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Biomedical subjects

S Jayaraman

Publications and source records attributed to S Jayaraman.

At least 37 records · Page 2Linked to original sources
PubMed2001

Proteins linked to a protein transduction domain efficiently transduce pancreatic islets.

The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused beta-galactosidase to the 11-amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-X(L) and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-alpha in a pancreatic beta-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient mice.

Animals↗
PubMed2001

Relationships among total lipid, lipid classes, and polychlorinated biphenyl concentrations in two indigenous populations of ribbed mussels (Geukensia demissa) over an annual cycle.

Two indigenous ribbed mussel (Geukensia demissa) populations were sampled approximately every four weeks during 1997 to investigate the relationships among concentrations of total lipid, lipid classes, and polychlorinated biphenyls (PCBs). One population was located in a highly contaminated area near a Superfund site (New Bedford Harbor, MA, USA), while the other population was located at a relatively clean site (West Island, Fairhaven, MA, USA). Mussel tissue total PCB concentrations (quantified as the sum of 18 congeners) from the contaminated site were two orders of magnitude greater than those at the clean site. Total lipid and triacylglycerol (TG) also were higher at the contaminated site. No significant relationship (p > 0.05) was observed between total PCBs and total lipid at either location; however, the correlations at both sites increased when total PCBs were compared with total TG or, to a lesser extent, total nonpolar lipid. Principal component analysis and linear discriminatory analysis indicated that the two mussel populations could be distinguished by the proportions of their lipid classes, particularly the concentration of nonpolar lipids, which consisted mainly of TG. These results suggest that the standard method of normalizing organism PCB concentrations to total lipid may not be appropriate as a routine practice, especially when the organism has a relatively low total lipid content (<6% dry wt in this study).

Animals↗
PubMed2001

Use of Ulva lactuca to identify ammonia toxicity in marine and estuarine sediments.

Toxicity identification evaluation (TIE) methods are being developed for use with whole sediments. Although a phase I TIE method has been developed to characterize ammonia toxicity in aqueous samples using the marine macroalga Ulva lactuca, the relationship between amphipod and mysid mortality and uptake by U. lactuca of bedded sediment ammonia had not been explored. Additionally, it was not known how interactions in whole sediments between metal and organic contaminants with U. lactuca and ammonia would affect TIE interpretation. The current study showed that ammonia toxicity to amphipods and mysids was reduced significantly in marine sediments in the presence of U. lactuca. The alga slightly affected metal concentrations but did not alter the concentration of organic contaminants in either overlying or interstitial waters. Conversely, ammonia uptake by U. lactuca was not affected by the presence of metal or organic contaminants in the sediments. When used with other TIE manipulations, U. lactuca can be utilized in a whole-sediment, phase I TIE to remove toxicity due to ammonia.

Ammonia↗
PubMed2001

HCO3- transport in relation to mucus secretion from submucosal glands.

