Showing papers by "University of California, San Francisco published in 1987"

Bilirubin is an antioxidant of possible physiological importance

Abstract: Bilirubin, the end product of heme catabolism in mammals, is generally regarded as a potentially cytotoxic, lipid-soluble waste product that needs to be excreted. However, it is here that bilirubin, at micromolar concentrations in vitro, efficiently scavenges peroxyl radicals generated chemically in either homogeneous solution or multilamellar liposomes. The antioxidant activity of bilirubin increases as the experimental concentration of oxygen is decreased from 20% (that of normal air) to 2% (physiologically relevant concentration). Furthermore, under 2% oxygen, in liposomes, bilirubin suppresses the oxidation more than alpha-tocopherol, which is regarded as the best antioxidant of lipid peroxidation. The data support the idea of a "beneficial" role for bilirubin as a physiological, chain-breaking antioxidant.

Development of a Short Acculturation Scale for Hispanics

Abstract: This article reports the development of a short (12-item) acculturation scale for Hispanics. Separate factor analyses of the responses of 363 Hispanics and 228 non-Hispanic whites produced three fa...

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

Abstract: We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.

Hispanic Familism and Acculturation: What Changes and What Doesn't?

Abstract: This investigation studied the effects of acculturation on attitudinal familism in 452 Hispanics compared to 227 white nonHispanics. Despite differences in the national origin of Hispanics, Mexican-, Central -and Cuban-Americans reported similar attitudes toward the family indicating that familism is a core characteristic in the Hispanic culture. Three basic dimensions of familism were found: Familial obligations, perceived support from the family and family as referents. The high level of perceived family support, invariable despite changes in acculturation, is the most essential dimension of Hispanic familism. Familial obligations and the perception of the family as referents appear to diminish with the level of acculturation, but the perception of family support doesn't change. Although these two dimensions of familism decrease concurrently with the level of acculturation, the attitudes of persons with high levels of acculturation are more familistic than those of white nonHispanics.

Functional inactivation of genes by dominant negative mutations

Abstract: Molecular biologists are increasingly faced with the problem of assigning a function to genes that have been cloned. A new approach to this problem involves the manipulation of the cloned gene to create what are known as 'dominant negative' mutations. These encode mutant polypeptides that when overexpressed disrupt the activity of the wild-type gene. There are many precedents for this kind of behaviour in the literature--some oncogenes might be examples of naturally occurring dominant negative mutations.

The Endocrinology and Developmental Biology of the Prostate

Abstract: I. Introduction MALE accessory sex glands such as the prostate, seminal vesicle, and bulbourethral gland have served in various capacities as models for investigating the action of androgens in regulating epithelial growth, RNA and protein synthesis, and secretory activity. The most extensively studied gland of this group is the prostate. This gland is found exclusively in mammals and produces many components of semen such as fructose, zinc ions, and various proteins important for the formation of the copulatory plug in rodents. The impetus for investigating regulation of prostatic growth and function stems in part from the many pathological complications which affect this gland. The prostate is the site of various types of inflammatory and infectious conditions (1) as well as benign and malignant proliferative changes in aging males. Despite extensive research, little of the pathogenesis or natural history of these diseases has been elucidated (2). Benign prostatic hypertrophy (BPH), a disease in which t...

Constitutive and Regulated Secretion of Proteins

Abstract: SORTING 257 CYTOSKELETON INVOLVEMENT WITH PROTEIN SECRETION 266 SECRETION IN EPITHELIAL CELLS .... 277 RECYCLING OF THE SECRETORY VESICLE MEMBRANE ..... . . 281

Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor.

