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Biomedical subjects

L A Snyder

Publications and source records attributed to L A Snyder.

At least 19 recordsLinked to original sources
PubMed2000

Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes.

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

Induction of HSV-gD2 specific CD4(+) cells in Peyer's patches and mucosal antibody responses in mice following DNA immunization by both parenteral and mucosal administration.

A DNA vaccine encoding glycoprotein D (gD) of herpes simplex virus type 2 (pHSV-gD2) was injected via parenteral and mucosal routes to determine the optimal route of delivery for immune stimulation. Generation of distal mucosal immunity following parenteral vaccination was also evaluated. While all routes of DNA vaccine administration resulted in systemic cellular and humoral responses, the intra-muscular (i.m.) and intra-dermal (i.d.) routes of delivery produced the highest responses. Furthermore, i.m. and i.d. routes produced mucosal humoral responses that were comparable to those obtained via mucosal routes. Specific pHSV-gD2 PCR signals were detected in the Peyer's patches (PP) within hours following vaccination and antigen specific IgA was detected in secretions and supernatants from gut fragment cultures. Furthermore, antigen specific CD4(+) cells were found in PP. Collectively these results suggest that the DNA vaccine stimulated a response in the PP, a major inductive site for mucosal responses.

Administration, Intranasal↗
PubMed1999

Dopamine D4/D2 receptor selectivity is determined by A divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments.

Conserved features of the sequences of dopamine receptors and of homologous G-protein-coupled receptors point to regions, and amino acid residues within these regions, that contribute to their ligand binding sites. Differences in binding specificities among the catecholamine receptors, however, must stem from their nonconserved residues. Using the substituted-cysteine accessibility method, we have identified the residues that form the surface of the water-accessible binding-site crevice in the dopamine D2 receptor. Of approximately 80 membrane-spanning residues that differ between the D2 and D4 receptors, only 20 were found to be accessible, and 6 of these 20 are conservative aliphatic substitutions. In a D2 receptor background, we mutated the 14 accessible, nonconserved residues, individually or in combinations, to the aligned residues in the D4 receptor. We also made the reciprocal mutations in a D4 receptor background. The combined substitution of four to six of these residues was sufficient to switch the affinity of the receptors for several chemically distinct D4-selective antagonists by three orders of magnitude in both directions (D2- to D4-like and D4- to D2-like). The mutated residues are in the second, third, and seventh membrane-spanning segments (M2, M3, M7) and form a cluster in the binding-site crevice. Mutation of a single residue in this cluster in M2 was sufficient to increase the affinity for clozapine to D4-like levels. We can rationalize the data in terms of a set of chemical moieties in the ligands interacting with a divergent aromatic microdomain in M2-M3-M7 of the D2 and D4 receptors.

Amino Acid Sequence↗
PubMed1991

Alternative transcripts of the rat and human dopamine D3 receptor.

A cDNA for the rat dopamine D3 receptor containing a 113 bp deletion has been isolated. The segment deleted, encompassing the first extracellular loop and third transmembrane domain, alters the reading frame, introducing 19 amino acids not found in the full length receptor followed by a premature stop codon. This novel mRNA encodes a 109 amino acid protein containing two putative transmembrane domains. A similar variant cDNA for the human D3 receptor has also been identified.

Amino Acid Sequence↗
PubMed1991

Distribution of dopamine D2 receptor mRNA splice variants in the rat by solution hybridization/protection assay.

We investigated the distribution of the two dopamine D2 receptor mRNA splice variants in the rat using a sensitive and quantitative solution hybridization/nuclease protection assay. In all brain and endocrine regions studied, both splice variants were detected and the mRNA of the longer form (D2L) was more abundant than that of the shorter form (D2S). The lowest percentages of D2S were found in the pituitary and adrenal glands.

Adrenal Glands↗
PubMed1991

Evidence for a new tumor-suppressor gene involved in gastrointestinal malignancies.

