analysis revealed that these two peaks represented the alpha and beta isozymes of PKC. In contrast, HEL cells produced only a single peak that contained the beta isozyme. None of the other PKC isozymes were detected in these fractions. The cytosol of platelets and HEL cells, however, were both found to contain the PKC-delta isozyme. Northern hybridization analyses and mRNA amplification by the polymerase chain reaction demonstrated the presence of mRNA encoding the alpha, beta, and delta PKC isozymes in platelets, but only the beta and delta isozymes in HEL cells. Phorbol myristate acetate (PMA), thrombin, or an endoperoxide analog induced the phosphorylation of the 47-kDa substrate of PKC (pleckstrin) found in platelets and HEL cells; preincubation of either HEL cells or platelets with PMA reduced the intracellular Ca2+ rise induced by thrombin. Thus, although both HEL cells and platelets contain PKC-beta and the recently described PKC-delta isozymes, the widely distributed alpha isozyme of PKC is absent in HEL cells; however, isozymes other than PKC-alpha are sufficient for some PMA-mediated functions that are similar to those seen in stimulated platelets.


Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains. Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.


Differential trans-activation of muscle-specific regulatory elements including the mysosin light chain box by chicken MyoD, myogenin, and MRF4. We have isolated cDNAs encoding a chicken homologue of MRF4 (cMRF4) in addition to chicken MyoD (CMD1) and myogenin (c-myogenin) described previously. In an attempt to understand the roles that cMRF4, CMD1, and c-myogenin play in chicken myogenesis, the effects of these factors on muscle-specific cis-elements identified in regulatory regions of myosin alkali light chain (MLC) genes were examined. The promoter analysis of some of MLC genes has revealed two sorts of muscle-specific positive regulatory elements to date, an enhancer located upstream of the adult type LC1 gene and a cis-element, termed an MLC box, conserved among promoters of various MLC genes. The LC1 enhancer was exclusively trans-activated by CMD1. Although c-myogenin also activated transcription driven by the LC1 promoter, it was suggested that c-myogenin requires a cis-element(s) other than the CMD1-responsive enhancer. Chicken MRF4 could not trans-activate any of the constructs containing the LC1 promoter. In contrast, the promoter of the embryonic L23 gene was trans-activated by all of the three factors. From deletion and mutation analysis, the MLC box was shown to be involved in their positive regulation. These results extend previous observations that individual myogenic regulatory factors exhibit different capabilities in transcriptional activation of muscle-specific genes by acting distinctively upon their regulatory elements.


Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase. In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.


Characterization of an associated microfibril protein through recombinant DNA techniques. The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.


Alterations in mRNA translation as a mechanism for the modification of enzyme synthesis during evolution. The ornithine decarboxylase model. The expression of renal ornithine decarboxylase (ODC) is very different between the two murine species Mus domesticus and Mus pahari. The latter species contains a reduced level of ODC protein in the kidney, yet a normal concentration of ODC mRNA, indicating an alteration in translation, protein degradation, or both. We describe two lines of experimental evidence indicating that the efficiency of ODC mRNA translation is decreased in M. pahari. First, in vivo measurements showed that the rate of biosynthesis of the ODC polypeptide is reduced in M. pahari relative to M. domesticus. Second, polysome analyses indicated that, on average, the ODC mRNA of M. pahari binds fewer ribosomes. These effects, which are kidney-specific, lend support to the notion that renal ODC expression in the two species differs at the level of mRNA translation. A full-length cDNA copy of the ODC mRNA of M. pahari was isolated, and its sequence was compared with that of M. domesticus. In the 5'-untranslated region, which is likely to play an important role in controlling translation, the M. pahari transcript contains several single-base changes, as well as a 12-base deletion. These changes confer a distinct predicted secondary structure to this region of the mRNA and may be involved in reduced translation. We conclude that during evolution, modifications that alter the biosynthesis of specific proteins can occur at the translational level; these modifications exert their effects on translation in a transcript- and tissue-specific manner.


Regulation of the expression of pp160, a putative insulin receptor signal protein, by insulin, dexamethasone, and 1-methyl-3-isobutylxanthine in 3T3-L1 adipocytes. pp160, a cytosolic protein with Mr of approximately 160,000, is phosphorylated on tyrosine in response to insulin and is considered to be involved in signaling from the insulin receptor. The expression of pp160 during the differentiation of 3T3-L1 fibroblasts to adipocytes and in adipocytes has been investigated using quantitative immunoblotting with antibodies against a peptide from pp160. Between day 6 and day 8 of differentiation induced by insulin, dexamethasone (Dex), and 1-methyl-3-isobutylxanthine (Mix), pp160 expression increased 10-20-fold over the amount present in confluent fibroblasts. Omission of either insulin or Dex resulted in reduced expression of pp160 and in incomplete adipogenesis. Chronic treatment of fully differentiated adipocytes for 24 h with either insulin, Dex, or Mix alone in the presence of serum resulted in a decrease in the expression of pp160 by 70-85%. Chronic exposure to insulin caused a significant increase in the apparent size of pp160 to 172 kDa. Alkaline phosphatase treatment lowered the Mr of pp160 from both insulin-treated and basal cells to 150,000. These results demonstrate that pp160 is expressed in 3T3-L1 adipocytes during the time when insulin receptors are expressed in large numbers and that the maintenance of pp160 concentrations in adipocytes can be regulated by insulin, Mix, and Dex. The decreased expression of pp160 caused by these factors may be related to postreceptor insulin resistance.