Abstract: We have detected nuclear localization signals within the 795 amino acid rat glucocorticoid receptor Using a transient expression assay, we monitored by immunofluorescence the subcellular distribution of receptor derivatives and beta-galactosidase-receptor fusion proteins Two distinct nuclear localization signals, NL1 and NL2, were defined NL1 maps to a 28 amino acid segment closely associated, but not coincident with the DNA binding domain; NL2 resides within a 256 amino acid region that also includes the hormone binding domain Most importantly, nuclear localization of fusion proteins containing either the full-length receptor or the NL2 region alone is fully hormone-dependent; similar results were obtained with the wild-type receptor, provided the analysis was performed in medium lacking serum and phenol red The rate of hormone-induced nuclear localization of an NL2-containing fusion protein is consistent with the rapid kinetics of hormone-regulated transcription mediated by the receptor Thus, hormonal control of nuclear localization contributes to the modulation of glucocorticoid receptor transcriptional regulatory activity

Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product.

Abstract: Human immunodeficiency virus-1 (HIV-1) gene expression is controlled by cellular transcription factors and by virally encoded trans-activation proteins of the HIV-1 tat and art/trs genes, which are essential for viral replication1,9–11. Tat trans-activates HIV-1 gene expression by interacting with the trans-acting response element (TAR) located within the HIV-1 long terminal repeat (LTR) (ref. 2). In transient expression assays, tat mediates its effects largely by increasing the steady-state levels of messenger RNA species that contain the TAR sequence at or near their 5′ ends2–4, suggesting a function for tat either in transcription or in subsequent RNA processing. The tat gene could also facilitate translation of mRNA containing the TAR sequence5–8. To determine the mechanism of trans-activation by tat, we analysed the structure and rate of synthesis of RNA species directed by the HIV-1 LTR in transient expression assays both in the presence and absence of tat. Although the rate of HIV-1 transcription initiation was not affected by tat, transcriptional elongation beyond position +59 was seen only in the presence of tat. Thus, tat trans-activates HIV-1 transcription by relieving a specific block to transcriptional elongation within the TAR sequence.

Insulin-like growth factor II receptor as a multifunctional binding protein.

Abstract: The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.

An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A.

Abstract: We report the development of a rapid nonradioactive technique for the genetic prediction of human disease and its diagnostic application to hemophilia A. This method is based on enzymatic amplification of short segments of human genes associated with inherited disorders. A novel feature of the procedure is the use of a heat-stable DNA polymerase, which allows the repeated rounds of DNA synthesis to proceed at 63 degrees C. The high sequence specificity of the amplification reaction at this elevated temperature permits restriction-site polymorphisms, contained in the amplified samples, to be analyzed by visual inspection of their digestion products on polyacrylamide gels. By means of this method, we have performed carrier detection and prenatal diagnosis of hemophilia in two families with use of the factor VIII intragenic polymorphisms identified by the restriction enzymes BclI and XbaI. Predictions can be made directly from chorionic villi, without previous DNA extraction, and fetal sex can be determined by amplification of sequences specific for the Y chromosome. Specific amplification of genomic sequences with heat-stable DNA polymerase is applicable to the diagnosis of a wide variety of inherited disorders. These include diseases diagnosed by restriction-site variation, such as Duchenne's muscular dystrophy and sickle cell anemia, those due to a collection of known mutations, such as beta-thalassemia, and those due to gene deletion, such as alpha-thalassemia.

Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth

Abstract: Angiogenesis, the formation of new capillaries, which is observed in embryonic and injured tissue and is particularly prominent in the vicinity of solid tumours1, involves the migration and proliferation of capillary endothelial cells. It is probably triggered by agents, such as basic fibroblast growth factor (bFGF), thought to be released from tissues adjacent to proliferating capillaries1. As well as being a potent inducer of cell division in capillary endothelial cells in vitro, bFGF can act as an angiogenic agent in vivo2. It is present in a wide variety of richly vascularized tissues including brain, pituitary, retina, adrenal gland, kidney, corpus luteum, placenta and various tumours1–3. So far, however, the normal bFGF-producing cell species in these tissues have not been identified2,4. We report here that capillary endothelial cells express the bFGF gene, that they produce and release bFGF and that bFGF derived from them can stimulate the proliferation of capillary endothelial cells. We conclude that bFGF can act as a self-stimulating growth factor for capillary endothelial cells, and that it is possible that the formation of new capillaries is induced by capillary endothelial cells themselves.

Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.

Abstract: On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.

Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers?

Abstract: Endothelial cells line the inner surface of blood vessels and act as the main barrier to the passage of cells and large molecules from the blood stream to the tissues. Recent interest in the part played by the endothelium in regulating vascular tone has focused on the synthesis and secretion of prostacyclin and an endothelium-derived relaxing factor. Endothelial cells respond to blood-borne agonist, but how the endothelium senses and responds to mechanical forces generated by the flow of blood under pressure is not known. Here we report patch-clamp recordings of ion channel activity from cell-attached membrane patches on aortic endothelial cells. In most of the patches examined, we observed unitary inward currents associated with the opening of a cation-selective channel (approximately 40 pS in standard saline). The channel is permeable to Ca2+ and its opening frequency increases when the membrane is stretched by applying suction through the patch electrode. The presence of mechanotransducing ion channels in endothelial cells may help explain how the endothelium mediates vascular responses to haemodynamic stresses.

Congenital malformations of the inner ear: a classification based on embryogenesis

Abstract: Approximately 20% of patients with congenital sensorineural hearing loss have radiographic abnormalities of the inner ear. A broad spectrum of anomalous patterns have been described, most of which have been lumped together under the term "Mondini's dysplasia." We feel that this grouping of many dissimilar entities under a single umbrella term is unwarranted. Based on a review of 63 patients with 98 congenitally malformed ears, we have been able to recognize a number of distinct anatomic patterns from their radiographic appearance. A remarkable similarity between these morphologies and the appearance of the inner ear at various stages of embryogenesis was found. This led us to propose a classification system based upon the theory that these deformities result from an arrest of development during varying stages of inner ear organogenesis.

Visual Motion Processing and Sensory-Motor Integration for Smooth Pursuit Eye Movements

Abstract: The function of smooth pursuit is to keep the fovea pointed at a small visual target that moves smoothly across a patterned background. Chemical lesions, single cell recordings, and behavioral measures have shown that the cortical motion processing pathways form the afferent limb for pursuit. Important areas include at least the striate cortex and the middle temporal visual area, and probably the medial superior temporal visual area and the posterior parietal cortex. We argue that the visual inputs are transmitted through a simple sensory motor interface in the pons, to the efferent limb in the brain stem and cerebellum. The efferent limb uses neural velocity memory to maintain pursuit automatically. We present evidence that the velocity memory is provided, at least in part, by eye velocity positive feedback between the flocculus of the cerebellum and the brain stem. Finally, we use a computer model to show how the maintenance of pursuit can be simulated on a millisecond time scale. The structure and internal elements of the model are based on the biological experiments reviewed in our paper. In the past five years, progress on the neural basis of pursuit eye movements has been rapid. Several areas of research have made substantial contributions, by using combinations of new and conventional methods. Many of the pathways that contribute to pursuit have been identified, and their physiological activity and functions are becoming understood. Continuing progress promises to yield an understanding of one specific form of visually guided movement, at the level of neuronal circuits and behavior, in the primate.

Regulation of ACTH secretion: variations on a theme of B.

Abstract: Publisher Summary This chapter discusses the regulation of function in the adrenocortical system. There are three major characteristics that describe most changes in activity of the system: (1) the circadian rhythm in basal activity, (2) stress-induced activation, and (3) corticosteroid feedback regulation. The first two may occur on very different timescales, and it is probably a consequence of the differing temporal demands that the third, corticosteroid feedback inhibition, is exerted by a variety of mechanisms over time. At any time of the day, the adrenocortical system can be activated by application of stressors. These include alteration of the value of a regulated variable or may be invoked by subjecting an animal to sudden disturbances of its environment, for example, noise, flashing lights, handling, strange environment, mild heat or cold. The adrenocortical system may be activated by traumatic stimuli or by psychological stimuli—in man, examinations, or mental arithmetic, or medical school admissions exams; in rats, unexpected decreases in food rewards.

Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.

Abstract: Potassium currents are crucial for the repolarization of electrically excitable membranes, a role that makes potassium channels a target for physiological modifications that alter synaptic efficacy. The Shaker locus of Drosophila is thought to encode a K+ channel. The sequence of two complementary DNA clones from the Shaker locus is reported here. The sequence predicts an integral membrane protein of 70,200 daltons containing seven potential membrane-spanning sequences. In addition, the predicted protein is homologous to the vertebrate sodium channel in a region previously proposed to be involved in the voltage-dependent activation of the Na+ channel. These results support the hypothesis that Shaker encodes a structural component of a voltage-dependent K+ channel and suggest a conserved mechanism for voltage activation.

Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro.

Abstract: 1 The actions of serotonin (5-HT) on pyramidal cells of the CA1 region of the rat hippocampus were characterized using intracellular recording in in vitro brain slices 2 5-HT typically evokes a biphasic response consisting of a hyperpolarization which is followed by a longer-lasting depolarization These effects on membrane potential are accompanied by a decrease in the calcium-activated after-hyperpolarization (ahp) 3 Detailed analysis using 5-HT antagonists and agonists indicates that the hyperpolarization is mediated by a 5-HT1A receptor Spiperone is the most effective antagonist of the response and the selective 5-HT1A agonist, 8-OHDPAT, behaves as a partial agonist at this receptor In agreement with the distribution of 5-HT1A binding sites, responses to 5-HT were most prominent in the stratum radiatum 4 The hyperpolarizing response is associated with a decrease in input resistance, is blocked by extracellular barium and intracellular caesium, is unaffected by the chloride gradient, and its reversal potential shifts with the extracellular concentration of potassium as predicted for a response mediated by a selective increase in potassium permeability 5 The depolarizing response and reduction in the ahp could be studied in isolation by blocking the hyperpolarizing response with either pertussis toxin or spiperone The pharmacology of these responses did not correspond to that of any of the 5-HT binding sites reported in CNS tissue Although the depolarization and blockade of the ahp have the same time course it is unclear if they are mediated by the same or different receptors 6 The depolarization most likely results from a decrease in resting potassium conductance However, neither a blockade of the M current nor the ahp current can account for the depolarization 7 Blockade of phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) did not enhance the depressant action of 5-HT on the ahp, making it unlikely that this action is mediated by cyclic AMP 8 Blockade of the ahp by 5-HT reduces spike frequency adaptation and counteracts the inhibitory action of 5-HT on 5-HT1A receptors This excitatory action outlasts the hyperpolarizing action 9 In summary 5-HT acts on at least two distinct receptors on hippocampal pyramidal cells, one coupled to the opening of potassium channels and a second coupled to a decrease in a resting potassium conductance and a decrease in the ahp

Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones.

Abstract: 1. Intracellular recording from hippocampal CA1 pyramidal cells in the slice preparation was used to analyse the pharmacological sensitivity of action potential repolarization and the hyperpolarizations that follow the action potential. The Ca2+-activated after-hyperpolarizations (a.h.p.s) could be divided into a fast a.h.p. with a time course of milliseconds, and a slow a.h.p. which lasted for a few seconds at a temperature of 30 degrees C. 2. The repolarization of the action potential is sensitive to the Ca2+ channel blocker Cd2+. This effect is simultaneous with a block of the fast a.h.p. which follows immediately upon the repolarization of the action potential. The slow a.h.p. was also blocked by Cd2+. 3. Low concentrations of the K+ channel blocker, tetraethylammonium (TEA; 200-500 microM), block the fast a.h.p. and slow down action potential repolarization. The slow a.h.p. was not affected by low concentrations of TEA. 4. The action potential repolarization and the fast a.h.p. are also reversibly sensitive to charybdotoxin. This agent had no effect on the slow a.h.p. 5. When EGTA or BAPTA were added to the normal recording electrolyte (KMeSO4), the generation of slow a.h.p.s was prevented. In addition, cells impaled with BAPTA-containing electrodes displayed broader action potentials and much reduced fast a.h.p.s compared to recordings made with electrodes containing KMeSO4 alone or with EGTA. 6. The slow a.h.p. can be eliminated by noradrenaline, 8-bromocyclic AMP or carbachol. Under these conditions there are no effects on the fast a.h.p. or on action potential duration. 7. Block of the fast a.h.p. with TEA or CTX (charybdotoxin) is associated with an increased frequency of the first few action potentials during a depolarization. This is a quite distinct effect from the greatly increased number of action potentials which results from block of the slow a.h.p. 8. The results support a conclusion that the fast a.h.p. is generated by the TEA- and voltage-sensitive Ca2+-activated K+ current, IC. This current is involved in spike repolarization and turns off upon the return to resting potential. Thus block of IC has no effect on the slow a.h.p. which is caused by a separate membrane current.

