Browsing by Subject "Other Medicine and Health Sciences"
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Item Clinical Diagnosis: A Manual of Laboratory Methods(W.B. Saunders Company, 1913-01-01) Todd, JamesWhile the original purpose of this book- to present Clearly and concisely the various laboratory methods which are of use in clinical medicine- has not been lost Sight of, its scope has been somewhat enlarged in the present edition. Each section has been carefully revised and much new Material has been added to every chapter. Among the many additions may be mentioned: the use of artificial light and the importance of numerical aperture in microscopic work; photomicrography with simple apparatus; The antiformin method for tubercle bacilli; detection and significance of albumin in the sputum; Tsuchiya's modification, of Esbach's test; the formalin test for ammonia And benedict's methods for sugar in urine; volume index Of red blood-corpuscles; wright and Kinnicutt's method of counting blood-platelets; Harlow’s blood-stain; a simple Technic for the diagnosis of typhoid fever by blood cultures; The Wassermann reaction, and Frothingham's Impression method in the diagnosis of rabies. Because of the growing importance of animal parasites, this chapter has been entirely rewritten and more than Doubled in extent. Two new chapters have been added: One upon bacteriologic methods, which supplements the Methods given in other portions of the book, and one upon preparation and use of vaccines, including therapeutic and diagnostic use of tuberculin.Item Determination of the Intracellular Levels of Cyclic ADP-Ribose in Cultured Human Cells Using a New Highly Sensitive Fluorescent HPLC Method(2002-05-01) Gill, Paramjit Kaur; Goldfarb, Ronald H.; Easom, Richard; Matthew, Porunelloor A.Determination of the Intracellular Levels of Cyclic ADP-Ribose in Cultured Human Cells Using a New Highly Sensitive Fluorescent HPLC Method Paramjit Kaur Gill*. Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, TX 76107-2699. Cyclic ADP-ribose (cADPR) is a second messenger that mobilizes intracellular stores of calcium in higher eukaryotic cells. The intracellular concentration of cADPR has previously been estimated to be in the femto- to nanomolar range. Therefore, there is a need for a highly sensitive assay to measure the levels of nucleotide in just a few million cells. Here, we have developed a highly, sensitive, specific, and reproducible fluorescent HPLC method to determine the intracellular concentration of cADPR in cultured cells. The procedure involves extraction of the nucleotide pool in 20% (W/V) TCA followed by the purification of soluble molecules containing two or more soluble molecules containing two or more riboses by boronate affinity chromatography. Purified nucleotides are then digested with phosphodiesterase to degrade all non-cyclic molecules, leaving cADPR intact. Contaminating products of phosphohydrolysis are then eliminated by a second boronate step and the pure preparation of cADPR obtained is converted to monomeric ADP-ribose with NADase (isolated from Bungarus fasciatus). After a third boronate purification, ADP-ribose is chemically derivitized to the etheno-adenine fluorescent form with chloroacetylaldehyde at 60°C, and the εADP-ribose formed is quantified by fluorescent-HPLC on a Partisil 10-SAX column. The specificity of our method was monitored by determining the yield at every step of the protocol with {32P]cADPR. Radiolabeled cADPR was synthesized from [32P]β-NAD+ and pure ADP-ribosyl cyclase from Aplysia californica. [32P]cADPR was subsequently purified by HPLC on a Partisil 10-SAX and a C-18 reverse phase column placed in tandem. While the recovery of a known amount of cADPR through each boronate step of the 4-day protocol was approximately 90%, the overall recovery throughout the procedure was between 30-40%. As expected, our mock incubations (negative controls) in the absence of phosphodiesterase or NADase treatment, as well as chloroacetaldehyde, yielded no εADP-ribose peak. Furthermore, spiking of a cell extract with commercially available cADPR resulted in the formation of a bigger fluorescent peak. Finally, our method indicated an intracellular concentration of cADPR in HeLa cells of 980 pmol of cADPR/10^8 cells. Considering HeLa cells have a larger cytoplasm compared to blood cells, our results agree well with those reported by DaSilva et al. who observed that the intracellular concentration of cADPR was 198 pmol/10^8 Jurkat cells, using a less sensitive chromatographic assay. Applications of this assay as a tool for biochemical investigation as to the role of cyclic ADP-ribose in the signal transduction events of rapamycin as an immunosuppressant and CD83 in B-CLL are discussed. *The laboratory study in this thesis were performed entirely in the laboratory of Dr. Rafael Alvarez-Gonzalez, in the department of Molecular Biology and Immunology and were performed entirely under his supervision. Dr. Alvarez served as my mentor from 1995-1998. Both Drs. Rafael Alvarez-Gonzalez and Ronald Goldfarb, Department Chairman, served as co-mentors from August 1999-March of 2000. From April 2001-July 2001, Dr. Goldfarb served as my mentor.Item North Texas Health & Science - 2011, Issue 1(University of North Texas Health Science Center at Fort Worth, 2011-01-01)Item North Texas Health & Science - 2012, Issue 1(University of North Texas Health Science Center at Fort Worth, 2012-01-01)Item North Texas Health & Science - 2012, Issue 2(University of North Texas Health Science Center at Fort Worth, 2012-06-01)Item North Texas Health & Science - 2013, Issue 1(University of North Texas Health Science Center at Fort Worth, 2013-01-01)