Receptor-mediated uptake and processing of vitamin D-binding protein in human B-lymphoid cells. Vitamin D-binding protein (DBP), a member of a multigene family including alpha-fetoprotein (AFP) and albumin, is a serum glycoprotein that reversibly binds and transports vitamin D and its metabolites to target cells. In this work, we demonstrate that normal and malignant human B-lymphocytes specifically bind and internalize DBP. Radioiodinated DBP (125I-DBP) was used to follow the uptake of the protein by Raji cells, a human pre-B-lymphoma cell line. Time course studies of DBP uptake by these cells exhibited a saturable profile at both 4 and 37 degrees C. The binding saturation curve obtained by incubating Raji cells at 4 degrees C with different concentrations (1.5 nM to 1.5 microM) of 125I-DBP showed two saturation plateaus; Scatchard analysis showed the presence of two groups of receptor sites with a Kd1 of 2.04 x 10(-7) M (n1 = 42,161 +/- 4,336 sites/cell) and a Kd2 of 1.01 x 10(-6) M (n2 = 198,000 +/- 48,000 sites/cell). After incubation of Raji cells at 37 degrees C with both fluorescein isothiocyanate (FITC) and horseradish peroxidase conjugates, DBP was internalized and could be localized in the cytoplasm. DBP-horseradish peroxidase conjugates were used to follow the uptake and to determine the endocytic pathway of the protein in Raji cells. The initial steps, contrary to those observed for AFP, did not apparently involve coated pits and vesicles. Small vesicles (approximately 50-60 nm) with electron-dense DBP-horseradish peroxidase reaction products were observed that could fuse with large endosomes. These endosomes appeared dispersed in the cytoplasm with some preferential localization in the Golgi centrosphere region. Pulse-chase experiments showed that only 10% of the uptaken protein was released in a nondegraded form. Accordingly, most DBP molecules accumulated in endosomes should be degraded in lysosomes, instead of being recycled back to the surface, as in the case of AFP. Contrary to malignant B-cells (Raji), the uptake ability for DBP of normal quiescent B-lymphocytes was very low. Specific binding and internalization of DBP-FITC by these cells were observed following mitogen-induced activation. Significant values of uptake were obtained at 37 degrees C after 72 h of incubation in the presence of pokeweed mitogen. The binding of DBP-FITC was partially inhibited in the presence of an excess of unlabeled protein. Taken together, the actual results suggest that DBP receptors are constitutively expressed by malignant B-cells and in a transitory form by normal B-lymphocytes undergoing mitogen-induced activation.(ABSTRACT TRUNCATED AT 400 WORDS)


Cloning and expression of a novel angiotensin II receptor subtype. Angiotensin II (AII) is a major regulator of cardiovascular function and fluid homeostasis. Recently, the cDNA for an AII receptor (AT1) was cloned from rat smooth muscle and bovine adrenal. To search for AII receptor subtypes, we amplified rat adrenal cortex cDNA by PCR using primers based on the AT1 receptor. The product was distinct from the AT1 receptor as indicated by restriction enzyme analysis and DNA sequencing. A full-length cDNA clone (2.2 kilobase pairs) encoding a novel AII receptor (AT3) was obtained by screening an adrenal cortex library. The AT3 cDNA encodes a Mr 40,959 protein with 95% amino acid identity to the rat smooth muscle receptor, but the overall nucleotide similarity is 71% due to low homology in the 5'- (58%) and 3'- (62%) untranslated regions. Expressed AT3 receptors in Xenopus oocytes and COS-7 cells mediate agonist-induced Ca2+ mobilization but are pharmacologically distinct from the AT1 receptors. AT3 mRNA is most abundant in the adrenal cortex and pituitary and differs from AT1 mRNA in its tissue distribution. The structural features of the AT3 receptor, including two additional potential phosphorylation sites for protein kinase C, could be related to the distinctive binding properties of the adrenal and vascular receptors and to their differential regulation during altered sodium intake.


Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers. Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.


FK506 binding protein associated with the calcium release channel (ryanodine receptor). The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.


Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3. Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.


Cloning and nucleotide sequence of the gene (citC) encoding a citrate carrier from several Salmonella serovars. The nucleotide sequence of the citC coding for the citrate carrier in several Salmonella serovars has been determined, and the amino acid sequence of the carrier protein was deduced. The predicted citrate carrier from Salmonella pullorum and Salmonella enteritidis consists of 446 amino acids with a molecular weight of 47,621, whereas that from Salmonella dublin is the same 446 amino acids with the slightly different molecular weight of 47,591, because 1 amino acid residue was substituted. The predicted proteins are highly hydrophobic (69% nonpolar amino acids). The hydropathy profile suggests that the proteins are composed of 11-12 hydrophobic membrane-spanning segments with two hydrophilic cores in the middle of the protein sequence. No homology in the nucleotide and amino acid sequences was found in the molecular structures of citA, citP, and tctI genes. The citC-coding citrate transport activity is Na(+)-dependent and specific for citrate only. The transcript from the citC gene was not detected in the total RNA from several Salmonella serovars except S. dublin in Northern blot analysis, although the promoter of the citC genes appeared to be functional in Escherichia coli and Salmonella typhimurium strains using the lacZ fusion assay. These results suggested that the citC gene-coding citrate carrier is probably a TctIII system such as that identified previously in S. typhimurium.


Role of sequences within the first intron in the regulation of expression of eukaryotic initiation factor 2 alpha. Resting human peripheral blood T cells synthesize proteins at very low rates and contain very low levels of eukaryotic initiation factor (eIF) 2 alpha mRNA. During mitogenic activation, the level of eIF-2 alpha mRNA increases at least 50-fold, an effect thought to be due primarily to intranuclear stabilization of the primary transcript (Cohen, R. B., Boal, T. R., and Safer, B. (1990) EMBO J. 9, 3831-3837). Analysis of sequences within the first intron revealed a region with homology to the "initiator" (Inr) sequence first described by Smale and Baltimore (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113). This Inr element is positioned 450 bases downstream of the eIF-2 alpha promoter and is oriented to generate an overlapping antisense transcript. Deletion or mutation of the Inr element results in a reproducible 5-8-fold increase in the activity of an eIF-2 alpha promoter-driven CAT reporter gene and a corresponding 2.5-fold decrease in activity of an antisense driven luciferase reporter gene in vivo in 293 cells. In vitro transcription analysis also reveals antisense transcripts which depend on an intact Inr element and whose 5' ends map to sequences surrounding the Inr consensus sequence. A potential role for double-stranded RNA generated by these overlapping divergent transcription units in the regulation of eIF-2 alpha gene expression in T cells is suggested.


