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Surviving Riyadh

Co-authored Articles

 

 

Enzymatic Basis for the Ca 2+-Induced Crosslinking of Membrane Proteins in Intact Human Erythrocytes  

 

Gerald E. Siefring, Alma B. Apostol, Pauline T. Velasco, Laszlo Lorand  

Journal: Biochemistry – BIOCHEMISTRY-USA, vol. 17, no. 13, pp. 2598-2604, 1978

Department of Biochemistry and Molecular Biology,

Northwestern University, Evanston, Illinois 60201


Abstract:

The accumulation of Ca²+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in ?-glutamyl-?-lysine crosslinks. Isolation of this isodipeptide, in amounts as high as 6 mol/105 g of protein, confirms the idea (Lorand, L…Weissmann, L.B., Epel, D. L…and Bruner-Lorand, J. (1976), Proc. Nat’l. Acad. Sci. U.S.A. 73, 4479) that Ca²+ -induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small. water soluble primary amines. Inasmuch as the amines are known to prevent Ca²+ -dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.

Keywords: erythrocytes,Ca²+,spectrin,protein,cells  

………………

Fiber Optic Biosensor for Detection of DNA Damage

 

Kim R. Rogers, Alma Apostol, J. Madsen,Charles W. Spencer

ABSTRACT

This paper describes a fiber-optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The hybridization pair consists of a biotin labeled 38 mer oligonucleotide immobilized to a streptavidin-coated optical fiber and a fluorescently-labeled near-complementary (two base mismatch) oligonucleotide reporter sequence. The hybridization-based assay detected 50nM labeled probe and could be run up to 10 times on the same fiber. Melting profiles were sensitive to high energy radiation and to 3-morpholinosydnonimine (SIN-1)-generated reactive decomposition products. The dynamic range of the assay for ionizing radiation extends from 20 to 1000cGy. Oxidative damage induced by SIN-1 was measured over a concentration range of 250µM to 3 mM.

Journal: Analytica Chimica- Anal CHIM ACTA, vol. 444, no. 1, pp. 51-60, 2001

Environmental Protection Agency Las Vegas, Nevada 89193

Keywords:fiber optic, DNA, damage, assay, oligonucleotide

……………….

Capillary Electrophoresis Immunoassay for 2, 4-Dichlorophenoxyacetic Acid  

 


Key Words: capillary electrophoresis, immunoassay, 2, 4-D, environmental Monitoring

Kim Rogers, Alma Apostol, William Brumley

Journal: Analytical Letters-ANAL LETT, vol. 33, no. 3, pp. 443-453, 2000

Environmental Protection Agency, Las Vegas, Nevada 89193

ABSTRACT

A capillary electrophoresis (CE) immunoassay format for 2, 4-dichlorophenoxyacetic acid (2, 4-D) is demonstrated. A fluorescent labeled 2, 4-D analog competes with the analyte of interest for a finite number of binding sites provided by anti-2, 4-D monoclonal antibodies. CE then provides a means of separating and measuring both the free and antibody-bound fluorescent tracer using laser-induced fluorescence detection. For this assay format, the amount of free tracer is a sensitive indicator for the concentration of analyte present in the sample. A sequential injection format allows the rapid analysis of a small number of samples. The dynamic concentration range for 2, 4-D in either buffer or river water is 5 ppb to 1000 ppb.

Keywords: capillary, electrophoresis, 2,4-D, antibodies, analyte

………………

Detection of Low Dose Radiation Induced DNA Damage Using Temperature Differential Fluorescence Assay  

 


Kim R. Rogers, Alma Apostol, Steen J. Madsen, Charles W. Spencer

Journal: Analytical Chemistry-ANAL-CHEM , vol. 71, no. 19, pp. 4423-4426, 1999

Environmental Protection Agency, Las Vegas, Nevada 89193

A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature –induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures of between 0.004 and 1 Gy were measured with doses as low as 0.008 Gy yielding significant responses. The double-strand, sensitive dye Pico Green was used as an indicator of DNA denaturation. Calibration plots indicate that fluorescence changes corresponding to amounts as alow as 1 ng of double stranded DNA (10 to the 6 copies for plasmid puc 19) are detected by this method.

Keywords: radiation, DNA damage, plasmid, Gy, Pico Green

…………………

Screening Assay For DNA Damage

 


Kim R. Rogers, Alma Apostol, and Kumar Ramanathan.

