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Biomedical subjects

G Pan

Publications and source records attributed to G Pan.

At least 91 records · Page 5Linked to original sources
PubMed1995

Homology requirements for ligation and strand exchange by the FLP recombinase.

The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae belongs to the integrase family whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. Ligation takes place when the 5'-OH generated during the cleavage step attacks the phosphotyrosine bond and reforms a phosphodiester bond. When the incoming 5'-OH is from the partner duplex, strand exchange occurs. The FLP recognition target (FRT) contains two inverted 13-base pair (bp) FLP binding sequences that surround an 8-bp core region. It has been shown that heterology in the core regions of the recombinase FLP recognition target sites can dramatically impair recombination. Therefore, it was of interest to study the homology requirements of the core sequence for FLP-mediated ligation. Using nicked duplex substrates containing mismatches in the core sequence, we have demonstrated that the FLP ligation reaction can tolerate mismatches at all positions in the 8-bp core except the position immediately adjacent to the cleavage site. Using half-FRT substrates that contain a single-stranded core sequence, we showed that 4 base pairs adjacent to the cleavage site in the core are required for FLP to execute ligation with a single-stranded oligonucleotide. FLP is also able to ligate the protruding single strand on a half-FRT site to the opposite strand to form a hairpin. We have studied the effect of the base composition of the protruding 8-nucleotide single strand upon the efficiency of hairpin ligation. These studies revealed the importance of intrastrand complementarity in the formation of hairpin by FLP. Hence we conclude that the homology in the position adjacent to the cleavage site is most important, and the degree of the homology required is dependent on the nature of the ligation assay.

Base Composition↗
PubMed1995

[Lactulose hydrogen breath test in small intestinal bacterial overgrowth].

Lactulose hydrogen breath test (LHBT) was evaluated in 21 patients suspected of having small intestinal bacterial overgrowth syndrome and 10 healthy volunteers as control. After dietary preparation and a 12-hour fast, subjects received 15g of lactulose mixed with 40% barium sulfate. The purpose of the barium meal was to reveal the position of lactulose in the intestines. End-expiratory samples of breath were taken at 15 minutes intervals at least for 4 hours. Breath hydrogen was measured with gas chromatography. A positive LHBT was defined as increase of hydrogen concentration in the breath more than 10 x 10(-6) above the baseline value before barium reached the sixth group of small intestine. Cultures were considered positive for bacterial overgrowth when anaerobic counts > or = 10(6) CFU/ml of aspirate. The procedure was carried out under sterile condition. Compared with the bacteriologic culture, LHBT has a sensitivity of 71.4%, specificity of 88.2% and accuracy of 80.6%. These results show that the LHBT is a simple, non-invasive and relatively reliable method for diagnosis of small intestinal bacterial overgrowth.

Adult↗
PubMed1994

Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site.

To further elucidate the mechanism of transcriptional initiation, we used synthetic oligonucleotides to prepare templates containing heteroduplex regions of varying size and location along the DNA of the adenovirus major late promoter. Unlike closed, linear DNA, or DNA with a downstream mismatch, DNA with a mismatch upstream of the initiation site only required the general factors TATA box-binding protein and transcription factor (TF) IIB to direct specific and accurate initiation in vitro by calf thymus RNA polymerase II. In the presence of TFIIF, initiation was possible on closed, linear DNA, but an upstream mismatch region still stimulated transcriptional initiation by more than 100-fold, leading to production of approximately 0.5 transcript/template in the absence of TFIIE, TFIIH, or ATP. The presence of a DNA mismatch was most effective in the -9 to -1 region; furthermore, stimulation by a mismatch did not require that the initiation site be included in the heteroduplex region. Efficient initiation at the immunoglobulin heavy chain promoter in the presence of TATA box-binding protein and TFIIB was also achieved when a mismatch region was introduced from -9 to +3. Our results suggest that initiation by RNA polymerase II in the absence of transcriptional activation is limited by melting of the promoter DNA upstream of the initiation site.

