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Biomedical subjects

L Lee

Publications and source records attributed to L Lee.

At least 307 records · Page 17Linked to original sources
PubMed1986

Centrally induced cardiovascular and sympathetic responses to adrenocorticotrophic hormone.

To investigate the effects of adrenocorticotrophic hormone on central cardiovascular regulation, an intracerebroventricular injection of the drug was given to male Wistar rats. With doses of 1 microgram and 10 micrograms per rat, blood pressure began to rise within 1 min, attaining a maximum value 5-10 min later. Both heart rate and abdominal sympathetic nerve activity increased simultaneously with the rise in blood pressure. Injection into the fourth ventricle or intravenous administration of the drug elicited no appreciable cardiovascular responses. These results suggest that endogenous adrenocorticotrophic hormone produced locally in the hypothalamus may participate in central cardiovascular regulation by increasing sympathetic outflow.

Action Potentials↗
PubMed1986

Hydrophobic binding domains of rat intestinal maltase-glucoamylase.

Rat intestinal microvillus maltase-glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Sephadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Sepharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with ethanol--formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26-28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase-glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.

Affinity Labels↗
PubMed1986

Immunochemical distribution of interphotoreceptor retinoid-binding protein in selected species.

An enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphotoreceptor retinoid-binding protein (IRBP) in tissues of monkey and other species using rabbit antiserum against monkey IRBP. A 1:7500 antiserum dilution was found optimal with the linear range extending to 250 micrograms IRBP/ml. The highest IRBP concentration was found in cannulation fluid of the monkey interphotoreceptor space, although vitreous and aqueous humors also contained IRBP. The presence of IRBP in the vitreous was confirmed by Western blot and 3H-retinol binding studies. The pineal gland of monkey and rat also was found to contain IRBP, as assessed by ELISA and immunocytochemistry; IRBP was below the limits of detection in turtle and chicken retina. IRBP levels were uniformly low in retinas of human cases with hereditary retinal degeneration, including retinitis pigmentosa (three cases) and choroideremia (two cases). The presence of IRBP in pineal as well as in the vitreous and aqueous humors may indicate a broader role for this putative retinoid-transport protein than previously suspected.

Animals↗
PubMed1986

Reappraisal of endocervical curettage in predicting cervical involvement by endometrial carcinoma.

Determining if there is cervical invasion by endometrial carcinoma is critical to both staging and therapy. The dependability of endocervical curettage (ECC) in the staging of endometrial cancer is seldom subject to review. This study appraised this dependability. The results indicated that the dependability of ECC in assessing cervical invasion by endometrial cancer is in serious question. Making the determination solely on the basis of ECC would result in the overtreatment of six of seven patients with positive ECC. In view of this high false-positive rate, direct visual inspection and biopsy of the endocervical canal with endoscopy might be warranted before definitive therapy.

Cervix Uteri↗
PubMed1985

Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells.

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.

Amidohydrolases↗
PubMed1985

Evidence for the phosphorylation of the type II insulin-like growth factor receptor in cultured cells.

The ATP pools of monolayer cultures of rat embryo fibroblasts and rat liver cells (BRL-3A2) were labeled with [32P]H3PO4. The type II insulin-like growth factor (IGF) receptor was purified by affinity chromatography on wheat germ lectin-Sepharose and IGF-II-Sepharose columns. A phosphorylated species having the expected size of the type II receptor (Mr = 220,000 without reduction, Mr = 260,000 with reduction) was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. IGF-II stimulated phosphorylation of the type II receptor in BRL-3A2 rat liver cells. Lability of the receptor phosphate bonds to alkaline pH suggests that the bulk of phosphorylation was occurring on serine residues.

Animals↗
PubMed1985

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas.

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina.

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.

Amino Acid Sequence↗
PubMed1985

Butyrate enhances the synthesis of interphotoreceptor retinoid-binding protein (IRBP) by Y-79 human retinoblastoma cells.

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) from Y-79 human retinoblastoma cells was investigated using immunocytochemistry and SDS-polyacrylamide gel electrophoresis. Indirect immunofluorescence of cells growing in monolayer culture for 11 and 13 days showed no significant IRBP staining although by SDS-polyacrylamide gel electrophoresis, a small amount of IRBP was detected in the culture medium, suggesting synthesis and extracellular secretion. Butyrate (2mM) treatment of cells starting on the eighth day of culture resulted in a dramatic increase of IRBP fluorescence 3-5 days after treatment. Treatment of cells in all conditions with 1 microM monensin for 3 h showed concentration of IRBP in the Golgi apparatus of about 10-20% of cells as proved by a double immunofluorescent technique, employing anti-IRBP antibody and wheat-germ agglutinin. Incubation of cells with either radiolabeled amino acids or glucosamine followed by analysis of cell cytosol and culture medium by SDS-polyacrylamide gel electrophoresis also confirmed that 1) IRBP is synthesized by the Y-79 cells and secreted into the medium and 2) its production is markedly increased by butyrate treatment. The enhancement of IRBP synthesis by butyrate suggests biochemical differentiation of Y-79 cells possibly into photoreceptor-like cells and offers a new system for studying the properties of this unique retinoid-binding protein and of factors that control its synthesis and secretion.

