Expression of a normal and variant Alzheimer's beta-protein gene in amyloid of hereditary cerebral hemorrhage, Dutch type: DNA and protein diagnostic assays

Amyloid fibrils deposited in cerebral vessel walls in Dutch patients with hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) are formed by polymerization of a 39-residue peptide similar to the beta-protein of Alzheimer's disease, Down syndrome, sporadic cerebral amyloid angiopathy and normal aging. Sequence analysis of genomic DNA in HCHWA-D patients demonstrated a point mutation, cytosine for guanine at position 1852 of the precursor beta-protein gene, which causes a single amino acid substitution (glutamine for glutamic acid) corresponding to position 22 of the amyloid protein. The normal allele was also present in these patients. To examine the expression of normal and variant beta-protein alleles in HCHWA-D we analyzed all the tryptic peptides obtained from several amyloid fractions from leptomeningeal vascular walls. Amino acid sequence of two peptides (T3a and T3b) with identical amino acid composition revealed that T3a had glutamine and T3b had glutamic acid at position 22. Thus both the normal and variant Alzheimer's beta-protein alleles are expressed in vascular amyloid in HCHWA-D and may be detected by tryptic peptide mapping. Moreover, we have developed a diagnostic assay for high risk populations and prenatal evaluation that is based on the existence of the mutation.     

Modifications of position 12 in parathyroid hormone and parathyroid hormone related protein: toward the design of highly potent antagonists

Truncated N-terminal fragments of parathyroid hormone (PTH), [Tyr34]bovine PTH(7-34)NH2, and parathyroid hormone related protein (PTHrP), PTHrP(7-34)NH2, inhibit [Nle8,18,[125I]iodo-Tyr34]-bPTH(1-34)NH2 binding and PTH-stimulated adenylate cyclase in bone and kidney assays. However, the receptor interactions of these peptides are 2-3 orders of magnitude weaker than those of their agonist counterparts. To produce an antagonist with increased receptor-binding affinity but lacking agonist-like properties, structure-function studies were undertaken. Glycine at position 12 (present in all homologues of PTH and in PTHrP), which is predicted in both hormones to participate in a beta-turn, was examined by substituting conformational reporters, such as D- or L-Ala, Pro, and alpha-aminoisobutyric acid (Aib), in both agonist and antagonist analogues. Except for N-substituted amino acids, which substantially diminished potency, substitutions were well tolerated, indicating that this site can accept a wide latitude of modifications. To augment receptor avidity, hydrophobic residues compatible with helical secondary structure were introduced. Incorporation of the nonnatural amino acids D-Trp, D-alpha-naphthylalanine (D-alpha-Nal), or D-beta-Nal into either [Tyr34]bPTH(7-34)NH2 or [Nle8,18,Tyr34]bPTH(7-34)NH2 resulted in antagonists that were about 10-fold more active than their respective 7-34 parent compound. Similarly, [D-Trp12]PTHrP(7-34)NH2 was 6 times more potent than the unsubstituted peptide but retained partial agonistic properties, although markedly reduced, similar to PTHrP(7-34)NH2. The antagonistic potentiating effect was configurationally specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid vesicle solubilization and reconstitution by detergents. Symmetrical analysis of the two processes using octaethylene glycol mono-n-dodecyl ether

The processes of liposome solubilization and reconstitution were studied by using n-dodecyl octaethylene glycol monoether (C12E8). The solubilization of large unilamellar liposomes prepared by reverse-phase evaporation was systematically investigated by turbidity, 31P nuclear magnetic resonance, and centrifugation experiments. The solubilization process is well described by the three-stage model previously proposed for other detergents, and our results further demonstrate the validity of some of the postulates related to this model. In stage I, the detergent distributes between the bilayers and the aqueous solution with a partition coefficient of 1.6 mM-1. In stage II, the detergent-saturated liposomes convert into mixed micelles, the conversion being complete by stage III where all the phospholipids are present as mixed micelles. The agreement between the three methods was excellent, and the results allowed quantitative determination of the effective detergent to phospholipid ratios at which the lamellar to micellar transformation begins and is complete, which amounted to 0.66 and 2.2 (mol/mol), respectively. Furthermore, compositional analysis determined from centrifugation experiments directly demonstrate that the properties of detergent-saturated liposomes and mixed micelles remain constant throughout most of stage II: the C12E8 to phospholipid ratios in the pelleted vesicles and in micelles are constant during stage II and similar to the ratios at which stage II was initiated and complete, respectively. On the other hand, bilayer formation upon detergent removal from mixed C12E8-phospholipid micelles by SM2 Bio-Beads is demonstrated to be the symmetrical opposite of bilayer solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary excretion of cGMP in response to atrial natriuretic peptide in dogs with acute pancreatitis

Previous studies have shown that when atrial natriuretic peptide (ANF) is given to anaesthetized dogs with hypovolemic acute pancreatitis, it will produce a diuresis and natriuresis but will not elevate the glomerular filtration rate (GFR). When the same dose of peptide is given to dogs equally hypovolemic (hemorrhage) but without pancreatitis, a brisk increment in GFR occurs. GFR will, however, rise in dogs with pancreatitis in response to other peptides, such as glucagon. In these studies we assessed cGMP excretion as a marker for ANF effect in both normal anaesthetized dogs and dogs with acute experimental pancreatitis. In each group, urinary output and sodium excretion increased significantly, but GFR rose only in the control group. Urinary excretion of cGMP rose equally and dramatically in both control and experimental animals. We conclude that GFR is prevented from rising in dogs with experimental pancreatitis following ANF, but this effect does not depend on depressed cGMP generation.     