Symposia Papers Presented Before the Division of Environmental Chemistry

American Chemical Society, San Diego, CA April 1-5 2001

U.S. Environmental Protection Agency

National Exposure Research Laboratory Las Vegas, NV 89193

SUMMARY

This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curve) of synthetic oligonucleotides. The hybridization pair consists of a biotin labeled 38-mer oligonucleotide immobilized to a streptavidin-coated fiber and a fluorescently-labeled near-complementary (2 base mismatch) oligonucleotide reporter sequence. The hybridization-based assay detected 50 nM labeled probe and could be run up to ten times on the same fiber. Melting profiles were sensitive to high energy radiation and 3-morpholinosydnonimide (SIN-1)-generated reactive decomposition products.  The  dynamic range of the assay for ionizing extends from 20cGy to 1000 cGy. Oxidative damage induced by SIN-1 was measured over a concentration range of 250 uM to 3 mM.

Keywords: assay, biosensor, fiber optic, oligonucleotides, sequence

………….

Current Applied Physics

Volume 3, Issues 2-3, April 2003, Pages 99-106

Environmental Protection Agency, Las Vegas, Nevada 89193

A fluorescence Based Assay for DNA Damage Induced by Radiation, Chemical Mutagens and Enzymes

 


Kumaran Ramanathan, Ronald K. Gary, Alma Apostol, Kim R. Rogers

Environmental Protection Agency

National Exposure Research Laboratory

944 E. Harmon Avenue, P.O. Box 93478, Las Vegas, NV 89119, USA

Department of Chemistry, University of Nevada Las Vegas, Las Vegas, NV 89154, USA

Abstract

A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) radiation, chemical mutagens or restriction enzymes produced an assay response. UV radiation at 254 nm (approximating UV-C) and 360 nm (approximating UV-A) were used to induce the damage in dsDNA. Chemical damage was induced using several compounds with known effects on nucleic acids. Restriction enzymes Hind III, Msp1, Sau 3A1 were used to cut the plasmid (pUC19) at specific sequences in addition to the non-specific endonuclease DNase I. The effects of these types of damage on repeated melting and annealing of dsDNA were observed in real time using several fluorescence indicator dyes. Low concentrations of dsDNA (between 10 and 100 ng/ml) and small volumes (20 µl) were required for this assay. Repeated measures yielded a coefficient of variation of 2% (CV%). In addition to measuring various DNA damaging agents, the potential application of this assay to study the efficiency of various sun blocking agents against UV-induced DNA damage is discussed.

Keywords: mutagen, dsDNA, UV, assay, restriction enzyme

……….

Physics and Astronomy

Journal of Biological Physics

Volume 15, Number 2 (1987), 29-32

 

Determination of the Density of Cells From Sedimentation Studies at 1G

 



G. H. Czerlinski, D. S, Reid, A. Apostol, K. D. Bauer and D. G. Scarpelli

Department of Molecular Biology and Pathology, Northwestern University Medical School, Chicago, Illinois 60611

Abstract

To obtain a possible correlation between cell density and cell size, the size of individual cells was measured under the microscope and their sedimentation velocity was measured; the density is obtained with Stoke's Law. Specifically, HeLa cells were sedimented in Joklik's medium at 30°C in a vertical glass tube with 2 mm×2 mm horizontal opening and cells observed with a horizontally aligned microscope. The overall mean density difference of HeLa cells at 30°C was 0.0316+–0.0044 g/cm3, resulting in a density of 1.0357 g/cm3 (with a density of 1.0040 g/cm3 for Joklik's medium at 30°C). Six size fractions had densities which were essentially the same within the errors of the mean densities of the fractions (from 0.0081 to 0.0202 g/cm3). The considerably varying deviations of individual densities from the mean suggested superimposed phenomena (see also Table I for microspheres of precise size).

Careful observation in balancing countercurrent flow revealed microconvection over 5 to 15 m regions, most likely based on small thermal differences in the horizontal plane.

Keywords: density, cells, IG, microscope, Stoke's law

……….

Foster Abstract Form, April, 1985

13th Internationa; Congress of Biochemistry

c/o Organisatie Bureau Amsterdom by Europaplein

1078 GZ Amsterdam

The Netherlands

Name of the author presenting the poster: George H. Czerlinski

Northwestern University

301 E. Chicago Avenue, W4-067

Chicago, Illinois 60611 U.S.A.