Amino Acids↗
PubMed1985

Structural studies of calcium-binding proteins using nuclear magnetic resonance.

Lanthanide-shifted 1H nuclear magnetic resonance (NMR) spectroscopy has been used to compare the structure in solution of the EF-hand calcium-binding domains of four parvalbumins (isoelectric pH[pI] 3.95, 4.25, and 4.37 from carp, and pI from buffalo fish). These four parvalbumins are shown by NMR to have very similar structures at the level of resolution typical of x-ray structures. At the higher resolution possible by the lanthanide NMR technique, specific differences are noted between the pI 3.95 isoprotein from carp and the other two carp isoproteins, and the buffalo fish parvalbumin is shown to be different from all three carp isoproteins. The differences are estimated to correspond to changes of the order of 0.2 A in the positions of some of the nuclei surrounding the EF calcium site.

Animals↗
PubMed1985

Dominantly inherited retinitis pigmentosa. Ultrastructure and biochemical analysis.

A 66-year-old white man had dominant retinitis pigmentosa. He developed progressive restriction of his visual field, night blindness, pallor of the optic discs, pigmentary retinopathy and posterior subcapsular cataracts. Postmortem examination of the eyes included electron microscopy and biochemical analysis of cyclic nucleotides and interphotoreceptor retinoid-binding protein (IRBP). Except for the fovea and periphery, the retina showed extensive gliosis and neuronal loss with loss of photoreceptor cells. The choriocapillaris was variably occluded in the regions of absent retinal pigment epithelium (RPE). In places, the pigment epithelium invaded the retina to the level of the internal limiting membrane. Biochemical analysis revealed that the interphotoreceptor retinoid-binding protein (IRBP), an important glycoprotein of the interphotoreceptor space, was virtually absent even in retinal areas where photoreceptor cells were still present. Cyclic nucleotide determinations indicated a decrease in the cyclic GMP concentration that reflected the general loss of photoreceptor elements. On the other hand the cyclic AMP levels in all retinal areas tested were abnormally elevated, indicating the possible involvement of this nucleotide in the pathogenesis of the disease.

Aged↗
PubMed1985

Oligomeric structure of the multifunctional protein CAD that initiates pyrimidine biosynthesis in mammalian cells.

The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240-kDa poly-peptide chain are folded into separate structural domains that have discrete functions. Previous studies suggested that CAD forms predominantly trimers. The trimers are found to be in slow equilibrium with hexamers and higher oligomers composed of multiples of three copies of the CAD polypeptide chain. However, quantitative chemical crosslinking studies of CAD with dimethyl suberimidate were used here to show a progressive conversion of monomer to crosslinked hexamer. High levels of the hexamer accumulate in the reaction mixture, suggesting that the major oligomeric form is hexameric, although residual amounts of smaller oligomers remain present. Larger oligomers may form by association of hexamers and are seen after longer crosslinking times. Sucrose gradient centrifugation shows a 20.8S species to be the slowest sedimenting peak, while the larger species sediments at 27.9S. Electron microscopic studies of rotary-shadowed preparations of CAD have confirmed that, while small amounts of other oligomeric forms are present, the CAD monomer is primarily associated into cyclic hexamers with an open planar appearance.

Animals↗
PubMed1985

Mapping of proteolytic and cyanogen bromide peptides from subunits of intestinal maltase-glucoamylase: evidence for significant homology.

Rat intestinal maltase-glucoamylase was purified in the presence of detergent and proteolytic inhibitors, and the 130000 and 145000 subunits were separated and isolated by preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic elution. Amino acid analyses were very similar, with a small excess of apolar amino acid residues in the 145000 subunit. Peptide mapping with Staphylococcus aureus V8 protease revealed eight similar peptide products for each, with apparent elongation of the five larger peptides in the 145000 subunit by a relative mass (Mr) of 2000-5000. alpha-Chymotrypsin maps showed at least eight identical cleavage products plus one large, shared product which was larger by a Mr of 5000 in the 145000 subunit. Cyanogen bromide cleavage of the 145000 subunit produced a single peptide of Mr 75000. A peptide of Mr 66000, also indicative of a central cleavage, was generated from the 130000 subunit, but a second cleavage into 43000 and 23000 segments was also evident. Several sets of antibodies formed against both antigens consistently gave reactions of identity without spurring on immunodiffusion. These results indicate extreme homology between the central segment of the 145000 subunit and the 130000 subunit. The cyanogen bromide cleavage results suggest, however, that the two central sequences are not absolutely identical and therefore that one subunit may not be a posttranslational derivative of the other.