The N terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids. Peptide 1-21, generated by cyanogen bromide cleavage of each of two highly purified components of human myelin basic protein, components 1 and 8, gave a series of peaks in the fast atom bombardment mass spectra with m/z 2299, 2327, 2355, 2383, and 2411, indicating additions of 42, 70, 98, 126, and 154 atomic mass units respectively with m/z 2327 and 2355 as the dominant species. The pentafluorobenzyl esters prepared from an acid hydrolysate analyzed by negative ion chemical ionization gas chromatography mass spectrometry confirmed that C6, C8, and C10 fatty acids were present. These data demonstrated (i) that the N terminus of a myelin basic protein is not simply acetylated but contains C2, C4, C6, C8, and C10 fatty acids with C4 and C6 as the dominant species, (ii) the two components studied (C-1 and C-8) showed different relative amounts of C2 and C8 in particular, and (iii) human myelin basic protein is the first protein to be reported with a complex N terminus consisting of several alkyl carboxylic acid species.


The high molecular weight Cat-301 chondroitin sulfate proteoglycan from brain is related to the large aggregating proteoglycan from cartilage, aggrecan. Monoclonal antibodies Cat-301 and Cat-304 recognize a neuronal cell surface-associated chondroitin sulfate proteoglycan (CSPG), which is expressed during critical periods of postnatal development in the mammalian central nervous system (CNS). In the present study we show that the CNS CSPG identified by Cat-301/304 is similar to aggrecan, the high molecular weight CSPG from cartilage. By Western blot analysis, cartilaginous tissues, which are rich sources of aggrecan, have a high concentration of a high molecular weight CSPG which is immunoreactive with Cat-301 and 304. The Cat-301 and 304 epitopes, however, are partially masked by chondroitin sulfate glycosamino-glycan and are unmasked by digestion of the antigen with chondroitinase ABC. Although the antigen from both cartilage and CNS can be purified by CsCl buoyant density gradient centrifugation, a standard technique for purifying aggrecan, most of the antigen from the CNS has a lower buoyant density than that of cartilage. This may be due, in part, to the paucity of keratan sulfate substitution on the CNS antigen compared with that of the cartilage antigen. Both the CNS and cartilage antigens bind to hyaluronic acid, a feature characteristic of aggrecan. The physiochemical, biochemical, and functional properties of the Cat-301/304 antigen from cartilage are identical to aggrecan. The CNS antigen is similar, but not identical, to the cartilage antigen, and may thus represent another member of the family of high molecular weight CSPGs which bind to and aggregate with hyaluronic acid.


Immunohistochemical demonstration of proliferating cell nuclear antigen in glioblastomas: pronounced heterogeneity and lack of prognostic significance. In order to evaluate a possible significance of the expression of proliferating cell nuclear antigen (PCNA) as clinically useful prognostic parameter, we retrospectively investigated a series of 40 glioblastomas by means of immunohistochemistry and compared the results to patient survival. All glioblastomas included in the study had been treated by operation, radiotherapy and intraarterial ACNU [3-(4-amino-2-methyl-5-pyrimidinylmethyl)-1-(2-chloroethyl)-1-nitr osourea] chemotherapy. Patient survival ranged from 2 months to 42 months (mean: 14.2 months). PCNA values varied widely, ranging from 0.5% to 75% (mean: 24.9%). Statistical analysis revealed no significant correlation between PCNA index and patient survival. Our study thus indicates that the expression of PCNA appears not to be a useful prognostic parameter for glioblastoma patients.


MACOP-B treatment in patients with Ki-1-positive large-cell anaplastic lymphoma. Nine adult patients with Ki-1-positive large-cell anaplastic lymphoma were treated with MACOP-B. Two suffered from relapsed disease and had previously received chemotherapy; a third patient had received a single dose of 100 mg/m2 cisplatin before initiation of MACOP-B. The stage of lymphoma was determined according to the Ann Arbor Conference criteria and was II in one, III in two and IV in six patients. All patients had constitutional symptoms. Five patients had achieved complete remission 4 weeks after termination of the protocol and there were two partial remissions. One patient died of massive pulmonary embolism during the 4th week of treatment; another patient, who had received MACOP-B as salvage therapy, died of progressive lymphoma 1 month after completion of the regimen. Maximal observed toxicities according to WHO were mucositis grade 3 (n = 3) and there were three cases with thromboembolic complications, including a fatal pulmonary embolism in a young patient. However, MACOP-B appears an effective, fairly well-tolerated and feasible therapy for patients with Ki-1-positive large-cell anaplastic lymphoma.


Comparative analysis of CA72-4, CA195 and carcinoembryonic antigen in patients with gastrointestinal malignancies. To establish further the clinical significance of the novel quantitative immunoradiometric assay system CA72-4 in patients with gastric and other digestive tract malignancies, the sera of a total of 208 subjects have been analysed and levels of carcinoembryonic antigen (CEA) and another promising new tumour marker, CA195, have been compared. Twenty patients had gastric (GC), 60 colorectal (CC), and 14 pancreatic carcinomas (CC); 94 patients had benign disorders, and 20 were healthy volunteers. CA72-4 elevations above normal (greater than 4 U/ml) were observed in 6 (30%) GC, 17 (28%) CC, and 8 (57%) PC patients. CA195 appeared more sensitive and was increased (greater than 10 U/ml) in 35% GC, 70% CC, and in 100% PC patients; CEA levels above normal (greater than 5 ng/ml) were noted in 35%, 45%, and 66% of patients respectively. CA72-4 had a rather high specificity and was increased in only 6/94 (6%) patients with benign diseases, whereas CA195 had a false positive rate of 23%, and CEA of 33%. Among 20 healthy donors, none had elevated levels of CA72-4 or CA195, but marginal elevations of CEA were noted in 3 smokers. Despite some advantage of the new tumour marker CA72-4 in terms of specificity, its value as a serodiagnostic test in gastrointestinal cancer patients seems inferior to that of CA195 and CEA.


The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide. The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.


Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle. The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.


Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen. FG human pancreatic carcinoma cells use integrin alpha v beta 5 as their primary vitronectin receptor since they fail to express integrin alpha v beta 3. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a beta 1 integrin-dependent manner. Transfection of FG cells with a cDNA encoding the integrin beta 3 subunit results in the surface expression of a functional integrin alpha v beta 3 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing beta 3 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with beta 3 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the beta 3 and beta 5 integrin subunits when associated with alpha v, promote distinct cellular responses to a vitronectin extracellular environment.