The role of HCO(3)(-) transport in relation to fluid secretion by submucosal glands is being studied in sheep, pigs, cats and humans. Optical methods have been developed to measure secretion rates of mucus volume from single glands with sufficient temporal resolution to detect differences in minute-by-minute secretion rates among glands. The ionic composition and viscoelastic properties of the uncontaminated gland mucus are measured with a combination of ratiometric fluorescent indicators, ion-selective microelectrodes, FRAP, and a miniaturized, magnetic force viscometer. Sheep glands secreted basally at low rates, showed small, transient responses to alpha- and beta-adrenergic agonists, and large responses to a cholinergic agonist, carbachol. Peak rates and temporal patterns of responses to carbachol differed markedly among glands. To assess the contribution of HCO(3)(-) transport to gland secretion, we either inhibited Na(+)/K(+)/2Cl(-) cotransporter (NKCC) with bumetanide or replaced HCO(3)(-) with HEPES and gassed with O(2). Bumetanide caused a small, non-significant inhibition of basal secretion, but removal of HCO(3)(-)/CO(2) significantly reduced basal secretion almost by half. Both bumetanide and removal of HCO(3)(-)/CO(2) reduced carbachol-stimulated secretion significantly, with HCO(3)(-) removal having the larger effect: a reduction to 33% of control (P<0.01). The remaining secretory response to carbachol was nearly eliminated by bumetanide. Sheep mucus pH measured with ion selective electrodes was about 0.4 log more acidic than the bath. In humans, we observed the same pattern of responses to agonists and antagonists as in sheep, and observed a mucus pH of 7.0 using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). We hypothesize that HCO(3)(-) transport is important in the formation of mucus secretion, but that most HCO(3)(-) is scavenged before the final mucus appears at the duct opening. Cystic fibrosis transmembrane conductance regulator's (CFTR) best understood function is as an anion channel, but increasing attention has been given to its role in HCO(3)(-) transport. By analogy with organ-specific CFTR effects on Cl(-) transport, it seems likely that the relative importance of CFTR in HCO(3)(-) transport will also vary across organs. Because lung disease is by far the greatest cause of mortality among people with cystic fibrosis, it is important to determine how loss of CFTR function causes lung disease. We are testing the hypothesis that loss of CFTR alters serous cell secretion in the lungs, and the corollary that such loss contributes to cystic fibrosis (CF) lung disease. CFTR is highly expressed in serous cells of submucosal glands and the Calu-3 serous cell model secretes HCO(3)(-). Human gland serous cells grown in culture and tested for fluid secretion under open circuit conditions showed reduced fluid secretion to all mediators. However, submucosal glands are complex organs containing at least 4 distinct regions and at least that many cell types, making it difficult to predict the consequences on whole-organ function from experiments with individual cell types. Therefore, we have resurrected long-neglected methods for studying whole-gland function, and have attempted to improve them in a variety of ways. We are refining these methods and increasing our understanding of gland function by studying tracheal glands from sheep, pigs and cats. As human tissues become available, they are studied with the best methods presently available. The key questions now being asked are: Is mucus secretion from submucosal glands altered in cystic fibrosis? If so, how is it altered and how does it contribute to CF lung disease? Answering the last question will require an understanding of how glands interact with other regions of the lung. In the context of this meeting, we present preliminary data on the role of HCO(3)(-) in gland mucus secretion.

Animals↗
PubMed2000

Enantio- and diastereodifferentiating cis,trans-photoisomerization of 2beta,3beta-diphenylcyclopropane-1alpha-carboxylic acid derivatives in organized media.

[reaction: see text] Four methods of asymmetric induction in the cis, trans-photoisomerization of 2beta, 3beta-diphenylcyclopropane-1alpha-carboxylic acid derivatives were studied. Best results (ca. 80% de) were obtained by irradiation of chiral esters, amides, and salts in NaY and LiY zeolites and in the pure crystalline state.

Journal Article↗
PubMed2000

Quenching mechanism of quinolinium-type chloride-sensitive fluorescent indicators.

Quinolinium based Cl- sensitive fluorescent indicators have been used extensively to measure intracellular Cl- activity. To define their fluorescence quenching mechanism, a series of N-methyl quinolinium derivatives were synthesized, including N-methylquinolinium (Q), 6-methylQ, 6-methoxyQ, 6-chloroQ, 3-bromoQ, 6-aminoQ and N-methylisoquinolinium. Stern-Volmer plots for quenching by Cl-, Br-, SCN-, I-, F-, OAc- and CO3(2-) from both intensity and lifetime measurements were linear. Bimolecular quenching rate constants (kq) decreased with increasing anion oxidation potentials and increased with increasing quinolinium reduction potentials. The free energy change for charge transfer (deltaG), calculated from indicator spectral and electrochemical properties, was found to correlate with log kq. These results suggest that quenching of quinolinium fluorescence in water by anions involves a charge-transfer quenching mechanism. Understanding the mechanism facilitates structure-based predictions of the anion sensitivities of quinolinium indicators to design improved Cl- indicators with tailored properties.

Chemical Phenomena↗
PubMed2000

Mechanism and cellular applications of a green fluorescent protein-based halide sensor.

