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Biomedical subjects

R Ramanathan

Publications and source records attributed to R Ramanathan.

At least 19 recordsLinked to original sources
PubMed1999

Real-time t(11;14) and t(14;18) PCR assays provide sensitive and quantitative assessments of minimal residual disease (MRD).

Non-Hodgkin's lymphoma (NHL) arises as a clonal transformation of normal B and T cell differentiation and is often characterized by a higher incidence of specific chromosomal translocations. We have developed real-time TaqMan PCR assays directed toward two of these tumor-associated DNA markers, the t(14;18)(q32;q21.3) at the major breakpoint region of the bcl-2 gene and the t(11;14)(q13;q32) at the bcl-1 major translocation cluster. During analysis of serial dilutions of t(14;18)-positive DNA, the t(14;18) real-time PCR was at least as sensitive as nested PCR and demonstrated enhanced quantitative potential. Moreover, in a blinded comparison of the t(14;18) real-time PCR and a clinically validated nested PCR protocol using 134 cell line and patient DNA samples, the real-time PCR detected the translocation in 30.0% more cases than nested PCR. Both the t(14;18) and t(11;14) real-time PCR assays were used to quantitate minimal residual disease (MRD) in an NHL clinical trial assessing the safety and efficacy of a tumor-purging protocol in autologous stem cell transplantation. The assays were also used to evaluate disease depletion in an ex vivo tumor spiking model in which normal peripheral blood was spiked with tumor cell lines and processed according to the clinical purging method. PCR data from both the clinical trial and the ex vivo model demonstrated a 4 to 6 log reduction in tumor cells during CD34+ and CD34+ Thy-1+ enrichment. Because the t(14;18) and t(11;14) real-time PCR assays are very sensitive, quantitative, rapid, and require no post-PCR manipulation, they may serve as practical alternatives to nested PCR.

Antigens, CD34↗
PubMed1999

Reverse aortic arch flow secondary to severe pulmonary hypertension in the neonate.

The purpose of this study is to determine the incidence of reverse aortic arch flow in newborn infants with pulmonary hypertension, and no other identifiable cause for flow reversal, being considered for extracorporeal membrane oxygenation (ECMO) 2) compare the left ventricular output, and 3) outcomes, of these infants, with those in the group of patients with normal arch flow. We reviewed the medical records and echocardiograms of all infants referred to our institution for extracorporeal membrane oxygenation support for pulmonary hypertension, between August 1994 and April 1996. Neonates with pulmonary hypertension and reverse aortic arch flow had significantly lower left ventricular output (p = 0.005), and had significantly higher mortality (p = 0.04), than those with normal aortic arch flow. They required significantly higher ventilatory support (p = 0.01) and tended to need more inotropic support. Reverse aortic arch flow in newborn infants with pulmonary hypertension, and no other cause for the reverse flow, indicates significantly decreased left ventricular output, and may be a marker for a more difficult clinical course and poor prognosis. In this group of patients with pulmonary hypertension, the survival was significantly higher in infants with no PDA or with pure left to right shunt across a PDA.

Aorta↗
PubMed1998

Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards.

The complementarity determining region III of the rearranged immunoglobulin heavy chain gene has been the target for tumor-specific PCR assays for the detection and follow-up of B-cell malignancies. Previously, these assays have relied on gel-based end point data collection methods (i.e., band densitometry) and, thus, have provided at best a semiquantitative assessment of tumor levels. We show the development of a novel, real-time TaqMan PCR assay to quantitate residual multiple myeloma cells in clinical samples after high-dose chemotherapy and autologous stem cell transplantation. We provide evidence that real-time PCR is reproducible, sensitive, and quantitative. In a 40-replicate PCR experiment targeting the beta-actin gene, the coefficient of variation for threshold cycle data was 1.6%, whereas it increased to 13.6% and 31%, respectively, for end point fluorescence and gel densitometry. Moreover, in an experiment directly comparing standard curves obtained from band densitometry and threshold cycle data, the standard curve constructed from threshold cycle data had a multiple R2 value of 1.00 and demonstrated a dynamic range >4 logs, compared with the 2-log linear range of gel densitometry. Finally, we show that when a complementarity determining region III-specific PCR primer is used in conjunction with a consensus primer for the immunoglobulin heavy chain joining gene, plasmid DNA can be used as a readily available and effective substitute for clonal plasma-cell genomic DNA when preparing standards. By applying real-time PCR to the analysis of clinical samples, we are able to quantitate levels of tumor involvement with unparalleled reproducibility and statistical confidence. Real-time PCR technology may well provide the accuracy and reliability necessary for minimal residual disease detection to have real prognostic significance.