Primary structure and biochemical properties of an M2 muscarinic receptor

Abstract: A partial amino acid sequence obtained for porcine atrial muscarinic acetylcholine receptor was used to isolate complementary DNA clones containing the complete receptor coding region. The deduced 466-amino acid polypeptide exhibits extensive structural and sequence homology with other receptors coupled to guanine nucleotide binding (G) proteins (for example, the beta-adrenergic receptor and rhodopsins); this similarity predicts a structure of seven membrane-spanning regions distinguished by the disposition of a large cytoplasmic domain. Stable transfection of the Chinese hamster ovary cell line with the atrial receptor complementary DNA leads to the binding of muscarinic antagonists in these cells with affinities characteristic of the M2 receptor subtype. The atrial muscarinic receptor is encoded by a unique gene consisting of a single coding exon and multiple, alternatively spliced 5' noncoding regions. The atrial receptor is distinct from the cerebral muscarinic receptor gene product, sharing only 38% overall amino acid homology and possessing a completely nonhomologous large cytoplasmic domain, suggesting a role for the latter region in differential effector coupling.

Structure, biochemistry, and assembly of epithelial tight junctions

Abstract: The zonula occludens (ZO), also referred to as the tight junction, forms the barrier to the diffusion of molecules and ions across the epithelial cell layer through the paracellular space. The level of electrical resistance of the paracellular pathway seems to depend on the number of strands in the ZO observed by freeze-fracture electron microscopy (EM). The ZO also forms the boundary between the compositionally distinct apical and basolateral plasma membrane domains because it is a barrier to the lateral diffusion of lipids and membrane proteins that reside in the extracytoplasmic leaflet of the membrane bilayer. In contrast to its appearance in transmission EM, the tight junction is not a fusion between the outer membrane leaflets of neighboring cells. Rather it consists of protein molecules, including the newly discovered protein ZO-1 and probably others, which bring the plasma membranes into extremely close apposition so as to occlude the extracellular space. Very little is known about the assembly of tight junctions, but several kinds of evidence suggest that they are very dynamic structures. Other elements of the epithelial junctional complex including the zonula adherens (ZA), the Ca2+-dependent cell adhesion molecule uvomorulin, or L-CAM, and actin filaments of the cytoskeleton may participate in the assembly of the ZO.

Transgenic mice with increased Cu/Zn-superoxide dismutase activity: animal model of dosage effects in Down syndrome.

Abstract: Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9- and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. Cu/Zn-superoxide superoxide dismutase activity is increased from 1.6- to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.

Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding

Abstract: Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity (approximately equal to 9.3 micrograms of G.R. LDL per ml were required to displace 50% of 125I-labeled normal LDL, vs. approximately equal to 3.0 micrograms of normal LDL per ml). Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated with an abnormal lipid composition or structure of the LDL: the chemical and physical properties of the particles were normal, and partial delipidation of the LDL did not alter receptor binding activity. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients.