Medicine and Health Sciences Commons^™

Fibril In Senile Systemic Amyloidosis Is Derived From Normal Transthyretin., Per Westermark, Knut Sletten, Bjorn Johansson, Gibbons G. Cornwell

Dartmouth Scholarship

The amyloid fibril in senile systemic amyloidosis (SSA), like that of familial amyloidotic polyneuropathy, is derived from transthyretin (TTR). SSA, however, is a common disease, affecting to some degree 25% of the population greater than 80 years old. In familial amyloidotic polyneuropathy, the amyloidogenesis has been considered to depend on point mutations leading to TTR variants. We show that the TTR molecule in SSA, on the other hand, has a normal primary structure. Factors other than the primary structure of TTR must therefore be important in the pathogenesis of TTR-derived amyloid.

Patterns Of Polyadenylation Site Selection In Gene Constructs Containing Multiple Polyadenylation Signals., Roger Denome, Charles Cole

Dartmouth Scholarship

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 …

K+ Efflux In Nih Mouse 3t3 Cells And Transformed Derivatives: Dependence On Extracellular Ca2+ And Phorbol Esters., Martin Lubin

Dartmouth Scholarship

In culture medium deficient in Ca2+, NIH mouse 3T3 cells lose K+, gain Na+, and stop growing. A marked increase in the rate of K+ efflux accounts for this loss; Na+, K+-ATPase pump activity increases but does not fully compensate for enhanced K+ efflux. Phorbol esters and cycloheximide inhibit K+ loss in Ca2+-deficient medium. Phorbol esters inhibit K+ efflux from human fibroblasts as well, even at physiological levels of Ca2+. Two cell lines derived from NIH-3T3, one transformed by a simian virus 40 deletion mutant, the other by the polyoma virus oncogene encoding the middle-sized tumor antigen, retain K+ and …

Dual Mechanisms Of Regulation Of Type I Iodothyronine 5'-Deiodinase In The Rat Kidney, Liver, And Thyroid Gland. Implications For The Treatment Of Hyperthyroidism With Radiographic Contrast Agents., Donald L. St Germain

Dartmouth Scholarship

Alterations in thyroid hormone status and the administration of radiographic contrast agents can markedly influence iodothyronine metabolism and, in particular, the activity of type I 5'-deiodinase (5'DI). In the present studies, the mechanisms responsible for these effects have been reassessed. As previously reported, the addition of iopanoic acid (IOP) to broken cell preparations resulted in a competitive pattern of 5'DI inhibition. However, the in vivo administration to rats of IOP or 3,3',5'-triiodothyronine (rT3) resulted in a noncompetitive pattern of inhibition of 5'DI in the liver, kidney, and thyroid gland, whereby marked decreases in maximal enzyme velocity (V max) were noted, …

Regulation Of Lymphokine Production And Human T Lymphocyte Activation By 1,25-Dihydroxyvitamin D3. Specific Inhibition At The Level Of Messenger Rna., William F.C. Rigby, Sylvia Denome, Michael W. Fanger

Dartmouth Scholarship

The steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (calcitriol), has been shown to inhibit T cell proliferation, primarily through inhibition of interleukin 2 (IL-2) production. In these experiments, we show that calcitriol also markedly inhibited production of the lymphokine, gamma interferon (IFN-gamma), by activated human T lymphocytes. Regulation of both IL-2 and IFN-gamma production as well as transferrin receptor (TfR) expression by calcitriol was apparent at the messenger RNA (mRNA) level as determined by Northern blotting. The decrease in IL-2 and IFN-gamma mRNA that occurred with calcitriol treatment was coordinate and not apparent up to 12 h after phytohemagglutinin stimulation, whereas decreased …

Regulation By Adrenal Corticosteroids Of Sodium And Potassium Transport In Loop Of Henle And Distal Tubule Of Rat Kidney., Bruce A. Stanton

Dartmouth Scholarship

Studies were conducted to examine the effects of adrenalectomy (ADX) and selective, physiological adrenal corticosteroid replacement on sodium and potassium transport by the superficial loop of Henle and distal tubule of rat kidney in vivo. In the loop of Henle, ADX inhibited sodium reabsorption by 33%. Whereas dexamethasone had no effect on reabsorption, aldosterone increased sodium transport to control levels. Thus, physiological levels of mineralocorticoids, but not glucocorticoids, control a fraction of sodium reabsorption in the loop of Henle. ADX also inhibited potassium reabsorption in the loop of Henle. Both dexamethasone and aldosterone reversed the inhibition, although only aldosterone increased …

Absence Of A Structural Basis For Intracellular Recognition And Differential Localization Of Nuclear And Plasma Membrane-Associated Forms Of Simian Virus 40 Large Tumor Antigen., Donald L. Jarvis, Charles N. Cole, Janet S. Butel

Dartmouth Scholarship

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- …

Evidence Against The Hypothesis That Prostaglandins Are The Vasodepressor Agents Of Pregnancy. Serial Studies In Chronically Instrumented, Conscious Rats., Kirk P. Conrad, Mary C. Colpoys

Dartmouth Scholarship

Renal hemodynamics increase dramatically during pregnancy, and pressor responsiveness to exogenous administration of vasoconstrictors is attenuated. We investigated whether or not vasodilatory prostaglandins mediate these phenomena. Trained, chronically instrumented, conscious pregnant rats were used. Control values of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were elevated at midgestation (P less than 0.01 and P = 0.05 from prepregnant means, respectively), and effective renal vascular resistance was decreased (P = 0.05). Indomethacin (4.5-6.5 mg/kg body weight [BW]) failed to decrease renal hemodynamics at this stage of pregnancy; in fact, it raised GFR somewhat further (P less than 0.05). …

Identification Of Sequences In The Herpes Simplex Virus Thymidine Kinase Gene Required For Efficient Processing And Polyadenylation., Charles N. Cole, Terryl P. Stacy

Dartmouth Scholarship

The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an S1 nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 …

Comparative Study Of Pituitary-Thyroid Hormone Economy In Fasting And Hypothyroid Rats., Donald L. St Germain, Valerie A. Galton

Dartmouth Scholarship

Starvation in laboratory rodents results in significant alterations in thyroid hormone economy characterized by decreased circulating levels of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) and a decline in serum thyrotropin (TSH) concentration. To investigate this apparent paradox, we have compared in fasted and hypothyroid animals the intracellular parameters mediating thyroid hormone action in the anterior pituitary gland. In vitro saturation analysis combined with quantitation of nuclear T3 content by radioimmunoassay allowed for characterization of pituitary nuclear T3 receptors and estimation of the endogenous fractional receptor occupancy. In rats, thyroidectomized 4 wk earlier, the 10-fold increase in serum TSH levels and decline …

Two Separable Functional Domains Of Simian Virus 40 Large T Antigen: Carboxyl-Terminal Region Of Simian Virus 40 Large T Antigen Is Required For Efficient Capsid Protein Synthesis., Joanne Tornow, Maryellen Polvino-Bodnar, George Santangelo, Charles N. Cole

Dartmouth Scholarship

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T …

Interleukin 2 Regulates Its Own Receptors., Kendall A. Smith, Doreen A. Cantrell

Dartmouth Scholarship

The cell surface density of high-affinity membrane receptors for the T-lymphocytotrophic hormone interleukin 2 (IL-2) determines the rate of T-cell-cycle progression. Since 10-fold greater numbers of IL-2 receptor molecules were found by using a radiolabeled monoclonal antibody reactive with IL-2 receptors (anti-Tac) compared with binding of IL-2, the functional relationship of the binding sites recognized by both of these ligands was assessed. In the presence of cycloheximide, IL-2 binding sites declined with a half-time (t1/2) of 2.6 hr, whereas the decay of anti-Tac binding sites was much slower (t 1/2 = 6.4 hr). Moreover, after limited membrane proteolysis, the half-time …

Interferon Gamma Blocks The Growth Of Toxoplasma Gondii In Human Fibroblasts By Inducing The Host Cells To Degrade Tryptophan., E. R. Pfefferkorn

Dartmouth Scholarship

Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The …

Recombinant Immune Interferon Increases Immunoglobulin G Fc Receptors On Cultured Human Mononuclear Phagocytes, Paul M. Guyre, Peter M. Morganelli, Renee Miller

Dartmouth Scholarship

Although recent studies suggest that interferons can increase the number of IgG Fc receptor (FcR gamma) sites on mouse macrophages, direct assessment of similar effects on human mononuclear phagocytes is lacking. We therefore measured the specific binding of 125I- and fluorescein-labeled IgG1 to human monocytes and leukemic cell lines after culture in vitro with highly purified human interferons. We report that natural and recombinant human gamma-interferon causes a dramatic (nearly 10-fold) increase in the number of FcR gamma on normal human monocytes and on the human cell lines HL-60 and U-937. Alpha and beta-interferons cause a modest but significant increase …

Iron And The Liver: Acute Effects Of Iron-Loading On Hepatic Heme Synthesis Of Rats. Role Of Decreased Activity Of 5-Aminolevulinate Dehydrase., H L. Bonkowsky, J F. Healey, P R. Sinclair, J F. Sinclair, S I. Shedlofsky, G H. Elder

Dartmouth Scholarship

Acute iron loading of rats, by intraperitoneal administration of iron-dextran (500 mg Fe/kg body wt 18-20 h before killing) decreased by 30% the rate of conversion of 5-amino-[14C]levulinate ([14C]ALA) into heme as measured with a recently described procedure for liver homogenates (1981. Biochem. J. 198: 595-604). The decrease in conversion of labeled ALA into heme caused by iron loading was shown to be due to a 70-80% decrease in activity of ALA dehydrase. The decrease in activity of ALA dehydrase caused by iron loading was not associated with a decrease in hepatic concentrations of GSH, nor could it be reversed …

Proteins Antigenically Related To The Human Erythrocyte Glucose Transporter In Normal And Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts., Donald W. Salter, Stephen A. Baldwin, Gustav E. Lienhard, Michael J. Weber

Dartmouth Scholarship

Antibody raised against the purified human erythrocyte glucose transporter specifically precipitated four proteins from normal and Rous sarcoma virus-transformed chicken embryo cells: a major protein of Mr 41,000 and minor proteins of Mr 68,000, 73,000, and 82,000. The Mr 41,000 and 82,000 proteins were found only in a membrane fraction, not in the soluble fraction, and displayed a heterogeneous mobility on NaDodSO4/polyacrylamide gel electrophoresis, suggesting glycosylation. The Mr 41,000 and 82,000 proteins were increased in amount after malignant transformation in direct proportion to the increase in hexose transport rate, and the increase was dependent on the expression of the src …

Detergent Extraction Of A Presumptive Gating Component From The Voltage-Dependent Sodium Channel, W J. Culp, D T. Mckenzie

Dartmouth Scholarship

A physiologically characterized radiolabeled neurotoxin complex obtained from venom of the scorpion Leiurus quinquestriatus has been used to identify detergent-solubilized presumptive sodium channel components in sucrose gradients. This toxin-binding component is found in extracts prepared from three sources of excitable membrane but appears to be absent from similar extracts prepared from nonexcitable membrane or from Torpedo californica membrane. Procedures that destroy the physiological activity of the Leiurus neurotoxin lead to a corresponding loss of toxin binding to the putative sodium channel component. The major component recognized by the Leiurus toxin sediments at 6.5 S. Scatchard analysis of quantitative binding experiments …

Rat Dendritic Cells Function As Accessory Cells And Control The Production Of A Soluble Factor Required For Mitogenic Responses Of T Lymphocytes., Wolfgang E. Klinkert, Jon H. Labadie, James P. O'Brien, Carl F. Beyer, William E. Bowers

Dartmouth Scholarship

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred …

Cellular Action Of Vasopressin In Medullary Tubules Of Mice With Hereditary Nephrogenic Diabetes Insipidus, Brian A. Jackson, Richard M. Edwards, Heinz Valtin, Thomas P. Dousa

Dartmouth Scholarship

Our previous studies (1974. J. Clin. Invest.54: 753-762.) suggested that impaired metabolism of cyclic AMP (cAMP) may be involved in the renal unresponsiveness to vasopressin (VP) in mice with hereditary nephrogenic diabetes insipidus (NDI). To localize such a defect to specific segments of the nephron, we studied the activities of VP-sensitive adenylate cyclase, cAMP phosphodiesterase (cAMP-PDIE), as well as accumulation of cAMP in medullary collecting tubules (MCT) and in medullary thick ascending limbs of Henle's loop (MAL) microdissected from control mice with normal concentrating ability and from mice with hereditary NDI.

Adenylate cyclase activity stimulated by VP or by …