Base Sequence↗
PubMed1998

The effects of IL-10 on proinflammatory cytokine expression (IL-1beta and IL-8) in hyaline membrane disease (HMD).

Deficient expression of the counterregulatory cytokine IL-10 by lung inflammatory cells may facilitate chronic inflammation and the pathogenesis of hyaline membrane disease (HMD), in premature infants. To determine if pathways which regulate proinflammatory cytokines in response to human recombinant IL-10 (rIL-10) were functional in the lungs of these neonates, bronchoalveolar lavage (BAL)-derived lung inflammatory cells (predominantly macrophages and neutrophils) from infants with HMD were cultured in the presence of lipopolysaccharide (LPS) and increasing concentrations of (rIL-10). The expression of IL-1beta and IL-8 protein was assessed 24 h later. IL-10 protein was also measured from the BAL aspirates of these newborns at 4-day intervals over the first month of life. In cell culture IL-1beta expression was inhibited by rIL-10 in a dose-dependent fashion while IL-8 expression was inhibited by higher concentrations of rIL-10. IL-10 protein was undetectable from BAL fluid of the premature infants sampled over 28 days. The results demonstrate that lung inflammatory cells, which do not express IL-10 in vivo, are capable of responding to rIL-10 in cell culture with reduction of IL-1beta and IL-8 expression. These data support the rationale for the development of rIL-10 as a potential anti-inflammatory agent in the treatment of HMD.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Bioavailability of intranasal promethazine dosage forms in dogs.

Intramuscular promethazine (PMZ) is used aboard the US Space Shuttle to ameliorate symptoms of space motion sickness. Bioavailability after an oral dose of PMZ during space flight is thought to be impaired because of gastrointestinal disturbances associated with weightlessness and space motion sickness. In an attempt to find an alternative dosage form for use in space, we evaluated two intranasal (i.n.) dosage forms of PMZ in dogs for absorption and bioavailability relative to that of an equivalent intramuscular dose. Promethazine (5 mg kg-1) was administered as two intranasal dosage forms and as an intramuscular (i.m.) dose to three dogs in a randomised cross-over design. Serial blood samples were taken and analysed for PMZ concentrations and the absorption and bioavailability of PMZ were calculated for the three dosage forms. PMZ absorption from the carboxymethyl cellulose microsphere i.n. dosage form was more rapid and complete than from the myverol cubic gel formulation or from an i.m. injection. Bioavailability of the microsphere formulation was also greater than that of the gel formulation (AUC 3009 vs 1727 ng h ml-1). The bioavailability of the two i.n. dosage forms (relative to that of the i.m. injection) were 94% (microsphere) and 54% (gel). The i.n. microsphere formulation of PMZ offers great promise as an effective non-invasive alternative for treating space motion sickness due to its rapid absorption and bioavailability equivalent to the i.m. dose.

Administration, Intranasal↗
PubMed1998

Mass spectrometric methods for distinguishing structural isomers of glutathione conjugates of estrone and estradiol.

Collisionally activated decompositions (CAD) of [M + H]+ ions from two sets (estrone and estradiol) of three isomeric glutathione (GSH) conjugates were studied by using five tandem mass spectrometric methods: (1) low energy (LE) CAD in an ion trap, (2) LE CAD in a triple quadrupole, (3) electrospray ionization (ESI)-source CAD in a tandem four sector, (4) high energy (HE) CAD of both ESI-produced and fast-atom bombardment (FAB)-produced ions in a tandem four-sector mass spectrometer, and (5) metastable-ion decompositions of FAB-produced ions. Four types of fragment ions are produced. The first type, formed from cleavage of the peptide backbone, gives rise to modified b2, modified y2, y2, and b1 ions. These fragments are observed with all the methods and show that the catechol estrogen attachment is at the cysteine moiety of the GSH. Internal fragment ions are the second type, and they also support that the modification is at cysteine. The third type involves fragmentation of the C-S bond to give an ion containing the steroid bonded to the sulfur. The fourth type of fragment ion is similar to the third but involves oxidation of the steroid ring and reduction of the GSH moiety; it is the most isomer specific of the four. The isomer-specific ions are of relatively low abundance in the product-ion spectra taken on the triple quadrupole and ion trap, but their abundances can be improved by increasing the collision energy. ESI source-CAD and the HE-CAD spectra of the isomers are the most distinctive because abundant product ions of all four types are seen in a single spectrum.