Regulation of alpha 2 beta 1-mediated fibroblast migration on type I collagen by shifts in the concentrations of extracellular Mg2+ and Ca2+. Extracellular Ca2+ can reverse the Mg(2+)-dependent, alpha 2 beta 1-mediated adhesion of WI38 human fibroblasts to type I collagen substrates. Affinity chromatography data also demonstrate that Ca2+ can specifically elute the fibroblast alpha 2 beta 1 integrin bound to type I collagen-Sepharose in Mg2+. In modified Boyden chamber migration assays, Mg2+ alone supports the alpha 2 beta 1-mediated migration of fibroblasts on type I collagen substrates, while Ca2+ does not. However, a twofold enhancement in migration was observed when combinations of the two cations were used, with optimal migration observed when the Mg2+/Ca2+ ratio was higher than one. Inhibitory mAbs directed against various integrin subunits demonstrate that these observed cation effects appear to be mediated primarily by alpha 2 beta 1. These data, together with reports that under certain physiological conditions significant fluctuations in the concentrations of extracellular Ca2+ and Mg2+ can take place in vivo, suggest that the ratio between these two cations is involved in the up- and downregulation of integrin function, and thus, may influence cell migratory behavior.


Development of low- and high-functioning autistic children. Aspects of developmental sequences and structures were assessed in low- and high-functioning autistic and non-autistic developmentally disabled children. Specific developmental issues examined included sequences, regressions and profiles. Classification into the high- or low-functioning groups was based on a full scale IQ cutoff of 50. In general, there were few differences in the sequences of development among the groups. However, the autistic children were more likely than non-autistic children to display developmental regressions and unevenness across developmental domains. These developmental peculiarities were more pronounced in the low- as compared to high-functioning autistic children. These findings are discussed with regard to issues of developmental processes, classification and autism.


Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum. We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.


Debate and argument: on modularity and development in autism: a reply to Burack. Burack (this issue) reopens questions first raised by my 1989a article in this Journal, on whether children with autism have a specific delay in the development of a theory of mind, or whether the data reflect deviance rather than delay. His paper is of considerable value in highlighting points for debate. In my response, I focus on three issues: First, the logical possibility that delay and deviance can occur together. Second, the evidence that autism may instantiate this possibility. Finally, the important question of the modularity of theory of mind.


High-performance liquid chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization. An on-column fluorometric derivatization method was developed for the determination of histamine and 1-methylhistamine (HMs) by high-performance liquid chromatography. The system for the derivatization consisted only of a commercially available single-plunger pump and a reversed-phase C18 column supported on synthetic polymer with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthalaldehyde as a derivatization reagent. It required no additional reaction system as for a post-column derivatization method. Injected HMs might be derivatized to a fluorophore on the inlet site of the high-performance liquid chromatographic column, followed by chromatography on the same column. Optimization of the on-column reaction conditions resulted in a simple and sensitive analytical method for the determination of HMs with excellent reproducibility and linearity of 0.05-5 micrograms/ml of both HMs. Application of this method to the determination of HMs in food samples resulted in a limit of quantification of 0.05 mg/100 g and in a greater than 95% overall mean recovery at a fortification of 0.1 mg/g of both HMs. This method was furthermore applicable to the determination of histamine released from rat peritoneal mast cells.


Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis. T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.


Epitope mapping of polyclonal and monoclonal antibodies against two alpha-bungarotoxin-binding alpha subunits from neuronal nicotinic receptors. Recently, cDNAs for alpha subunits of two different neuronal alpha-bungarotoxin-binding proteins (alpha BgtBP) were isolated from chick brain, designated alpha BgtBP alpha 1 and alpha BgtBP alpha 2. These are now also referred to as subunits alpha 7 and alpha 8, respectively. Expression studies in Xenopus oocytes have indicated that alpha 7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain alpha BgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences alpha 7(327-412) and alpha 8(293-435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequences are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified alpha BgtBPs and the fusion proteins indicates that sequence segments 290-420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured alpha BgtBPs isolated from chick brain also recognize subunit-specific sequential epitopes within the sequence segment 290-420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbs characterized in these studies will provide useful probes for further studies of alpha BgtBP structure and histological localization.


Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells. Myelin proteolipid protein (PLP) contains one established antigenic epitope within the 139-151 amino acid sequence, with encephalitogenic activity for SJL mice (PLP139-151). In the current study, the processing and presentation of PLP by subsets of splenic antigen-presenting cells (APC) were examined by comparing their capacity to stimulate PLP-responsive T-cell clones, two of which are specific for PLP139-151, and one which is not specific for this peptide. In order to study whether PLP requires processing before its presentation by APC, PLP-pulsed and fixed APC were shown to stimulate PLP-specific T cells. However, the addition of PLP to unpulsed, fixed APC resulted in the absence of T-cell stimulation, while the ability of these fixed APC to bind antigenic peptide and efficiently present it to T cells, was demonstrated by their ability to use a synthetic peptide for the stimulation of the T cells. In order to study potentially different processing efficiencies among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC activities were studied separately. The non-adherent (NAd) APC were unable to stimulate PLP139-151 specific T-cell clones with PLP as antigen. In contrast, a T-cell clone specific for a separate, but unidentified epitope on PLP was stimulated by NAd APC efficiently. In addition, stimulation of PLP139-151-specific T-cell clones by NAd APC did occur when the synthetic peptide instead of intact PLP was used as antigen, indicating a defect in PLP processing by the NAd APC.(ABSTRACT TRUNCATED AT 250 WORDS)


Virus-reactive and autoreactive T cells are accumulated in cerebrospinal fluid in multiple sclerosis. Elevated numbers of B cells--plasma cells secreting antibodies to measles and mumps virus, and to myelin associated glycoprotein (MAG), one of several putative myelin autoantigens--have previously been reported in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS), while it is unknown if corresponding T cell reactivities occur. We have defined the T cell reactivities to measles and mumps virus and to MAG in an immunospot assay which is based on the detection of secretion of interferon-gamma (IFN-gamma) by single cells upon stimulation with specific antigen in short term cultures. Patients with MS had higher numbers of MAG-reactive T cells in blood compared to controls, while no differences were observed for measles or mumps virus-reactive T cells. In CSF, elevated numbers of MAG-reactive T cells and also of measles- and mumps-reactive T cells were found in patients with MS compared to other neurological diseases. A strong accumulation of antigen-reactive T cells was observed in the MS patients' CSF compared to blood. The magnitude of these T cell reactivities did not correlate with clinical MS variables. The T cell repertoire in MS thus includes, besides myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein, also MAG and, in addition, measles and mumps virus. It is not clear whether these T cell reactivities accumulated in the CSF have importance for the pathogenesis of MS or reflect phenomena secondary to myelin damage, or result from both these alternatives.