Animals↗
PubMed1993

Identification of the functional domains of the FLP recombinase. Separation of the nonspecific and specific DNA-binding, cleavage, and ligation domains.

The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae binds to its recognition target (FRT) site, induces a bend in the DNA, and promotes DNA cleavage and strand ligation. We have subjected this protein to limited proteolysis and have purified three polypeptides: P13 (13 kDa), P21 (21 kDa), and P32 (32 kDa). These peptides are derived from the following regions of FLP: P13, amino acids 2-123; P21, amino acids 148-346; P32, amino acids 124-423. In this report, we show that P13 binds to DNA nonspecifically and P32, like P21, binds to the FRT site specifically. A single molecule of P32 is able to induce a bend in the DNA of 55 degrees, similar to that induced by intact FLP (63 degrees). P13 enhances the binding of P21 or P32 to the FRT site. Both P32 and P21 can catalyze DNA ligation in combination with P13. P32 can cleave and covalently attach to the FRT site in combination with P13, whereas P21 cannot. These results suggest that FLP contains two DNA-binding domains. A nonspecific DNA-binding region is located in the NH2-terminal 123 amino acids, whereas the region that imparts specific DNA-binding resides in amino acids 148-346. Only the regions in P13 and P21 are needed for ligation activity, but those in P13 and P32 are required for cleavage.

Base Sequence↗
PubMed1993

Ligation of synthetic activated DNA substrates by site-specific recombinases and topoisomerase I.

The FLP protein of the 2-microns plasmid of Saccharomyces cerevisiae is a conservative site-specific recombinase that is involved in the amplification of the plasmid. This recombination reaction proceeds via the covalent attachment of the protein to the 3'-phosphoryl group at the site of the breaks through a phosphotyrosine linkage. We have recently developed an assay that measures FLP-mediated strand ligation independent of FLP-mediated cleavage and covalent attachment to the DNA. The substrate for ligation was produced by FLP-induced cleavage of the FLP recognition site followed by digestion with Pronase and was shown to contain (at least) a tyrosine residue at the 3'-PO4 terminus adjacent to the FLP cleavage sites. We have now synthesized artificial substrates that bear a tyrosine residue on the 3'-PO4 of an appropriate oligonucleotide and find that this substrate is ligated as efficiently as the previous ligation substrates that were isolated after FLP cleavage of the substrate. Analogous substrates for other members of the integrase family of recombinases (lambda integrase protein, P1-Cre protein) as well as for mammalian topoisomerase I are also active as ligation substrates with their cognate protein. This class of activated substrates should be useful in the study of breakage and reunion reactions involving DNA.

Animals↗
PubMed1993

Mutations of the FLP recombinase gene that cause a deficiency in DNA bending and strand cleavage.

We have used site-directed mutagenesis to change several amino acids of the C-terminal portion of the FLP recombinase of Saccharomyces cerevisiae. These residues are absolutely conserved among the six FLP-like proteins from various yeast strains. We have examined the ability of the altered proteins to catalyze recombination in vivo and in vitro and to perform various partial steps of the reaction in vitro. Two of the mutations produced a partial defect in DNA binding but the remainder resulted in normal binding. All of these mutations caused impairment of the ability of the protein to induce the type II bend of the FRT site, and some of these proteins were also defective in DNA strand cleavage. None of the mutations affected the ability of the proteins to perform synapsis between two FRT sites, but some were defective in strand ligation. Interestingly, some mutant proteins showed impairment of the initial stages of the recombination reaction on a linear substrate and yet they maintained the ability to resolve a Holliday intermediate in the reaction. We conclude that this conserved region of the FLP protein is important for the early stage(s) of the recombination reaction.

Amino Acid Sequence↗
PubMed1993

Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

Amino Acid Sequence↗
PubMed1992

Ligation activity of FLP recombinase. The strand ligation activity of a site-specific recombinase using an activated DNA substrate.