We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the absence of Cl(-), which increased to 7.86 at 150 mM Cl(-). At pH 7.5, YFP-H148Q fluorescence decreased maximally by approximately 2-fold with a K(D) of 100 mM Cl(-). YFP-H148Q had a fluorescence lifetime of 3.1 ns that was independent of pH and [Cl(-)]. Circular dichroism and absorption spectroscopy revealed distinct Cl(-)-dependent spectral changes indicating Cl(-)/YFP binding. Stopped-flow kinetic analysis showed a biexponential time course of YFP-H148Q fluorescence (time constants <100 ms) in response to changes in pH or [Cl(-)], establishing a 1:1 YFP-H148Q/Cl(-) binding mechanism. Photobleaching analysis revealed a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers. The anion selectivity sequence for YFP-H148Q quenching (ClO(4)(-) approximately I(-) > SCN(-) > NO(3)(-) > Cl(-) > Br(-) > formate > acetate) indicated strong binding of weakly hydrated chaotropic ions. The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the tri-amino acid chromophore. YFP-H148Q transfected mammalian cells were brightly fluorescent with cytoplasmic/nuclear staining. Ionophore calibrations indicated similar YFP-H148Q pH and anion sensitivities in cells and aqueous solutions. Cyclic AMP-regulated Cl(-) transport through plasma membrane cystic fibrosis transmembrane conductance regulator Cl(-) channels was assayed with excellent sensitivity from the time course of YFP-H148Q fluorescence in response to extracellular Cl(-)/I(-) exchange. The green fluorescent protein-based halide sensor described here should have numerous applications, such as anion channel cloning by screening of mammalian expression libraries and discovery of compounds that correct the cystic fibrosis phenotype by screening of combinatorial libraries.

3T3 Cells↗
PubMed2000

Statins and cardiovascular diseases: the multiple effects of lipid-lowering therapy by statins.

Cholesterol lowering involving different therapies improves the clinical outcome of patients. To define the underlying pathomechanism, we studied whether treatment with statins was associated with changes in blood thrombogenicity, endothelial dysfunction and soluble adhesion molecule levels. Fifty hypercholesterolemic patients were treated with pravastatin (40 mg/day, n=24) or simvastatin (20 mg/day, n=26). Lipid profile and blood thrombogenicity were assessed in all patients before and after 3 months of cholesterol reducing therapy. Blood thrombogenicity was assessed as thrombus formation, perfusing non-anticoagulated blood directly from the patients' vein through the Badimon perfusion chamber (shear rate 1690/s). Endothelial-dependent vasomotor response was tested by laser-Doppler flowmeter. Soluble adhesion molecule level were measured by ELISA. Total and LDL cholesterol were reduced in the two treatment groups by statin therapy. Statin therapy was associated with a significant reduction in blood thrombogenicity and endothelium-dependent vasoresponse. No differences were observed between simvastatin or pravastatin treatment. Lipid lowering by statins had no effect on plasma levels of fibrinogen, sL-selectin, sP-selectin and sICAM-1 antigen. Cholesterol lowering by both statins reduced the increased blood reactivity and endothelial dysfunction present under hypercholesterolemia. The multiple effects of lipid lowering therapy by statins may explain the benefits observed in recent epidemiological trials.

Anticholesteremic Agents↗
PubMed2000

Regulation of T cell apoptosis.

Proliferative expansion of lymphoid cells is required for effective immune responses against invading microorganisms, but after the infection is controlled, the expanded effector cells must be eliminated to prevent non-adaptive accumulation of cells. Higher vertebrates have developed extensive networks of signal transduction pathways to ensure controlled activation and expansion of cells during immune responses and apoptotic deletion of lymphoid cells that are no longer needed at the end of immune responses. Extracellular signals received by cell surface receptors that trigger intracellular signaling cascades are essential elements that control both processes. These signal transduction pathways converge to regulate cell fate at both transcriptional and post-transcriptional levels. Here we review the role of pathways, especially those triggered by TNF receptor-related molecules, that determine the fate of T cells during development and activation. In addition, we introduce the possibility that these same pathways may be abnormally programmed and so lead to immune cell accumulation during inflammatory diseases such as asthma.

Animals↗
PubMed1999

Resistance to Fas-mediated T cell apoptosis in asthma.

Over activation of CD4+ T cells in the peripheral blood and airway tissues is characteristic of asthma; therefore, we investigated whether activated T cells from asthmatic subjects have altered apoptotic potential through the Fas death receptor. We found that mitogen-stimulated peripheral blood T cells of asthmatic subjects expressed cell surface Fas, but failed to undergo the normal degree of apoptosis after Fas receptor ligation. T cells from asthmatics exhibited normal apoptotic responses to gamma-irradiation (dependent on IL-1 converting enzyme family proteases), ceramide, and mitogen challenge, suggesting functional integrity of the apoptotic pathway. Furthermore, the defect in Fas-dependent apoptosis was overcome by prestimulation with allogeneic accessory cells instead of mitogen. Taken together, the findings suggest that selective resistance to Fas-dependent apoptosis reflects altered Ag-driven, accessory cell-dependent signaling and that ineffective activation of Fas signal transduction may contribute to T cell-dependent immunoinflammation in asthma.

