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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells were dissociated from normal rat pituitaries by a combination of mechanical agitation and enzymatic action, seeded into culture flasks and grown in monolayer culture. When such cells were exposed to an extract of the hypothalamic stalk-median eminence area (HSME) a dose-dependent secretion of ACTH was observed. A 2-h exposure to Ca-free media significantly reduced the HSME-stimulated release of ACTH but the measured levels were still greater than the unstimulated controls. When the 45Ca2+ uptake into the cultured cells was measured both control and HSME-stimulated cells yielded identical results (60-80 nmol Ca/mg cell protein). Upon removal of the calcium associated with the surface coat it was found that HSME actually decreased the cellular uptake of calcium. Since variations in uptake can result from changes in influx or efflux as well as from variations in pool size or turnover times of calcium exchange with intracellular compartments, a series of isotope washout experiments were performed. Neither HSME nor theophyline affected the rate constant of calcium efflux from what is believed to be the cytosol pool to the extracellular media. Both agents, however, prompted a shift of intracellular calcium into a more tightly bound compartment. The data suggest that the calcium required for pituitary hormone secretion is derived primarily from an intracellular rather than extracellular origin. It may be that, via the action of cyclic AMP, such calcium can be mobilized from intracellular stores and shifted to a more tightly bound compartment where it can participate in the intracellular processes associated with secretion.
Mol Cell Endocrinol 1977 Feb
PMID:Involvement of intracellular calcium in hormone secretion from pituitary cells. 19 67

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.
Mol Biol Cell 1992 Jun
PMID:Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum. 149 69

Previous studies allow the construction of three distinct models of the binding of G and arginine within the active site of the Tetrahymena self-splicing preribosomal precursor RNA. These models (base triple, axial I and axial II) are now distinguished by measurements on the specificity of RNAs with nucleotide substitutions at positions spanning the site. Because the semi-conserved unpaired nucleotide 263 has no effect on substrate or inhibitor selection by the Tetrahymena RNA we conclude that the axial I model is improbable. In contrast, data with substituted RNAs and nucleoside analogs suggest that nucleotide 265 makes a hydrogen bond with the substrate. Accordingly the active site appears axial because substrate contacts exist at more than one nucleotide on the 5' side of the P7 helix. The effects of this hydrogen bond are observable in cases where the donor or acceptor is on the RNA, whether nucleotide 265 is a purine or pyrimidine, or whether nucleotide 265 is mispaired, wobble paired or normally paired. This pattern is consistent with the axial II model. Molecular dynamics and energy minimization calculations lead to the same conclusions as these site-directed substitutions; the base triple and axial I models are unstable dynamically. Under thermal agitation, the third model site (axial II) is transformed to a related, but more stable structure, axial III. The axial III active site is characterized by the extrusion of the conserved bulged base 263 from the P7 helix, a semi-pocket for G base formed by stacking of nucleotide 262, and formation of all bonds to the G base originally proposed for both the base triple and axial II sites. Because of these hydrogen bonds the axial III site is also consistent with data on enzymatic specificity. The axial III model indicates an unforeseen capacity for pocket formation within the groove of an RNA helix, suggests that the site may be unusually flexible, and bears on a hypothesis concerning the origin of the genetic code.
J Mol Biol 1991 Dec 20
PMID:An axial binding site in the Tetrahymena precursor RNA. 176 61

Cultured cells provide isolated systems for both biochemical and morphological studies. Previous methods of processing cell culture specimens for electron microscopy (EM) have been limited to sectioning either a monolayer or centrifuged cell suspensions which are not morphologically intact. In our improved method, N-butylglycidyl ether is added to cell cultures (2-5 min with agitation) following in situ fixation (3.0% glutaraldehyde in 0.1 M Pipes, pH 7.2, for 20 min, osmium tetraoxide 4% for 20 min). A thin pliable "sheet" of cells floats free from the plastic culture device and can be manipulated (centrifuged or folded) to obtain a vast number of morphologically intact cells for examination. We have examined several cell types (vascular smooth muscle, lung, liver, and endothelial cells) grown in two types of plastic culture flasks (Nunc and Falcon). This new method provides excellent EM morphology, maximizes the number of cells examined, and allows determination of cell orientation since a remnant of the dissolved flask remains loosely bound to the bottom of the cells.
J Ultrastruct Mol Struct Res 1986 Jan
PMID:Improved transmission electron microscopy (TEM) of cultured cells through a "floating sheet" method. 377 79

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Bangham's report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).
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PMID:Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation. 397 Sep 8