Rapid Determination Of The Density of Live Cells

 


G. H. Czerlinski, A. Apostol, K. D. Bauer and D. G. Scarpelli

Department of Molecular Biology and Pathology

Northwestern University Medical School,

Chicago, Illinois U.S.A.

In cellular growth, differentiation and transformation it is sometimes desirable to know the (gravitational) density of cells (for eventual sorting). This may easily be done under microscope with horizontal optical axis. The objective is focused onto capillary square glass tube aligned vertically and in parallel to a mm-scale. A cell suspension is sucked into the tube with the aid of a rubber bulb and the sedimenting cells are timed as they pass over a defined distance. Prior determination of density, and viscosity of the medium and of the mean radius of the cells allows to apply Stokes Law, leading to the density of the cells.

To test the above concept, a suspension of 100,000 Hela cells in 1 ml Joklik’s MEM + 5% FBS was used. A mean cell radius of 0.001 cm had been determined before. Density and viscosity of the suspending medium were 1.00476 g/ml and 0.00886 g/cm/s, resp. A typical cell required 10 s to sediment over 0.1 cm (23C). Stokes Law then results in a density of 1.411 g/ml for the typical cell.

A thermostated device is now being developed together with countercurrent flow to stabilize the location of the cells, which could then be photographed at high magnification. One could then attribute specific densities to specific cells. If the cells are replaced by defined microspheres (of known density), the device could be used as a rapid viscometer.

Keywords: density, cells, viscosity, sorting, viscosity

………..

Purification of Flood Water

 


P. L. Rubio, A. Coronel, H. de Leon, Alma B. Apostol

National Science Development Board (NSDB)

National Institute of science and Technology (NIST)

National Water and Air Pollution Control Commission (NWAPCC)

Manila, Philippines

April, 1973

ABSTRACT

A feasible method of purifying flood water would prove handy in a disastrous flood. The method should be able to supply purified flood water for drinking to about fifty to one hundred families in relocation centers. The authors opined that a feasible method makes use of the following materials: Three drums of 55-gallon capacity, 4 grams of aluminum sulfate, a bag of sand, 57 grams of Biogent 236 or chlorine, and 200 ml Chemfloc or coagulant. Water samples from each drum were analyzed chemically and bacteriologically. The purified water from the third drum showed that it was suitable for drinking and suitable for household purposes. After chlorination, water from the third drum showed zero growth of bacteria. The absence of bacterial colonies in tests plates meant that the water was fit for drinking and household purposes. Unlike the murky water in the first drum, the water flowing out of the third drum was clear and had an acceptable smell. It was no different from water flowing out of the taps for drinking and other household purposes. The experiment of purifying flood water was compared with boiling method of purifying water. It was found that mere boiled water still have the murky color, offensive smell, and impurities making boiled flood water unacceptable for drinking and household purposes.

Keywords: flood water, purification, NIST, coagulant, NSDB

..........

Unpublished Collaborative Projects, Los Angeles, California

 



Franciskus J. Walther, MD, PhD

Div. of Neonatology and Pediatric Pulmonology

Childrens Hospital Los Angeles, Los Angeles, CA 90027

October, 1988

Protection from Pulmonary Oxygen Toxicity By PEG- Conjugated Antioxidant Enzymes

 


Premature infants are born before full maturation of the surfactant and antioxidant enzyme systems in their lungs. Surfactant deficiency is the main cause of the respiratory distress syndrome in premature infants and often needs to be treated with mechanical ventilation and high inspired oxygen concentrations. Oxygen treatment increases the production of free oxygen radicals and may damage the cells lining the lung alveoli. Intracellular antioxidant enzymes such as superoxide dismutase and catalase, detoxify free oxygen radicals.

Respiratory distress syndrome combined with reduced antioxidant enzyme activity puts the premature infant at high risk for bronchopulmonary dysplasia, the most common chronic lung disease in infancy. Oxygen toxicity may be prevented by pre-treatment with antioxidant enzymes. Attachment of antioxidant enzymes to polyethylene glycol (PEG) permits intracellular access. This study investigates the effects of the administration of PEG-conjugated superoxide dismutase to pneumocytes from adult rats. If this approach is successful, intratracheal administration of antioxidant enzymes may become an option for the prevention of bronchopulmonary dysplasia in premature infants.

..........