Estradiol↗
PubMed1998

Structure and fragmentation mechanisms of isomeric T-rich oligodeoxynucleotides: a comparison of four tandem mass spectrometric methods.

Understanding the product-ion spectra of T-rich tetradeoxynucleotides is a starting point in the development of a mass spectrometric scheme to determine the mutagenicity of individual types of DNA damage. We obtained product-ion spectra for electrospray-produced ions that were activated in the ion source (electrospray ionization-source collision-activated-dissociation) and by high-energy collisions in the MS/MS mode of a four-sector instrument. We also activated singly and doubly charged ions by low-energy collisions in an ion-trap mass spectrometer and investigated post source decompositions of matrix-assisted laser desorbed ions in a time-of-flight mass spectrometer. The various methods of extracting structural information give remarkably consistent results. The difference in the relative abundances of wn and dn ions of the singly charged oligonucleotides and the formation of [a3-B3] ions, where B3 is the base on the third position, are effective for identification and distinction of pairs of isomeric tetranucleotides. A sufficient number of tetramers and pentamers were studied to enable us to propose a charge-remote mechanism for the formation of site-specific [an-Bn] ion.

DNA Damage↗
PubMed1998

Metabolic activation and formation of DNA adducts of hexestrol, a synthetic nonsteroidal carcinogenic estrogen.

Hexestrol (HES), a synthetic nonsteroidal estrogen, is carcinogenic in Syrian golden hamsters. The major metabolite of HES is its catechol, 3'-OH-HES, which can be metabolically converted to the electrophilic catechol quinone, HES-3',4'-Q, by peroxidases and cytochrome P450. Standard adducts were synthesized by reacting HES-3',4'-Q with dG and dA to produce the adducts 3'-OH-HES-6'(alpha, beta)-N7Gua and HES-3',4'-Q-6'-N6dA, respectively. When HES-3',4'-Q was reacted with calf thymus DNA, 3'-OH-HES-6'(alpha,beta)-N7Gua was identified by HPLC and tandem mass spectrometry as the depurinating adduct, with minor amounts of stable adducts. 3'-OH-HES was bound to DNA after activation by horseradish peroxidase, lactoperoxidase, or rat liver microsomes. The depurinating adduct 3'-OH-HES-6'(alpha, beta)-N7Gua was identified in these systems at levels of 65, 41, and 11 micromol/mol of DNA-P, respectively. Unidentified stable adducts were observed in much lower amounts and were quantified by the 32P-postlabeling method. Similarly to 3'-OH-HES, the catechol metabolites of the natural steroidal estrogens estrone (E1) and estradiol (E2), namely, 2-OHE1, 4-OHE1, 2-OHE2, and 4-OHE2, can be oxidized to their corresponding quinones by peroxidases and cytochrome P450. The quinones of the carcinogenic 4-OHE1 and 4-OHE2 have chemical and biochemical properties similar to those of HES-3',4'-Q. The results suggest that formation of HES-3',4'-Q may be a critical event in tumor initiation by HES and that HES is an excellent model compound to corroborate the hypothesis that estrogen-3,4-quinones are ultimate carcinogenic metabolites of the natural steroidal estrogens E1 and E2.

Animals↗
PubMed1998

Synthesis and structure elucidation of estrogen quinones conjugated with cysteine, N-acetylcysteine, and glutathione.

