Biomedical subjects

Z Fathi

Publications and source records attributed to Z Fathi.

25 records · Page 2Linked to original sources

PubMed1992

Molecular genetic analysis of two distinct receptors for mammalian bombesin-like peptides.

The mammalian bombesin-like peptides are known to be growth factors for certain cells with high-affinity bombesin receptors and have been implicated as autocrine growth factors influencing the pathogenesis and progression of a subset of human small-cell lung carcinomas. Thus, antagonists that interfere with bombesin receptor-ligand interaction might prove to be of value in treatment of gastrin-releasing peptide (GRP)-responsive tumors. A precise definition of the structure and properties of the bombesin receptors found on human lung cancer cells would provide important information for the design and rational application of such antagonists. Recently, we isolated cDNA clones encoding two distinct receptors for the mammalian bombesin-like peptides, GRP, and neuromedian B (NMB). The two receptors show 56% amino acid identity, encode seven putative transmembrane domains, and are members of the G-protein-coupled receptor superfamily. Ligand-binding studies show that while both receptors can be activated by either GRP or NMB, one receptor has a higher affinity for GRP than for NMB (GRP-R), while the other has a higher affinity for NMB than for GRP (NMB-R). A different spectrum of antagonists is needed to block responses from the two different receptors. These studies indicate that it will be critical in future studies to define which bombesin receptor subtypes are present on a given tumor to optimize the potential therapeutic benefit of antagonists in blocking growth.

Amino Acid Sequence↗

PubMed1992

Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors.

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.

3T3 Cells↗

PubMed1991

Genetic and molecular biological characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component.

The gene affected by five previously isolated temperature-sensitive (ts) mutants (ts 10, ts 18, ts 38, ts 39, ts 44) of vaccinia virus strain WR constituting a single "normal" complementation group has been characterized. Marker rescue and DNA sequence analysis show that the five members of the complementation group map in an open reading frame, ORF 18R, which spans the HindIII I-G junction and has the capacity to encode a 77.6-kDa protein. The nucleotide sequence change responsible for temperature sensitivity in each of the five mutants was determined. Two of the mutants, ts 38 and ts 44, have the identical nucleotide change and may therefore be sisters. Northern blot analysis demonstrates that ORF 18R is transcribed at both early and late times during infection. Two distinct early transcripts have been observed which are 5' coterminal and which contain a 518 nucleotide 5' untranslated region. The long early transcript spans the entire 18R gene while the 3' end of the shorter early transcript maps to an early transcription termination signal contained within the 18R coding sequence. The 5' ends of the late transcripts have been mapped to a family of AUG proximal sites using both S1 nuclease and primer extension analysis. Primer extension analysis also identifies additional late 5' ends which map between nucleotides -500 and -1000 relative to the ORF 18R AUG.

Amino Acid Sequence↗

PubMed1991

Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component.

Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.

Animals↗

PubMed1991

A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase.

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-beta-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the HindIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpo132. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.

Blotting, Southern↗

PubMed1991

Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

Cell Line↗

PubMed1986

Efficient targeted insertion of an unselected marker into the vaccinia virus genome.

A method is described by which an unselected marker can be inserted into the vaccinia virus genome. Cells were infected with defective virus (either isatin-beta-thiosemicarbazone dependent or temperature sensitive) and then cotransformed with a mixture of full-length wild-type genomic vaccinia virus DNA and a cloned vaccinia DNA molecule containing an allele for phosphonoacetic acid resistance. After incubation under conditions which are nonpermissive for the defective virus but which do not select for incorporation of phosphonoacetic acid resistance, the virus yields were assayed for the presence of all markers involved. Phosphonoacetic acid resistance was inserted into an otherwise wild-type genome with an efficiency of 25-33%. This represents an increase in efficiency of 150-to 3000-fold relative to controls. The procedure should be extremely useful for engineering the vaccinia virus genome.

DNA, Viral↗