Phospholipid Concentrations in Tracheal Aspirate Fluid in Infants Requiring Extracorporeal Membrane Oxygenation

 


Abstract submitted to Western Society for Pediatric Research

Franciskus J. Walther, MD, PHD

Authors: KC Bui, A. Apostol, D. Warburton, FG Walther

Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, California

October 5, 1989

Extracorporeal membrane oxygenation (ECMO) is used to treat infants greater than 2,000 g with life-threatening respiratory failure. Lung compliance is initially very low and improves dramatically while weaning from ECMO. We measured the total amount of surfactant phospholipids and the disaturated phosphatidylcholine (DSPC) content in tracheal aspirates from 8 infants (weight 3.54-4.30 kg, gestational age 38-43 weeks) who were placed on ECMO for respiratory failure secondary to meconium aspiration syndrome (n=6) or pneumonia (n=2) at a mean postnatal age of 40 h. Suction material was obtained in a standardized way every 4 h and samples were pooled over 24 h periods. Samples were collected throughout the period of ECMO treatment. Total phospholipids were estimated by phosphorus assay following chloroform:methanol extraction and DSPC by osmic acid precipitation. Comparison of tracheal aspirates obtained at initiation of ECMO showed minimal amounts of phospholipids and DSPC versus samples collected at the time of discontinuation of ECMO. Phospholipid and DSPC contents demonstrated an abrupt increase prior to weaning from ECMO. We conclude that infants qualifying for ECMO treatment secondary to life-threatening respiratory failure often exhibit deficiency. Surfactant replacement therapy should be considered in these infants.

Keywords: Surfactant, Respiratory Failure, Meconium Aspiration Syndrome,

Pneumonia

…………

Augmentation of Catalase Activity in Type II Pneumocytes

 


Abstract submitted to; Western Society for Pediatric Research Oct. 1989

FJ Walther, A Apostol, D Warburton, HJ Forman,

Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, CA

Excess free oxygen metabolites overwhelm endogenous antioxidant defenses but may be counteracted by supplementing the intracellular activity of catalase. Conjugation of catalase with polyethylene glycol (PEG) extends the short half-life of catalase and permits intracellular access by endocytosis. Type II pneumocytes were isolated from male 250-300 g pathogen-free rats  following the protocol of Dobbs et al.(1986),and were plated at a final density of 1.5-2 x 10cells /well. After 20 h incubation test solutions were added and the plates were incubated for 24 h. Intracellular catalase activity was measured by the rate of reduction of H2(02) substrate at 240 nm. Incubation of 1x 106 cells with 0.125-1 mg of native catalase increased intracellular enzyme activity by less than 25% in comparison with controls, but addition of comparable amounts of PEG-catalase produced a 2 to 3-fold increase in intracellular enzyme activity. FITC-PEG catalase was located intracellularly under fluorescence microscopy. Uptake of PEG-catalase started ti plateau at 8-12 h. Exposure to H2(02) and t-butyl hydroperoxide led less LDH release in cells  treated with PEG-catalase. We conclude that supplementation with PEG-catalase enhances the activity of catalase in type II pneumocytes and increases their resistance to oxidant stress.

Keywords: Free Oxygen, Radicals, Polyethylene Glycol, Antioxidant Enzyme  

…………

Pulmonary Antioxidant Enzyme Activity in Preterm Lambs Exposed to Corticosteroid and Thyrothropin-Releasing Hormone in Utero

 


Abstract submitted to Western Society for Pediatric Research 1989

FJ Walther, A Apostol, M Ikegami, D Warburton, D Polk

Department of Pediatrics, University of Southern California School of Medicine and King-Drew and Harbor-UCLA Medical Centers, Los Angeles, CA

Forty-three twin lamb fetuses of fetuses of 121±1 days gestation were catherized and received intravenous saline (n=8), 0.75 mg/kg/h cortisol (ST) (n=15), 10 ug thryrothropin releasing hormone (TRH) every 12 h for 6 doses (n=9), or ST+TRH for 70 h. before delivery at 128±1 days (n=11). After delivery, the lambs were ventilated for 75 min. Superoxide dismutase, catalase, and glutathione peroxidase activity and DNA content were measured in fetal lung tissue using standard assays. Mean ± SEM catalase activity was 83±6 U/mg DNA in the controls, 83±6 U/mg DNA in the TRH group, 135±13 U/mg DNA in the ST + TRH group (p<0.005), and 153±11 U/mg DNA in the ST group (p
Conclusion: prenatal exposure to ST increased pulmonary antioxidant enzyme activity, whereas TRH alone or TRH added to ST provided no additional benefit. Increased antioxidant enzyme activity may be part of the beneficial effect of prenatal steroids.