Tolerance to acetylcholine receptor induced by AChR-coupled syngeneic cells. Intravenous administration of acetylcholine receptor (AChR)-coupled syngeneic spleen cells induced AChR-specific tolerance in Lewis rats. Injection of 20 x 10(6) AChR-coupled spleen cells resulted in a significant reduction of the antibody response to AChR when the animals were subsequently immunized with AChR (up to 66% inhibition). Tolerance induced by AChR-coupled cells was antigen-specific: challenge with AChR plus an unrelated antigen, ovalbumin (OVA), resulted in significantly reduced serum antibody titers to AChR but normal or enhanced titers to OVA, compared to controls. Lymphocytes from these rats showed substantially reduced proliferative responses in vitro to AChR, and normal or enhanced responses to OVA. To investigate the role of Ia antigens in tolerance induction, AChR was coupled to Ia-positive or Ia-negative spleen cells. Injection of AChR-coupled Ia-positive cells induced antigen-specific tolerance to AChR; challenge with AChR plus OVA resulted in reduced serum antibody titers and lymphoproliferative responses to AChR, and normal or enhanced titers and cellular responses to OVA. Injection of AChR-coupled Ia-negative cells did not induce tolerance to AChR. These results demonstrate that AChR, which is known to be highly immunogenic, can be rendered tolerogenic without denaturation or linkage to toxic substances. The possible mechanisms of tolerance induced by AChR-coupled spleen cells are discussed.


Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes. The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear cells (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.


Parents' developmental perceptions and expectations for their high-risk infants. This study examines the relationship between developmental outcomes of high-risk infants and parental perceptions and expectations. Parents of 209 consecutive high-risk infants were asked to provide their development interpretations and expectations before their infants received standard developmental assessments between April and October 1989. Moderate correlations between parents and professional assessments of motor and language skills were noted (p less than .05 to p less than .01). Most agreements occurred when infants were assessed as normal by professionals. Disagreements were common and occurred in all areas of development. These mismatches were not associated with gestational age at birth, neonatal complications, poverty, or estimates of parental experience. Professionals should take seriously any expressed developmental concerns by parents of high-risk infants. Expressed developmental concerns, however, cannot be relied on for developmental screening of high-risk infants.


Intracranial olfactory neuroblastoma: evidence for olfactory epithelial origin. To determine the possible histogenesis of the intracranial variant of olfactory neuroblastoma. Four specimens from three cases of intracranial olfactory neuroblastoma were studied by light microscopy and immuno-histochemistry, and electron microscopy in two cases. Light microscopical examination showed small cell tumour with additional features of epithelioid cells in one case and ganglion cells in another. Olfactory and Homer-Wright rosettes were present. All the specimens showed a uniform positive reaction to neurone specific enolase, S-100, and cytokeratin antibodies. Glial fibrillary acidic protein was absent. The salient electron microscopic features were the presence of cell junctions, cytoplasmic intermediate filaments, basal bodies and cytolasmic processes. Dense cored vesicles were absent. The results strongly support the view that intracranial olfactory neuroblastomas are of olfactory epithelial origin and differ from conventional neuroblastomas.


Use of monoclonal antibody E48 in diagnosing transitional cell carcinoma of urinary bladder. To investigate the localisation of the E48 epitope and to determine the use of monoclonal antibody E48 for the identification of transitional cell carcinomas (TCC); and to determine if antigenic expression was affected by different standard fixation methods. Biopsy specimens were labelled with E48 for immunoelectron microscopy. One hundred and nineteen tissue samples from 47 bladder carcinomas were tested for reactivity with E48, using fresh frozen, sublimate formalin, and formalin fixed tissue. Thirteen undifferentiated bladder tumours and 10 undifferentiated prostatic carcinomas were incubated with E48 and prostate specific antigen. Reactivity to E48 was found in all grade 1 and 2 carcinomas and most (83%) grade 3 tumours. At the ultrastructural level, expression was mainly associated with desmosomes and the cytoplasmic membrane. The reactivity of E48 was generally strong in fresh frozen tissue samples and remained preserved in fixed tissue samples. Ten of the 13 bladder carcinomas expressed E48; all prostatic tumours were totally negative. E48 is a sensitive marker for transitional cell carcinoma and suitable for differentiation between urothelial and prostatic undifferentiated carcinoma. It can be used in routinely processed, formalin fixed, biopsy specimens.


Integration of neurobiology and psychopathology in a unified model of suicidal behavior. Suicidal behavior is the result of a combination of factors that span the domains of psychopathology, genetics, early life experience, family interactions, social stress, physical illness, and neurobiology. To develop predictive and explanatory models of suicidal behavior it is necessary to consider all of these domains. A stress-diathesis model is proposed that classifies risk factors for suicidal behavior into those that are trait-dependent and those that are state-dependent. The timing of suicidal behavior is determined by state-dependent factors. The relationship of psychopathologic factors such as severity of depression or anxiety to suicide will be discussed. Biologic changes that correlate with suicide may be either state- or trait-dependent. Particular emphasis will be given to changes in the serotonin system and how these may represent a constitutional risk factor as opposed to a state-dependent risk factor for suicidal behavior.


Prediction of suicidal behavior from biologic tests. Biochemical studies related with suicidal behavior have mainly dealt with monoaminergic and corticosteroidal measures. We used some of these measures in a study of 61 suicide attempters who, except for occasional doses of benzodiazepines, had been medication free for a mean of 16 days. The monoamine metabolites 5-hydroxyindoleacetic acid, homovanillic acid, and 3-methoxy-4-hydroxyphenylglycol were measured in lumbar cerebrospinal fluid (CSF). We found that violent suicide attempters (N = 18) had 5-hydroxyindoleacetic acid concentrations below the median of all patients, whereas the concentrations of 3-methoxy-4-hydroxyphenylglycol were mainly above the median. We found no significant differences between violent and nonviolent (N = 43) attempters concerning CSF homovanillic acid, 24-hour urinary norepinephrine-epinephrine and cortisol, activity of monoamine oxidase in platelets, or post-dexamethasone plasma cortisol. Four patients completed suicide, and 3 of them had CSF 5-hydroxyindoleacetic acid concentrations at or below the median. All completed suicides had CSF 3-methoxy-4-hydroxyphenylglycol concentrations above the median. Urinary measures and platelet monoamine oxidase activity of completed suicides were in the higher concentration ranges. Patients who repeated suicidal behavior after the index investigation had low 24-hour urinary cortisol levels more often than those who did not repeat. Because our subgroups of patients are small, we cannot draw any firm conclusions about the value of our CSF and urinary biochemical findings predicting suicidal behavior. However, our CSF findings in violent suicide attempters are similar to those observed in other studies.


Distribution of substance P-immunoreactive elements in the preoptic area and the hypothalamus of the rat. The localization and morphology of neurons, processes, and neuronal groups in the rat preoptic area and hypothalamus containing substance P-like immunoreactivity were studied with a highly selective antiserum raised against synthetic substance P. The antiserum was thoroughly characterized by immunoblotting; only substance P was recognized by the antiserum. Absorption of the antiserum with synthetic substance P abolished immunostaining while addition of other hypothalamic neuropeptides had no effect on the immunostaining. The specificity of the observed immunohistochemical staining pattern was further confirmed with a monoclonal substance P antiserum. The distribution of substance P immunoreactive perikarya was investigated in colchicine-treated animals, whereas the distribution of immunoreactive nerve fibers and terminals was described in brains from untreated animals. In colchicine-treated rats, immunoreactive cells were reliably detected throughout the preoptic area and the hypothalamus. In the preoptic region, labeled cells were found in the anteroventral periventricular and the anteroventral preoptic nuclei and the medial and lateral preoptic areas. Within the hypothalamus, immunoreactive cells were found in the suprachiasmatic, paraventricular, supraoptic, ventromedial, dorsomedial, supramammillary, and premammillary nuclei, the retrochiasmatic, medial hypothalamic, and lateral hypothalamic areas, and the tuber cinereum. The immunoreactive cell groups were usually continuous with adjacent cell groups. Because of the highly variable effect of the colchicine treatment, it was not possible to determine the actual number of immunoreactive cells. Mean soma size varied considerably from one cell group to another. Cells in the magnocellular subnuclei of the paraventricular and supraoptic nuclei were among the largest, with a diameter of about 25 microns, while cells in the supramammillary and suprachiasmatic nuclei were among the smallest, with a diameter of about 12 microns. Immunoreactive nerve fibers were found in all areas of the preoptic area and the hypothalamus. The morphology, size, density, and number of terminals varied considerably from region to region. Thus, some areas contained single immunoreactive fibers, while others were innervated with such a density that individual nerve fibers were hardly discernible. During the last decade, knowledge about neural organization of rodent hypothalamic areas and mammalian tachykinin biochemistry has increased substantially. In the light of these new insights, the present study gives comprehensive morphological evidence that substance P may be centrally involved in a wide variety of hypothalamic functions. Among these could be sexual behavior, pituitary hormone release, and water homeostasis.


A projection from the parabigeminal nucleus to the pulvinar nucleus in Galago. A projection from the parabigeminal nucleus (Pbg) to the striate-recipient zone of the pulvinar nucleus in the prosimian Galago was identified by anterograde and retrograde transport methods. In addition to the pulvinar nucleus, Pbg projections were found to terminate in layers 4 and 5 of the dorsal lateral geniculate nucleus and the central lateral nucleus. All three of these structures project to the superficial layers of the striate cortex. Similarities between the Pbg in mammals and the nucleus isthmi in nonmammals in connections and neurochemistry reinforce the idea that these two nuclei are homologous.


Polyaxonal amacrine cells of rabbit retina: size and distribution of PA1 cells. Type 1 polyaxonal (PA1) amacrine cells have been identified previously in rabbit retina, and their morphological characteristics have been described in detail in the preceding paper. Like other polyaxonal amacrine cells they bear distinct dendritic and axonal branching systems, the latter of which originates in two to six thin, branching axons which emerge from or near to the cell body. Unlike other types of polyaxonal amacrine cells, however, their branching is stratified at the a/b sublaminar border and their cell bodies are most often displaced interstitially in the inner plexiform layer (IPL). This report emphasizes quantitative features of the population of PA1 cells, documented in Golgi-impregnated and Nissl-stained retinas, and provides further evidence in Nissl preparations for the amacrine-cell nature of polyaxonal amacrine cells. The cell bodies of Golgi-impregnated PA1 amacrine cells are relatively large: 12-15 microns in equivalent diameter over the range extending from the visual streak 6 mm into ventral retina. Over the same range, dendritic trees are 400-800 microns in equivalent diameter, but they are much smaller than the axonal arborizations, which extend up to and perhaps beyond 2 mm from the cell body. Interstitial cell bodies appropriate to PA1 cells have been identified in Nissl-stained, whole-mounted rabbit retinas. In the plane of the retina, these are comparable in area to smaller medium-size ganglion cells, but their very pale Nissl staining, high nuclear/cytoplasmic ratio, and absence of nucleolar staining are all characteristics of amacrine cells. Interstitial displacement of presumed PA1 cells is rare in the visual streak, and the frequency of interstitial cells reaches a peak between 1 and 2 mm ventral to the streak. Counts in Nissl-stained retinas and estimates from nearest neighbor analyses in these and in Golgi-impregnated retinas indicate a density of PA1 cells in the range of 15-16 cells/mm2 at about 2 mm ventral to the streak, when an estimated 25% shrinkage of the material is taken into account. Dendritic field overlap, based upon this estimate, is calculated to be about fourfold, while a lower bound to estimates of the overlap of axonal arborizations is nearly an order of magnitude higher. Many similarities are noted in a qualitative and quantitative comparison of PA1 amacrine cells in rabbit and monkey retinas. In assessing the contribution of the structural organization of PA1 amacrine cells to their possible functional role(s), it is notable that their appearance conforms not to amacrine cells as commonly viewed, but to a more conventional model of neuronal dynamic polarization.(ABSTRACT TRUNCATED AT 400 WORDS)