Amino Acids↗
PubMed1985

Characteristics of Crimean-Congo hemorrhagic fever virus (Xinjiang strain) in China.

Virus strains isolated from blood of patients during a hemorrhagic fever outbreak in 1968 in southern Xinjiang, China, from Hyalomma asiaticum and from sheep, were found to be identical or closely related to Crimean-Congo hemorrhagic fever (C-CHF) virus by complement fixation and indirect immunofluorescence tests with convalescent sera of patients and with C-CHF reference antibody. The virus was inactivated by ether and acid. Viral synthesis was not suppressed by 5-iododeoxyuridine suggesting an RNA-containing genome. The buoyant density in sucrose was 1.16-1.18 g/cm3. The particle weight was estimated at 3.26 +/- 0.46 X 10(8). The diameter of the virus particles was 85-105 nm.

Antibodies, Viral↗
PubMed1984

Synthesis of interphotoreceptor retinoid-binding protein (IRBP) by monkey retina in organ culture: effect of monensin.

Whole monkey retinas were incubated in short-term organ culture with either radiolabeled amino acids or glucosamine. Soluble retinal proteins and proteins in the culture medium were analyzed by SDS-poly-acrylamide gel electrophoresis. Fluorography showed that the interphotoreceptor retinoid-binding protein (IRBP), a 146,000 Mr glycoprotein localized in the extracellular matrix, is synthesized by the neural retina and rapidly secreted into the medium. Secretion is blocked by 10-5M monensin. No significant IRBP synthesis was observed in the pigment-epithelium-choroid complex. IRBP is thus the major component synthesized and secreted by the neural retina into the interphotoreceptor space. This, and its affinity for retinoid makes it a prime candidate for an extracellular retinoid transport vehicle.

Animals↗
PubMed1984

Zein synthesis in the embryo and endosperm of maize mutants.

Zein synthesis in the developing (22 and 50 days postpollination) endosperm and embryo of maize (Zea mays L.) double mutants, brittle-1; opaque-2 and brittle-2;opaque-2, were compared and correlated with sucrose concentration and RNase activity in order to test the hypothesis that high sucrose concentrations may prevent the interaction between zein polyribosomes and endoplasmic reticulum and make the zein mRNAs more susceptible to hydrolysis by high RNase activity, resulting in a severe reduction in zein synthesis. The double-mutant combinations of opaque-2 with each of the starch-deficient mutants, brittle-1 and brittle-2, maintained not only a high sucrose concentration in the endosperm but also a higher RNase activity than either one of the single mutants alone. Consequently, these double mutants severely suppressed the synthesis of two major zein components in their endosperms. In contrast to the endosperm system, embryos of the double mutants produced amounts of zein (and electrophoretic patterns) similar to that of the opaque-2 embryo, and their embryos contained levels of sucrose and RNase activity comparable to that of the o2 and normal control. These results are consistent with the notion that a posttranscriptional degradation of zein mRNAs by RNase, rather than a specific transcriptional block, is involved in the endosperm to suppress zein synthesis in these double mutants.

Amino Acids↗
PubMed1984

Prognostic significance of morphology of tumor and retroperitoneal lymph nodes in epithelial carcinoma of the ovary. I. Correlation with lymph node metastasis.

The prognostic indices based on a morphologic study of tumor and retroperitoneal lymph nodes in 63 patients with epithelial carcinoma of the ovary are reported. The purpose of the study was to identify those variables most frequently related to nodal involvement. The cases in the series consisted of 11 Stage I, 10 Stage II, 34 Stage III, and 8 Stage IV. Histologic distribution was 60.4% serous type, 11.1% mucinous, 6.3% endometrioid, 6.3% clear cell, and 15.9% unclassified. All patients had maximal surgery and selective biopsy of para-aortic and pelvic lymph nodes. The results showed statistically significant variables associated with nodal metastasis in both primary tumor and regional lymph nodes. The indices in primary tumor were grade of tumor, vascular invasion, lymphocytic infiltration, and stromal fibrosis; those in lymph node were type of lymph node reaction, sinus histiocytosis, and fibroblastic proliferation. The nodes with lymphocyte depletion were associated with nodal spread in 81.3% of cases. It is concluded that morphologic study of tumor and lymph node could identify prognostic factors predicting regional nodal metastasis in ovarian carcinoma.

Female↗