Catechol estrogen quinones (CE-Q) have been implicated as ultimate carcinogenic metabolites in estrogen-induced carcinogenesis. CE-Q may covalently bind to DNA to initiate cancer. These quinones can also be conjugated with glutathione, a reaction that prevents damage to DNA by CE-Q. The glutathione conjugates are then catabolized through mercapturic acid biosynthesis to cysteine and N-acetylcysteine conjugates. This may be the most important detoxification pathway of CE-Q. The chemical synthesis and characterization of these conjugates are the first essential steps to better understand their function in biological systems. Eighteen conjugates were synthesized by reaction of estrone-3,4-quinone (E1-3, 4-Q), estradiol-3,4-quinone (E2-3,4-Q), estrone-2,3-quinone (E1-2, 3-Q), or estradiol-2,3-quinone (E2-2,3-Q) with various sulfur nucleophiles, RSH, in which R is the cysteine, N-acetylcysteine, or glutathione moiety. Reactions of E1-3,4-Q and E2-3,4-Q produce regiospecifically 4-OHE1-2-SR and 4-OHE2-2-SR, respectively, in almost quantitative yield. E1-2,3-Q and E2-2,3-Q react regioselectively and quantitatively to form 2-OHE1(E2)-1-SR and 2-OHE1(E 2)-4-SR, in which the 1-isomers are always the major products. The ratio between 1 and 4 isomers is 3.5 for cysteine, 2.7 for N-acetylcysteine, and 2.5 for glutathione. The synthesized conjugates will be used as standards in the identification of these compounds formed in biological systems.

Acetylcysteine↗
PubMed1998

Covalent binding of catechol estrogens to glutathione catalyzed by horseradish peroxidase, lactoperoxidase, or rat liver microsomes.

Oxidation of catechol estrogens (CE) leads to the reactive electrophilic CE quinones. Reaction of CE-3,4-quinones with DNA has been implicated in tumor initiation. One pathway to prevent this reaction is conjugation of CE quinones with glutathione (GSH). Four CE, 4-hydroxy estrone (4-OHE1), 4-hydroxyestradiol (4-OHE2), 2-OHE1, and 2-OHE2, were conjugated with GSH after oxidation catalyzed by horseradish peroxidase (HRP), lactoperoxidase (LP), or rat liver microsomal cytochrome P450. This reaction is a free-radical chain autoxidation that produces very high yields of products. Six mono-GSH conjugates, 4-OHE1(E2)-2-SG, 2-OHE1(E2)-1-SG, and 2-OHE1(E2)-4-SG, and four di-GSH conjugates, 4-OHE1(E2)-1,2-bisSG and 2-OHE1(E2)-1,4-bisSG, were identified and quantified. These di-GSH conjugates were also obtained quantitatively from oxidation of mono-GSH conjugates by the same enzymes. HRP and LP gave very similar product profiles. Phenobarbital- and 3-methylcholanthrene-induced microsomes with either NADPH or cumene hydroperoxide as cofactor oxidized 4-OHE2 to form similar amounts of GSH conjugates. Enzymatic oxidation of 2-OHE1(E2) in the presence of GSH produced more 2-OHE1(E2)-4-SG than the 1-isomer. This contrasts with the direct reaction of E1(E2)-2,3-Q and GSH, in which the 1-isomer is formed more abundantly than the 4-isomer (Cao, K., Devanesan, P. D., Ramanathan, R., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1998) Chem. Res. Toxicol. 11, 909-916). Competitive enzymatic oxidation of equimolar 4-OHE2 and 2-OHE2 in the presence of an equimolar amount of GSH yielded more 2-OHE2 conjugates than 4-OHE2 conjugates, despite E2-3,4-Q being more reactive with GSH than E2-2,3-Q. These results suggest that 2-OHE2 is a better substrate than 4-OHE2 in the catalytic oxidation to quinones, despite the greater reactivity of E2-3,4-Q, compared to E2-2,3-Q, with GSH.

Animals↗
PubMed1998

Differential regulation of IL-8 by IL-1beta and TNFalpha in hyaline membrane disease.

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1beta and TNFalpha was studied in a short-term culture system. In vivo, IL-8 and IL-1beta protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1beta antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNFalpha antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1beta and anti-TNFalpha antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-1beta antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1beta, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1beta and TNFalpha regulate IL-8 expression in the lungs of preterm infants.

Antibodies, Blocking↗
PubMed1998

Relief of urinary tract obstruction in tuberculosis to improve renal function. Analysis of predictive factors.