Polyaxonal amacrine cells of rabbit retina: PA2, PA3, and PA4 cells. Light and electron microscopic studies with a functional interpretation. Polyaxonal (PA) amacrine cells are a new class of amacrine cell bearing one to six branching, axon-like processes that emerge from the cell body or dendritic trees within 50 microns of the cell body. These slender processes of uniform caliber branch at right angles and in many respects closely resemble the axons of Golgi type II cells found elsewhere in the brain. Of the four types of polyaxonal amacrine cell that we have recognized in rabbit retina, two have been described previously in brief communications. One of these, the PA1 amacrine cell with its interstitially displaced cell body, located in the inner plexiform layer (IPL), has been analyzed extensively in two preceding reports. This paper concerns PA2, PA3, and PA4 amacrine cells. Type 2 polyaxonal (PA2) amacrine cells, identified in Golgi preparations of whole-mounted rabbit retinas, have smaller cell bodies (9-14 microns) than the other three types and these are always displaced to the ganglion cell layer (GCL) or the inner border of the inner plexiform layer (IPL). The dendritic fields of PA2 cells are also smaller than those of other PA amacrine cells, and most of their sparse dendritic branching is narrowly stratified at the border of strata (S) 4 and 5. Some members of this more heterogeneous amacrine cell "type" are bistratified, however, and more highly branched with terminal branches rising to end in S1. PA2 amacrine cells bear a scattering of small dendritic spines and may also exhibit complex dendritic appendages arising at the ends of terminal branches in proximal regions of the dendritic tree. PA2 cells emit one to three axons from the proximal dendritic tree, and about half of the cells bear a single axon. Type 3 polyaxonal (PA3) amacrine cells resemble PA1 cells in the large size of their cells bodies (11-16 microns) and dendritic fields, but differ from the latter in placement of cell bodies, which is in the GCL, and dendritic and axonal stratification, which is multistratified, ranging from S4 to S1, with a concentration in S3 or S4 and a variable contribution to S1. PA3 cells differ from PA1 cells in several other respects, including dendritic branching which occurs at higher frequency and is biased toward temporal retina, and in characteristic bristling dendritic spines, clustered in the intermediate regions of the dendritic tree, that are longer, more variable in appearance and more tightly clustered than the small, uniform spines of PA1 cells that are clustered on proximal dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)


Galanin-like innervation of rat submandibular and sublingual salivary glands: origin and effect on acinar cell membranes. The distribution and source of a galanin-like innervation of rat salivary glands has been examined. Additionally, submandibular and sublingual acinar cell membrane responses to galanin or a cholinergic agonist were studied. Galanin-immunoreactive fibers were observed throughout the submandibular and sublingual glands in association with ducts and acini. A subset of submandibular ganglion cells expresses galanin immunoreactivity. Parasympathectomy resulted in a marked decrease in galanin immunoreactivity in the glands. Sympathectomy resulted in marked reduction of dopamine beta-hydroxylase immunoreactivity with no appreciable change in galanin immunoreactivity. Retrograde labeling experiments demonstrated that galanin-immunoreactive sensory neurons in the trigeminal ganglion do not innervate the submandibular or sublingual gland. These results indicate that the galanin-like innervation of rat salivary glands is derived from parasympathetic nerves to the glands. Since rat sublingual glands contain largely mucous acini while rat submandibular gland acini are seromucous, electrophysiological responses to galanin and the muscarinic agonist, bethanechol, were compared. Agonist-induced voltage shifts varied between the two glands. The galanin-induced response at the level of the resting membrane potential in submandibular acinar cells was a hyperpolarization, while that in sublingual acinar cells was a depolarization. There was also a greater voltage dependence to the galanin-induced submandibular response than to the sublingual response. Differences were also noted in the acinar cell response to cholinergic stimulation between these glands. These results demonstrate the existence of a galanin-like innervation to salivary glands that may be functionally relevant. Moreover, the results challenge the idea that agonist-induced membrane responses are similar among acinar cells of different glands.


Distribution of neurotensin immunoreactivity within the human amygdaloid complex: a comparison with acetylcholinesterase- and Nissl-stained tissue sections. In a previous study, we reported marked depletion of neurotensin-immunoreactivity (NT-IR) within selected regions of the amygdala of patients with Alzheimer's disease. The significance of these observations was partly obscured largely because we lacked a thorough understanding of the innervation pattern of neurotensin in the normal human amygdala. Accordingly, in the present study, we used a polyclonal antibody against neurotensin to characterize the distribution and morphology of neurotensin-immunoreactive neuronal elements within the human amygdaloid complex. NT-IR occurred in a topographic manner that respected the cytoarchitectural boundaries of the amygdaloid subregions as defined by Nissl staining and acetylcholinesterase histochemistry. Most NT-IR in the amygdala was contained within beaded fibers and dot-like puncta. Within the subnuclei of the amygdala, immunoreactive neuritic elements were most dense within the central nucleus followed by the medial nucleus and intercalated nuclei. The anterior amygdaloid area, basal complex, paralaminar nucleus, cortical nucleus, cortical-amygdaloid transition area, and amygdalohippocampal area contained moderate densities of immunoreactivity. The accessory basal and lateral nuclei exhibited scant NT-IR. Immunoreactive neurons were found only within the anterior amygdaloid area and the central, medial, intercalated, and lateral capsular nuclei. The distribution of NT-immunoreactive processes and cell bodies within selected regions of the amygdala provides an anatomical substrate that may explain, in part, the neuromodulatory actions of neurotensin upon autonomic, endocrine, and memory systems.