Keywords: Cortisol, Superoxide Dismutase, Catalase, Glutathione

……….

Augmentation of Superoxide Dismutase Activity in Type II Pneumocytes

 


Abstract submitted to Western Society for Pediatric Research

FJ Walther, D Warburton, AB Apostol, HJ Forman

Developmental Lung Biology Research Center, Childrens Hospital of Los Angeles, University of Southern California School of Medicine, Los Angeles, CA

Excessive production of free oxygen radicals overwhelms endogenous antioxidant defenses to produce cell injury and may be counteracted by supplementating the intracellular activity of superoxide dismutate (SOD), catalase, or glutathione peroxidase. Native SOD has a short half-life and does not penetrate across cell membranes. Covalent conjugation of SOD with polyethylene glycol (PEG) extends the half-life of SOD and adds a surface-active group that is used extensively to induce fusion for cell hybridization.

Type II pneumocytes were isolated from male 250-300 g pathogen-free rats following the lung lavage, elastase digestion, mechanical dissociation, and IgG differential adherence protocol of Dobbs et al, Type II pneumocytes were plated at a final density of 1.5-2 x 106 cells/well. After 20-24 h. incubation the cells were washed, test solutions were added, and the plates returned to the incubator for 24 h. Cells were washed, lysed with Triton X-100, and SOD activity was measured with the xanthine oxidase/cytochrome c assay. Incubation of 1 x 106 type II pneumocytes with 500-600 U of native SOD did not change cellular enzyme activity in comparison with controls (234±22 U SOD/mg DNA), but addition of 500-600 U of PEG-SOD produced a 9-fold increase in cellular activity (2,145±317 U SOD/mg DNA).

We conclude that PEG-conjugation to SOD enhances the internalization and activity of SOD in cultured type II pneumocytes.

Keywords: SOD, catalase, glutathione, type II pneumocytes, PEG-SOD

 ..........

 

 Antioxidant Activity of Lung Surfactants

 

Abstract submitted to The American Pediatric Society, The Society for Pediatric Research

 

Frans J. Walther, Alma Apostol, David Warburton, and H. William Taeusch

 

University of Southern California School of Medicine and King/Drew Medical Center, Department of Pediatrics, Los Angeles, California

 

Intratracheal instillation of exogenous surfactant reduces the severity of respiratory distress syndrome and may also provide antioxidant activity. We compared mean (±SD) superoxide dismutase (SOD) and catalase (CAT) activity (n≥6) in Surfactant-TAÒ (chloroform:methanol extracted cow surfactant), Exosurf®(protein-free surfactant), natural cow and sheep surfactants, and in human surfactant protein A (SP-A):

 

                  CAT, IU/mg PL,        SOD, IU/mg PL,      protein, %    

 

 

 

Surfactant-TA           .7±.2                 0.0                      1.2

 

Exosurf                    .7±.2                 0.0                      0.0

 

Cow surfactant      12.9±1.3               0.0                      8.7

 

Sheep surfactant    32.5±1.9           7.1+3.3                  34.5

 

Chloroform:methanol extraction of natural cow and sheep surfactant reduced their CAT activity to .8±.6 and .9±.3 IU/mg PL and their protein content to 0 and 11.2%, respectively. SP-A had a CAT activity of 9.0±1.6 IU/mg and a SOD activity of 1.1±.3 IU/mg.

 

We conclude that natural surfactants contain CAT but not SOD activity. The absence of CAT activity in extracted and protein-free surfactants, together with the low CAT activity of SP-A suggests that the CAT activity of natural surfactant is contributed by the presence of other proteins in lung lavage. The antioxidant activity of natural surfactant would not be expected to provide clinically significant protection against oxygen injury.

 

 

Keywords: CAT, SOD, surfactant, natural surfactant, protein

 

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Enzymatic Basis for the Ca 2+-Induced Crosslinking of Membrane Proteins in Intact Human Erythrocytes   
Gerald E. Siefring, Alma B. Apostol, Pauline T. Velasco, Laszlo Lorand  
Journal: Biochemistry – BIOCHEMISTRY-USA, vol. 17, no. 13, pp. 2598-2604, 1978
Department of Biochemistry and Molecular Biology,
Northwestern University, Evanston, Illinois 60201