Several approaches to surface membrane stripping have been applied to the adult schistosome. Membrane removal was evaluated by the use of different extrinsic and intrinsic markers of which alkaline phosphatase proved to be the most reliable. After initial studies employing incubation of worms in buffer alone, Triton X-100 or freeze/thaw, the last method was chosen for development. The final method applies a single freeze/thaw step to adult worms in balanced salt solution followed by short bursts of agitation on a vortex mixer to release the tegument. Differential and density gradient steps subsequently yield a final membrane pellet enriched over 130 times in surface alkaline phosphatase. The method has been characterized during its development using electron microscopy and enzyme markers for contaminant worm fractions.
Mol Biochem Parasitol 1983 Oct
PMID:Tegument surface membranes of adult Schistosoma mansoni: development of a method for their isolation. 666 62

Electrophoretic analysis of a 60 min reaction between E. coli endotoxin and the amoebocyte lysate showed that the coagulation reaction was complete by 15 min, with the conversion of coagulogen (21 kDa) to coagulin (17 kDa). Coincident with this observation was the maximal activities at 15 min, of Factor C and proclotting enzyme. On agitation of the coagulin gel clots, bioactive endotoxin was recovered. Densitometric scan of the electrophoretically-resolved proteins showed that the sum of coagulogen and coagulin remained almost constant at various time intervals of the coagulation reaction. Electrophoresis serves as a convincing and visually discernible method of studying the kinetics of coagulation, and defining the onset and completion of gelation. Furthermore, it is a useful means of examining the integrity of fresh lysate preparations based on the presence or absence of the 17 kDa coagulin band.
Biochem Mol Biol Int 1993 Mar
PMID:Electrophoretic analysis of endotoxin-activated gelation reaction of Carcinoscorpius rotundicauda amoebocyte lysate. 849 May 76

The secondary structure of the bovine growth hormone releasing factor analog, [Ile2, Ser8.28, Ala15, Leu27, Hse30] bGRF(1-30)-NH-Ethyl, acetate salt (U-90699F) was studied in solution by Fourier transform infrared and Raman spectroscopies. Spectroscopic studies revealed that concentrated aqueous solutions of U-90699F (100 mg ml-1) undergo a secondary structure transition from disordered coil/alpha-helix to intermolecular beta-sheet. Disordered coil and alpha-helical structure were grouped together in the infrared and Raman studies since the amide I vibrations are close in frequency and overlap in assignments was possible. Before the conformational transition, the facile exchange of the peptide's amide hydrogens for deuterium indicated that the majority of amide hydrogens were readily accessible to solvent. The kinetics of the conformational transition coincided with an increase in solution viscosity and turbidity. An initiation phase preceded the conformational transition during which only minor spectral changes were observed by infrared spectroscopy. The initiation phase and reaction kinetics were consistent with a highly cooperative nucleation ultimately leading to a network of intermolecular beta-sheet structure and gel formation. Increased temperature accelerated the conformational transition. The conformational transition was thermally irreversible but the beta-sheet structure of aggregated or gelled peptide could be disrupted by dilution and agitation.
Spectrochim Acta A Mol Biomol Spectrosc 1997 Oct
PMID:Spectroscopic studies on the conformational transitions of a bovine growth hormone releasing factor analog. 937 14

Whilst embedded within the gut wall the inaccessibility of the enteric nervous system (ENS) is a major drawback for the establishment of an in vitro model for the human ENS. Using a method which combines collagenase digestion, mechanical agitation and manual dissection it was possible to dissect myenteric plexus from human colon of patients at all ages, from newborn to old-age. While complex networks of ganglia and their interconnecting strands could be isolated from newborn gut, the adult tissue allowed only single or small groups of ganglia to be dissected coherently. Pieces of plexus were cultivated on glass coverslips or on plastic sheets respectively. Explants from newborn or older patients displayed different growth patterns. The cytological behaviour of the newborn explants is characterized by an intensive neurite outgrowth. After 1 to 2 days in culture, glial cells start to leave the explant while proliferating and forming a dense cellular carpet. The axons appear partly arranged in bundles, expanding above the glial carpet. Single neurites can leave the carpet and attach directly on the substrate. Semithin sections and EM studies reveal the existence of numerous neurons within the ganglia. The characteristic dense arrangement of neurons, glial cells and neuropil of the myenteric ganglia in vivo was only partly conserved in newborns while in cultured adult ganglia the cells were sparsely scattered throughout the explant with large clefts between the single cells. The ganglia of the adults do not show any considerable neurite outgrowth, which correlates with the low amount of neurons within the ganglia. The migratory behaviour of the adult glial cells is rather moderate, also the proliferation rate compared to the newborn cultures. In general single cells leave the explant without forming an glial carpet as seen in the newborn cultures. The described method delivers an in vitro paradigm for the study of human myenteric plexus. It underlines the differences in growth pattern, neurite outgrowth and glial proliferation from newborn and older children, as well as from adult patients, thus establishing a base line for future studies.
Cell Mol Biol (Noisy-le-grand) 1997 Dec
PMID:Human newborn and adult myenteric plexus grows in different patterns. 948 42

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented.
Mol Gen Genet 2000 Mar
PMID:Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless. 1077 49


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