Astrocytes in cat visual cortex studied by GFAP and S-100 immunocytochemistry during postnatal development. A monoclonal antibody to glial fibrillary acidic protein (GFAP) and a polyclonal antiserum to the S-100 protein were used to study the expression of these astrocytic proteins in the postnatal visual cortex of the cat. Three changes in antigen expression of these astroglial markers could be distinguished over development. First, the density of cells in the white matter, which are heavily labelled with both antibodies from birth until adulthood, diminishes after the third postnatal weeks. By intracellular filling with Lucifer Yellow the reduction of the cell density can be attributed to the disappearance of large astrocytes with a morphology of transforming radial glia, present only in early postnatal development. Second, heavily labelled, large cells present in the grey matter at the seventh postnatal day have disappeared by the fifth postnatal week. On the basis of their morphology these cells can also be classified as radial glial cells. Finally, astroglial cells of the adult-like stellate form appear to be labelled in the cortical layers between the third and seventh postnatal weeks. While the density of these cells and the S-100 immunoreactivity of the cell bodies is adult-like at the fourth postnatal week, there is a gradual increase of the staining intensity with the GFAP antibody up to the seventh postnatal week. This developmental period is paralleled by the appearance of S-100-positive astrocytic processes. The gradual expression of GFAP immunoreactivity and the increased expression of S-100 is interpreted as reflecting the time course of astrocytic maturation. A possible relation of the maturation of astrocytes and cortical development, both of which are prominent in the time period between the third and seventh postnatal week, is discussed.


Distribution of neurons expressing neurokinin B in the rat brain: immunohistochemistry and in situ hybridization. Neurokinin B (NKB) belongs to the family of neuropeptides named tachykinins. Members of this family such as substance P or neurokinin A have been proposed to function as neurotransmitters or neuromodulators. Searching for possible sites of action of NKB in the central nervous system, we have now investigated its distribution within the rat brain by immunohistochemical techniques and in situ hybridization. For immunohistology two different antisera directed against amino acid sequences within preprotachykinin B were used. One antiserum had been raised against a synthetic derivative of NKB; the other one was directed towards the amino acids 50-79 of preprotachykinin B, which are referred to as peptide 2. Essentially the same distribution of immunoreactive perikarya was obtained with both antisera and it closely corresponded to the cellular localization of preprotachykinin B mRNA. Neurons containing NKB immunoreactivity and mRNA were present in many areas including cerebral cortex, hippocampal formation, amygdaloid complex, bed nucleus of the stria terminalis, ventral pallidum, habenula, medial preoptic area, arcuate nucleus, and lateral mammillary bodies. Dense immunoreactive fibers were observed in various parts of the brain and were most prominent in the olfactory bulb and tubercle, the lateral olfactory tract, medial hypothalamus, around blood vessels of the median eminence and interpeduncular nucleus, amygdaloid nuclei, stria terminalis, subbrachial nucleus, and medial geniculate nucleus. Fibers of less intense staining were seen among other brain areas in the substantia nigra, the reticular formation, and the area of the nucleus of the solitary tract. Surgical lesion of the fasciculus retroflexus revealed that the dense fiber network observed in the interpeduncular nucleus originates from the ventral and dorsal parts of the medial habenula. Our data suggest a widespread and distinct distribution of neurons expressing NKB within the central nervous system, suggesting possible neuromodulatory roles of this neuropeptide for various brain functions.


Distribution of neuropeptide Y, substance P, and choline acetyltransferase in the developing visual system of the pigeon and effects of unilateral retina removal. The distribution of three neuroactive substances, neuropeptide Y, substance P, and choline acetyltransferase, was studied by immunocytochemical methods in central visual regions of adult, developing, and ablated pigeon brains. In normal adult brains, neuropeptide Y-positive cells and processes were present in the nucleus pretectalis, the nucleus of the basal optic root, the nucleus of the marginal optic tract, and the visual Wulst. Substance P-positive cells and processes were found in the optic tectum and in the visual Wulst. Stained fibers and terminal-like processes, but no cells, were also observed in several visual thalamic nuclei. Choline acetyltransferase-positive cells and processes were located in the optic tectum, visual Wulst, the nucleus isthmo opticus, nucleus isthmi and certain visual thalamic nuclei. Cholinergic fibers and processes, but no cells, were present in the nucleus principalis precommissuralis, the supraoptic decussation, and the nucleus lentiformis mesencephali, pars magnocellularis. In the course of development, the distribution of immunoreactivity for all three substances was found to vary. These changes often involved either progressive increases or decreases in the density of labeled cells, neuropil and/or terminal-like profiles. Experiments with retina ablated pigeons clearly demonstrated that changes in the normal pattern of immunoreactivity distribution only occurred if the retina was removed immediately after hatching, i.e., before retinofugal connections have been established. The adult pattern of immunoreactivity for all three substances appears to be reached at about the same time that the anatomical and functional maturation of the pigeon visual system is completed. The present results suggest that this temporal correlation reflects the important role that retinal afferents play in the development of these putative peptidergic and cholinergic systems.


Rapid growth of astrocytic processes in N. magnocellularis following cochlea removal. Removal of the cochlea or pharmacological blockade of eighth nerve activity in young postnatal chickens results in rapid transneuronal cell death and atrophy in neurons of n. magnocellularis. The present experiments were designed to examine the influence of afferent input on astrocyte structure in n. magnocellularis. Young chickens were subjected to unilateral cochlea removal. At times ranging from 5 minutes to 72 hours later, the brainstems were histologically processed with a polyclonal antibody against glial fibrillary acidic protein (GFAP). A second group of chick brainstems was impregnated by a Golgi method 6 hours after unilateral cochlea removal and impregnated three-dimensional reconstructions were made of glial cells in n. magnocellularis (NM). Analyses of GFAP positive processes in NM revealed an observable increase in the number of astrocytic processes at the borders of the nucleus within 30 minutes of cochlea removal and a twofold increase in GFAP + glial processes by 6 hours. A secondary increase in the number and density of GFAP + processes occurred between 24 and 72 hours following cochlea removal, during the period of axonal degeneration, and transneuronal cell atrophy and death. Analyses of astrocytes impregnated by the Golgi method revealed that individual glial cells had increased their total process length and the number of processes by approximately twofold by 6 hours after cochlea removal. These results suggest that the structure of astrocytes is rapidly and dramatically influenced by the level of excitatory activity in a neuronal system. Furthermore, the similarity of results obtained with GFAP immunohistochemistry and three-dimensional reconstruction of astrocytes provides evidence that the short-term changes observed following cochlea removal represent the actual growth of glial processes. We speculate that modulations in glial processes as a function of afferent activity may act to influence synaptic efficacy.


CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used. The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response.


Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2. One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.