Inactivation or loss of tumor-suppressor genes is believed to lead to the development or progression of malignancies. To determine whether a tumor-suppressor gene is located on chromosome 8, DNA was extracted from tumor and normal tissue of colorectal, gastric, and pancreatic specimens, and allele loss was investigated by Southern hybridization techniques with the chromosome 8 probe D8S7. Twenty-five percent of pancreatic carcinomas, 50% of gastric carcinomas, and 50% of colorectal carcinomas were found to have lost an allele on chromosome 8. These findings suggest the presence of a tumor-suppressor gene on chromosome 8, which is involved in colorectal carcinoma, gastric carcinoma, and pancreatic carcinoma. Definition of the frequency with which this tumor-suppressor gene is involved in gastrointestinal malignancies will await the study of many patients who are classified as informative and the use of multiple probes for chromosome 8.

Adenocarcinoma↗
PubMed1980

Drosophila nucleosomes contain an unusual histone-like protein.

Mononucleosomes prepared from Drosophila melanogaster nuclei contain the four core histones H2A, H2B, H3, and H4 plus an additional histone-like, acid-soluble, chromosomal protein. It is probably the protein designated D2 by Alfageme et al. [Alfageme, C.R., Zweidler, A., Mahowald, A. & Cohen, L.H. (1974) J. Biol. Chem. 249, 3729-3736]. D2 elutes with histone H2A from a Bio-Gel P-100 column, but can be distinguished electrophoretically from H2A and from the other standard Drosophila core histones. The amino acid composition of D2 resembles the compositions of H2A and H2B. However, peptide mapping reveals that D2 is not a simple sequence variant of either H2A or H2B. D2 is present in nuclei from embryos and adult heads, and thus is not restricted to a narrowly defined developmental period. It is present in D. melanogaster and D. virilis, and thus appears to be conserved during the evolution of Drosophila. D2 is present in D. melanogaster chromatin with an approximate frequency of one molecule per five nucleosomes, and must therefore be associated with a subset of nucleosomes. The function of this protein is not known. Its presence in nucleosomes, evolutionary conservation, and comparatively large abundance all suggest that it is an important nucleosomal element. It will be interesting to learn whether this histone-like protein is encoded in a subset of the Drosophila histone gene cluster or is encoded separately.

Amino Acids↗
PubMed1978

Correlation between phosphorylated H1 histones and satellite DNAs in Drosophila virilis.

Drosophila virilis DNA contains satellites I, II, and III. D. novamexicana DNA contains satellite I. D. virilis H1 histone contains subfractions a, b, c, d, and e; D. novamexicana H1 contains subfractions a, b, and c. Therefore, satellites II and III might be correlated with H1d and H1e. To test the validity of this correlation, the H1 histones of polytene nuclei, which contain less than 1% satellite DNA, were analyzed. Polytene nuclei of D. virilis contain substantially decreased levels of H1c and H1e and marginally decreased levels of H1d. Polytene nuclei of D. novamexicana contain decreased levels of H1c.H1c is correlated with satellite I (common to D. virilis and D. novamexicana); H1e is correlated with satellites II and III; H1d is not correlated with any satellite DNA, because its level is virtually unchanged in polytene cells lacking detectable amounts of satellite DNA. Alkaline phosphatase digestion of the H1 histones reveals that H1c is the phosphorylated form of H1b and H1e is the phosphorylated form of H1d. Therefore, the under-replication of satellite DNAs is correlated with the decreased phosphorylation of H1 histones. In vitro, D. virilis H1 histones preferentially bind D. virilis DNAs in the progression III greater than II greater than I greater than main band, whereas D. virilis core histones do not preferentially bind any D. virilis DNA. As an extension of these results, we suggest that phosphorylated H1 histones bind D. virilis satellite DNAs in vivo and are involved in the compaction of heterochromatin.

Alkaline Phosphatase↗