The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed exclusively in osteoblasts, osteocytes and odontoblasts with markedly elevated expression found in X-linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors. Because these syndromes are associated with abnormalities in mineralization and renal phosphate excretion, we examined the effects of insect-expressed full-length human-MEPE (Hu-MEPE) on serum and urinary phosphate in vivo, 33 PO 4 uptake in renal proximal tubule cultures and mineralization of osteoblast cultures. Dose-dependent hypophosphatemia and hyperphosphaturia occurred in mice following intraperitoneal (IP) administration of Hu-MEPE (up to 400 μg kg -1 31 h -1 ), similar to mice given the phosphaturic hormone PTH (80 μg kg -1 31 h -1 ). Also the fractional excretion of phosphate (FEP) was stimulated by MEPE [65.0% (P < 0.001)] and PTH groups [53.3% (P < 0.001)] relative to the vehicle group [28.7% (SEM 3.97)]. In addition, Hu-MEPE significantly inhibited 33 PO 4 uptake in primary human proximal tubule renal cells (RPTEC) and a human renal cell line (Hu-CL8) in vitro (V max 53.4% inhibition; K m 27.4 ng/ ml, and V max 9.1% inhibition; K m 23.8 ng/ml, respectively). Moreover, Hu-MEPE dose dependently (50-800 ng/ml) inhibited BMP2-mediated mineralization of a murine osteoblast cell line (2T3) in vitro. Inhibition of mineralization was localized to a small (2 kDa) cathepsin B released carboxy-terminal MEPE peptide (protease-resistant) containing the acidic serineaspartate-rich motif (ASARM peptide). We conclude that MEPE promotes renal phosphate excretion and modulates mineralization.

A pheromone biosynthesis activating neuropeptide (PBAN) hormone that controls sex pheromone production in female moths was identified from the brain-subesophageal ganglion complexes of the adult corn earworm, Heliothis zea. PBAN has 33 amino acid residues and a molecular weight of 3900. Its amino acid sequence has no significant homology with any of the fully characterized peptide hormones. The synthetic peptide, at a dose of between 2 and 4 picomoles, induced production of a normal quantity of sex pheromone in ligated H. zea females. The peptide also induced pheromone production in six other species of moths, thus indicating that this or similar peptides may be responsible for the regulation of pheromone production in moths.

SuntmaryThe activation of T lymphocytes, both in vivo and in vitro, induces the expression of CD69. This molecule, which appears to be the earliest inducible cell surface glycoprotein acquired during lymphoid activation, is involved in lymphocyte proliferation and functions as a signal transmitting receptor in lymphocytes, natural killer (NK) cells, and platelets. To determine the structural basis for CD69 function, the cDNA coding for CD69 was isolated by a polymerase chain reaction-based strategy using oligonucleotides deduced from peptide sequences of the purified protein. The isolated cDNA exhibited a single open reading frame of 597 bp coding for CD69, and predicted a 199-amino acid protein of type II membrane topology, with extracellular (COOH-terminal), transmembrane, and intracellular domains. The CD69 clone hybridized to a 1.7-kb mRNA species, which was rapidly induced and degraded after lymphocyte stimulation, consistent with the presence of rapid degradation signals at the 3' untranslated region. Transient expression of the polypeptide encoded by CD69 cDNA in COS-7 cells demonstrated that it presented properties comparable to native CD69 protein. The CD69 gene was regionally mapped to chromosome 12 p13-p12 by both somatic cell hybrid DNA analysis and fluorescence in situ hybridization coupled with GTG banding (G bands by trypsin using Giemsa). Protein sequence homology search revealed that CD69 is a new member of the Ca2+-dependent (C-type) lectin superfamily of type II transmembrane receptors, which includes the human NKG2, the rat NKR-P1, and the mouse NKR-P1 families of NK cell-specific genes. CD69 also has structural homology with other type II lectin cell surface receptors, such as the T cell antigen Ly49, the low avidity immunoglobulin E receptor (CD23), and the hepatic asialoglycoprotein receptors. The CD69 protein also shares functional characteristics with most members of this superfamily, which act as transmembrane signaling receptors in early phases of cellular activation.