The FLP protein of the 2-microns plasmid of yeast belongs to the integrase family of site-specific recombinases whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. We have made an activated DNA substrate and have shown that FLP can promote efficient strand ligation without forming a covalent intermediate with the DNA substrate. The strand ligation activity of FLP is independent of its ability to cleave DNA. Since site-specific recombinases are members of the larger class of topoisomerases, these findings may be generally applicable to other members of this class of enzymes.

Base Sequence↗
PubMed1990

Indirect inhibition of myocyte RNA and protein synthesis by interleukin-1.

Soluble mediators of the inflammatory response may directly influence myocardial function and metabolism in the absence of myocardial cell necrosis. Previous reported experimental data have demonstrated that the monokine interleukin-1 (IL-1) can produce myocardial depression and may influence muscle protein metabolism. To further investigate this hypothesis, IL-1 was added to neonatal rat cardiac muscle cell (MC) cultures with and without additional rat cardiac non-muscle cells (NMC). Incorporation of 3H-uridine or 14C-phenylalanine into acid-insoluble material was utilized as a measure of RNA or protein synthesis. IL-1 in concentrations of up to 500 units/ml had no effect on MC RNA or protein synthesis. When NMC were added to the MC culture, IL-1 exhibited a concentration-dependent inhibition of both RNA and protein synthesis, with effects apparent at concentrations as low as 5 units/ml. Supernatants from IL-1-treated NMC cultures exerted a dose dependent reduction on the incorporation of radiolabeled precursor into MC cultures, suggesting production of a soluble substance mediating the IL-1 effect. Supernatants from IL-1 treated rat skin fibroblasts or rat skeletal muscle myoblasts increased MC radiolabeled precursor incorporation slightly, in contrast to the decrease seen with NMC supernatant. Furthermore, IL-1 treated NMC supernatant had no inhibitory effect on skeletal myoblasts. We conclude that IL-1 decreases protein and RNA synthesis in MC cultures through a second mediator elaborated from the NMC population.

Animals↗
PubMed1990

Osteosarcoma: MR imaging after preoperative chemotherapy.

The authors reviewed 76 magnetic resonance (MR) images of 38 patients with osteosarcoma treated with preoperative chemotherapy (intraarterial cisplatin with or without systemic chemotherapy). Histologic maps of the surgical tumor specimens in 33 cases were correlated with either late-chemotherapy or postchemotherapy MR images. There were four MR patterns--dark, mottled or speckled, homogeneous, and cystic--that corresponded to different amounts of tumor matrix, granulation tissue, hemosiderin deposits, fluid-filled cysts, and residual viable tumor. Nested foci of residual viable tumor could not be specifically identified, although tumor progression or skip metastases were accurately depicted in four patients. Other findings included (a) peritumoral edema in the soft tissues and intramedullary space that shrank with chemotherapy, (b) chemotherapy effect in the surrounding soft tissues, (c) a dark rim around the extramedullary component of the tumors corresponding to a collagenous capsule continuous with the periosteum, (d) development of metaphyseal hemorrhages and bone marrow infarcts, and (e) intramedullary vascular channels.

Adolescent↗
PubMed1990

Stimulatory role of substance P on gonadotropin release in ovariectomized rats.

Substance P (SP) has been shown to be present in the hypothalamus and anterior pituitary. To evaluate a possible physiological role of endogenous SP in the control of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release, specific antiserum against SP (anti-SP) was injected intraventricularly (3 microliters into the third ventricle) or intravenously (50 or 200 microliters) into conscious, ovariectomized (OVX) rats. Third ventricular injection of the antiserum induced a significant decrease in both plasma LH and FSH levels when compared to values in control animals injected with normal rabbit serum (p less than 0.01 and p less than 0.025, respectively). The effect was observed within 10 mi and levels remained suppressed for 60 min. In contrast, intravenous injection of large doses of anti-SP had no effect on the release of both hormones. In order to confirm the stimulatory effect of SP itself, synthetic SP was injected intravenously and intraventricularly into estrogen-primed (E-primed), OVX rats. Synthetic SP dramatically stimulated LH release, but not FSH release when injected either intravenously or intraventricularly at doses of 10 and 50 micrograms (p less than 0.001, p less than 0.005 vs. control, respectively). To investigate any direct action of SP on gonadotropin release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats. SP did not affect the release of gonadotropins in vitro. These results indicate that endogenous hypothalamic SP exerts a tonic stimulatory hypothalamic control of basal gonadotropin release in OVX rats.