OBJECTIVE: To assess the effect of the relief of obstruction on the ultimate function of the affected renal unit in urinary tuberculosis, and to identify predictors of functional recoverability. PATIENTS AND METHODS: Of a total of 82 patients with urinary tuberculosis presenting over 7 years, 38 with evidence of upper tract obstruction were analysed. All patients were assessed with pre-operative excretory urography, urinary mycobacterial cultures and serum chemistry. Patients with subnormally functioning kidneys were assessed with baseline renal scans. Preliminary intervention in the form of JJ stenting or percutaneous nephrostomy (PCN) was carried out in patients with reasonable renal function. Function was reassessed after 4 weeks to detect evidence of improvement and factors which could affect the outcome were determined. RESULTS: Thirty-eight patients had documented upper tract obstruction, of whom six had bilateral obstruction (total of 44 renal units). Ten renal units were not functioning at presentation, with a mean (SD) glomerular filtration rate (GFR) of 3.0 (5.73) mL/min, and no preliminary intervention was performed. In the remaining 34, preliminary intervention was carried out before definitive surgery (JJ stenting in 14, PCN in 15 and PCN followed by antegrade JJ stenting in five); 21 of these renal units were salvaged but 13 were lost despite overcoming the obstruction. Three of the 13 units deteriorated from having acceptable pre-treatment GFRs to becoming non-functional. Good renal cortical thickness, a low grade of renal involvement (Semb 1 or 2), the presence of more distal disease in the form of ureteric stricture and a GFR of > 15 mL/min were good predictors of renal recovery after diversion. CONCLUSIONS: The loss of some renal units seems inevitable in patients with urinary tuberculosis, despite advances in chemotherapy. Having pre-operative predictors of renal recovery may ensure optimal patient selection, thereby reducing the number of procedures and economic burden on the patient who does not require intervention.

Adult↗
PubMed1998

Aneurysm of the ductus arteriosus: a congenital lesion.

The objective of this study was to evaluate the clinical, radiological, and echocardiographic findings in 11 neonates with aneurysm of ductus arteriosus presented in our institutions between 1993 and 1996, and to postulate a new theory for the pathogenesis of this lesion. Medical records, radiographic studies, and echocardiograms were reviewed. All infants underwent follow-up echocardiograms every 2 to 3 days until the aneurysm spontaneously resolved or surgery was performed. The infants were predominantly term males; six had evidence of fetal distress, two were diagnosed prenatally by fetal echocardiogram, chest X ray evidenced mediastinal mass in six patients. The first echocardiogram showed structurally normal heart with an aneurysmal patent ductus arteriosus. In eight patients the aneurysm completely resolved by 5 to 10 days. One infant underwent surgical resection of the aneurysm after observation for 11 days with no change in size. Thrombosis of the aneurysm was noted in two patients; both underwent surgery. Increasing reports of ductal aneurysms in infants may reflect the availability of high-resolution echocardiography and more frequent use of echocardiography in the neonatal intensive care unit. Spontaneous resolution occurred in the majority of cases as in previous reports. We postulate that, at least in some cases, aneurysm of the ductus arteriosus is a congenital lesion that may represent poststenotic dilation of the ductus due to turbulent flow through a stenotic segment at its pulmonary artery end during fetal life. The presence of aneurysm of the ductus arteriosus should be excluded in selected cases of fetal distress, by fetal echocardiography.

Aortic Aneurysm, Thoracic↗
PubMed1998

A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake.

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM, is induced during competence development in wild-type H. influenzae but not in RJ248.

DNA Transposable Elements↗
PubMed1997

Monitoring recombinant protein drugs: a study of insulin by H/D exchange and electrospray ionization mass spectrometry.

The increasing emergence of new protein- and peptide-based drugs makes necessary the development of rapid and sensitive methods to check consistency between and within batches of biotechnology pharmaceuticals to ensure product quality. We evaluated electrospray ionization mass spectrometry in combination with H/D isotopic exchange as a potential tool, taking as examples for this case study the four insulins used for treating insulin-dependent diabetes. Two (bovine and porcine) are produced naturally, and two are produced by recombinant biotechnology techniques [recombinant human (r-human) and its human insulin analog (LysPro)]. The extent of H/D exchange at a given time was measured with less than 2 micrograms (< 350 pmol) of sample and was sufficient for discriminating among the different insulins. After 60 min, bovine, porcine, r-human, and LysPro insulins exchanged on average 25, 28, 30, and 38 amide protons, respectively. After prolonged incubation with D2O for 24 h, bovine and porcine insulins exchanged 31 protons, whereas r-human and LysPro insulins exchanged 34 and 43 amide protons, respectively. The differences in H/D exchange are protein signatures that relate to differences in conformation and folding. The extent of exchange distinguishes among the insulin types and assures the consistency of batch preparations for a given insulin.

Animals↗