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.

Type beta transforming growth factor (TGF-beta) has been purified 200 000-fold from bovine kidneys. This peptide is characterized by its ability to induce anchorage-dependent normal rat kidney cells to grow in soft agar in the presence of epidermal growth factor (EGF); TGF-beta is not mitogenic for cells grown in monolayer culture. Purified TGF-beta does not compete with EGF for binding to membrane receptors. The concentration of TGF-beta required to elicit a half-maximal response for formation of colonies greater than 3100 micron2 in the soft agar assay is 2-3 pM (55 pg/mL) when assayed in the presence of 0.8 nM EGF (5 ng/mL). The four-step purification procedure which includes chromatography of acid--ethanol tissue extracts on polyacrylamide sizing gels, cation exchange, and two steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 micrograms/kg. Amino acid analysis of purified TGF-beta shows 16 half-cystine residues per mole. Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypeptide chains cross-linked by disulfide bonds. In the absence of beta-mercaptoethanol, the colony-forming activity is associated with a single silver-staining band of molecular weight 25 000; in the presence of beta-mercaptoethanol, the TGF-beta is converted to an inactive species that migrates as a single band of molecular weight 12 500-13 000. Sequence analysis indicates that at least the first 15 N-terminal amino acids of the two TGF-beta subunits are identical.

By comparing the HPLC elution profiles of peptides isolated from different brain regions, two cerebellumspecific species have been identified. The peptides were isolated by sequential chromatography on reverse phase, followed by ion-pairing HPLC using alkane sulfates as pairing reagents. The sequences of both peptides have been determined by gasphase Edman degradation and carboxypeptidase Y analysis and were confirmed by synthesis. The larger peptide, termed cerebellin, is a hexadecamer of primary amino acid sequence NH2 -Ser -Gly -Ser -Ala -Lys -Val -Ala -Phe -Ser-Ala -Ile -ArgSer-Thr-Asn-His-OH. The second peptide is a pentadecamer with a primary sequence identical to residues 2-16 of cerebellin, the nomenclature of which is des-Serl-cerebellin. Subcellular fractionation of cerebellum followed by ion-pairing HPLC analysis shows that both cerebellin and des-Serl-cerebellin are enriched between 2.5-and 4-fold in the P2 crude synaptosomal fraction, suggesting their sequestration in some subcellular particle in vivo.A major goal of developmental neurobiology has been to define the cellular, molecular, and genetic mechanisms that underlie the orderly assembly and maturation of the mammalian nervous system. One fruitful approach to the study of neurodevelopment has been to identify cell-specific markers, usually by immunological techniques, and to assess the expression of these substances in model neural systems (1)(2)(3)(4)(5). However, the general procedure of detecting and monitoring marker expression with antibodies does have a number of limitations. First, only antigenic molecules may be studied. Second, immunocytochemical data are often difficult to quantitate. Finally, subtle modifications of the antigenic determinants, such as glycosylation, may result in ambiguous interpretation of data.In an attempt to circumvent some of the above difficulties, we have tried to characterize brain regions by their native peptide composition. Since the cerebellum has been pointed out to be a particularly useful neural structure for the investigation of the forces regulating neuronal development (6), we have first applied this approach to the rat cerebellum. We report here the isolation and complete primary structures of two related cerebellum-specific peptides that will be shown in further publications to be significant markers for neuronal development in the mammalian cerebellum.MATERIALS AND METHODS Preparation of Tissue Samples for HPLC Analysis. Six-to 8-week-old CD, Sprague-Dawley-derived, rats (Charles River Breeding Laboratories) were decapitated, and their brains were rapidly removed, dissected, and immediately frozen in liquid nitrogen. Prior to HPLC analysis, frozen tissues were homogenized in 10 vol of 6 M guanidine hydrochloride using a Polytron (Brinkmann model PT205T probe) at maximum setting for 30 sec at 4TC. The homogenate was diluted with an equal volume of 0.2 M pyridine/1 M acetic acid and then centrifuged at 25,000 x g for 30 min. The sample was desalted by passing 20 ml of the supernatant over a...