Animals↗
PubMed1989

Subtotal cystectomy and antirefluxing hepaticoduodenostomy for choledochus cyst in children.

Total cystectomy with Roux-en-Y hepaticojejunostomy for choledochus cyst often causes malabsorption and other problems as a result of jejunal biliary diversion bypassing the duodenum. Restoration of near normal anatomy can only return the normal physiological harmony and assure normality. A simple antirefluxing hepaticoduodenostomy has proved successful.

Anastomosis, Roux-en-Y↗
PubMed1989

Role of substance P in suppressing growth hormone release in the rat.

To evaluate a possible physiological role of endogenous substance P (SP) in the control of growth hormone (GH; somatotropin) secretion, a specific antiserum against SP (anti-SP) was injected intraventricularly (3 microliters into the third cerebral ventricle) in unanesthetized unrestrained normal male rats. Control rats received an equivalent volume of normal rabbit serum (NRS). Intraventricular injection of the NRS lowered plasma GH concentrations significantly. The lowering was detected on first measurement at 10 min after injection and was maximal at 30 min. This was followed by a return toward the initial levels. Third ventricular injection of antiserum significantly increased plasma GH in comparison with control animals injected with NRS. The effect was observed within 10-20 min, and levels remained elevated for the 120-min duration of the experiment. To confirm the possible inhibitory role of endogenous SP on GH release, 3 microliters of 0.9% NaCl (saline) alone or saline containing a specific antagonist of SP, [D-Pro2,D-Trp7,9]SP, was injected into the third ventricle of normal male rats. The antagonist also increased plasma GH significantly (P less than 0.005) within 5 min compared with values in the saline-injected control group. Levels remained elevated for 30 min but had returned toward control values 60 min after injection. In contrast, synthetic SP significantly decreased plasma GH when injected intravenously or intraventricularly compared with plasma GH in the control saline-injected group. To investigate a possible direct action of SP on GH release from the anterior pituitary gland, we incubated synthetic SP with dispersed anterior pituitary cells for 1 hr. The release of GH from incubated anterior pituitary cells was not affected at any dose of SP (10(-9) to 10(-6) M) tested. These data strongly indicate that endogenous SP has a physiological inhibitory role in the control of GH secretion at the level of the hypothalamus in the male rat.

Animals↗
PubMed1988

Traumatic epidural hematoma of the cervical spine: diagnosis with magnetic resonance imaging. Case report.

A traumatic epidural hematoma of the cervical spine is reported in a 13-year-old girl. The patient recovered spontaneously over several days without surgical intervention. The diagnosis was made on magnetic resonance (MR) imaging, which also demonstrated subsequent resolution of the hematoma. The etiological factors of spinal epidural hematomas are reviewed and the utility of MR imaging in differentiating other causes of acute spinal cord injury is emphasized.

Adolescent↗
PubMed1987

Clot embolic stroke in the vertebrobasilar system of rabbits: a transfemoral angiographic technique.

Previous animal models of thromboembolic stroke have focused upon the carotid artery system. An alternative approach utilizing the vertebrobasilar system in rabbits is presented here. Left vertebral artery catheterization was accomplished relatively simply in this model using transfemoral angiographic techniques. Twenty-four-hour-old clot emboli with calculated volumes in the range of 0.007-0.11 cc were delivered by this route and found to lodge at the juncture of the basilar tip and the Circle of Willis in 11 of 13 rabbits. Intra-arterial infusion of streptokinase in one animal resulted in angiographically complete clearing of clot. Certain technical advantages in this particular approach are discussed.

Animals↗