Reconstitution of the immunopurified 49-kDa sodium-dependent bile acid transport protein derived from hepatocyte sinusoidal plasma membranes

Reconstitution, using phosphatidylcholine liposomes in conjugation with immunological purification procedures, has been used to establish directly the identity of the hepatocyte Na(+)-dependent bile acid transport protein. Octyl glucoside-solubilized sinusoidal plasma membranes were shown to form proteoliposomes exhibiting taurocholate transport properties which were similar to those of plasma membrane vesicles, namely, Na(+)-dependence and marked inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and by taurochenodeoxycholate. Proteoliposomes formed from plasma membrane proteins depleted of the putative 49-kDa bile acid transport protein by immunoprecipitation with monoclonal antibody 25D-1, which specifically recognizes this protein (Ananthanarayanan, M., von Dippe, P., and Levy, D. (1988) J. Biol. Chem. 263, 8338-8343), showed a 94% reduction in mediated transport capacity. Proteoliposomes containing total membrane protein also demonstrated Na(+)-dependent alanine transport. The addition of taurochenodeoxycholate or the removal of the 49-kDa protein by monoclonal antibody 25D-1 immunoprecipitation had no effect on the uptake of alanine, thus confirming the specificity of these procedures. When only the immunoprecipitated 48-kDa protein was used in the reconstitution system, a 2200% increase of taurocholate uptake was observed. These results definitively establish that this 49-kDa sinusoidal membrane protein is the sole essential component of the Na(+)-dependent bile acid transport system.     

Anti-tumor effects of an antiserum raised in syngeneic mice to a tumor-specific T suppressor factor

Summary DBA/2 mice inoculated with either cells from the syngeneic P815 tumor or tumor cell membrane extracts develop T suppressor cells which suppress the in vitro generation of cytotoxic T lymphocytes with specificity for the tumor. A soluble suppressor factor with similar properties can be isolated from suppressor cell-enriched populations. It can be highly purified by appropriate immunoadsorption. Antisera to this suppressor factor raised in either DBA/2 or C57BL/6 mice can specifically absorb out suppressor factor and eliminate suppressor cells in the presence of complement. The in vivo effects of these antisera were tested for their ability to modulate the growth of P815 tumors in DBA/2 mice. It was found that the antiserum raised in syngeneic (DBA/2) but not allogeneic (C57BL/6) mice was able to significantly slow the rate of tumor growth and to prolong survival in treated mice. The antiserum was effective in this way only if it was administered early in the course of tumor growth. It was shown that this effect was not attributable to the presence in the serum of antibodies directed to antigens present on P815 cells, and it therefore appears to be due to interference with the function of T suppressor cells arising early in the immune response to the tumor cells.     

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.     

The Comb-footed spider genera Theridion, Achaearanea and Anelosimus of Israel (Araneae: Theridiidae)

Israeli thendiid spiders of the genera Theridion, Achaearanea and Anelosimus have been revised. A relative richness in species is presented providing thereby updated information on the little known Mediterranean spider fauna. All type and non-type material previously described from the Middle East, deposited in several European collections has been re-examined, along with species from adjacent regions considered pertinent to the study undertaken. Altogether 21 species are recognized. Systematic, ecologic and all available zoogeographic information on taxa treated are discussed along with recent, pertaining literature. The presence of seven species formerly reported from Israel has been confirmed and the occurrence of another four species unknown hitherto from this region, has been proved. Some of these have never been adequately described or illustrated.
Ten new species are described: Theridion ochreolus, T. agaricographus, T hierwhonticus, T. jordanensis, T. negebensis, T. gekkonicus, T. dafnensis, T. vallisalinarum, T. pustiliferus and Anelosimus giladensis. The male of Theridion melanostictum is described for the first time. Keys, illustrations of diagnostic characters and records of distribution are provided for each species, all readily applicable also in adjacent countries. These may provide clues for better understanding of zoogeographic patterns of the Palearctic fauna, including those of the Old World Desert belt extending south and east of the Mediterranean region.     

A comparison of the ability of serum and monoclonal antibodies to gastric inhibitory polypeptide to detect immunoreactive cells in the gastroenteropancreatic system of mammals and reptiles

Summary A monoclonal antibody raised to gastric inhibitory polypeptide (GIP) has been compared with conventional rabbit and guinea-pig antisera to GIP. Four staining methods were tested and of these the peroxidase antiperoxidase (PAP) method proved to give the best results with both the mouse and rabbit antibodies. The monoclonal antibody, when used to stain pancreatic tissue, gave negative results whereas a distinct population of gut endocrine cells was readily demonstrable, suggesting that GIP is not a constituent of the mammalian pancreas. The monoclonal antibody was found to be the most sensitive for immunocytochemistry achieving the titre of 1:10⁶ in rat gut. A C-terminal specific antibody, with a high affinity and avidity to GIP, it was clearly the preferred antibody for immunocytochemical studies.