Adolescent↗
PubMed1999

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging.

The fluorescence of quinolinium-based Cl- indicators such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl- by a collisional mechanism without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl--sensitive moiety [excitation wavelength (lambdaex) 350 nm, emission wavelength (lambdaem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl--insensitive moiety because of its different spectral characteristics (lambdaex 380 nm, lambdaem 546 nm), insensitivity to Cl-, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl--sensitive indicators were alpha-MQ-alpha'-dimethyl-AQ-xylene dichloride and trans-1, 2-bis(4-[1-alpha'-MQ-1'-alpha'-dimethyl-AQ-xylyl]-pyridinium)ethyl ene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl- sensitive; Stern-Volmer constants 82 and 98 M-1) and 565 nm (Cl- insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 microM, 10 min). Changes in Cl- concentration in response to Cl-/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl- indicators for ratiometric measurement of Cl- concentration.

3T3 Cells↗
PubMed1999

Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells.

Limitations of available indicators [such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl(-) are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl(-) and/or I(-) sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(beta-D-ribofuranosylamino)-pyrido[2, 1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400-470 and 490-560 nm, molar extinction 11,100 M(-1). cm(-1) (424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I(-) by a collisional mechanism (Stern-Volmer constant 60 M(-1)) and is not affected by other halides, nitrate, cations, or pH changes (pH 5-8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I(-) (50% quenching at 38 mM I(-)). LZQ was used to measure forskolin-stimulated I(-)/Cl(-) and I(-)/NO(-)(3) exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype.

1-Methyl-3-isobutylxanthine↗
PubMed1999

The Wearable Motherboard: a flexible information infrastructure or sensate liner for medical applications.

Research on the design and development of a Sensate Liner for Combat Casualty Care has led to the realization of the world's first Wearable Motherboard or an "intelligent" garment for the 21st Century. This Georgia Tech Wearable Motherboard (GTWM) provides an extremely versatile framework for the incorporation of sensing, monitoring and information processing devices. The principal advantage of GTWM is that it provides, for the first time, a very systematic way of monitoring the vital signs of humans in an unobtrusive manner. Appropriate sensors have been "plugged" into this motherboard using the developed Interconnection Technology and attached to any part of the individual being monitored, thereby creating a flexible wearable monitoring device. The flexible data bus integrated into the structure transmits the information to monitoring devices such as an EKG Machine, a temperature recorder, a voice recorder, etc. The bus also serves to transmit information to the sensors (and hence, the wearer) from external sources, thus making GTWM a valuable information infrastructure. GTWM is lightweight and can be worn easily by anyone--from infants to senior citizens. GTWM has enormous potential for applications in fields such as telemedicine, monitoring of patients in post-operative recovery, the prevention of SIDS (sudden infant death syndrome), and monitoring of astronauts, athletes, law enforcement personnel and combat soldiers.

Ambulatory Care↗
PubMed1998

Tamoxifen-induced light and electron microscopic changes in the rat testicular morphology and serum hormonal profile of reproductive hormones.

The effects of oral administration of tamoxifen at doses of 40 and 200 micrograms/kg/day on testicular histology, testicular ultrastructure and serum hormonal profile were studied. The drug was administered to adult male rats over a period of 90 days and the effect was assessed at 10-day intervals. The morphometry, microscopic structures of the testis, including ultrastructure and daily sperm production rate, were evaluated. The hormone profiles of luteinizing hormone (LH), follice-stimulating hormone (FSH), testosterone, and estradiol were studied. The testes from treated animals showed disorganization of tubular elements with increased intercellular space. At day 50, the changes were extensive including presence of phagosomes. Morphometric studies showed a reduction in the spermatid and spermatozoan population (69.3%) with no changes in tubular diameter. The mean Leydig cell area was significantly lowered at day 50, at both doses. The daily sperm production rate was reduced as compared with controls. An array of degenerative changes were revealed by ultrastructural studies. The changes were extensive at day 50 at both doses. The characteristic features were lost in most of the cells with phagolysosomes becoming abundant. The cytoplasm of the cells was dense with poorly defined cytoplasmic organelles. Circulating LH levels were not modified at the 40 micrograms/kg/day dose but at 200 micrograms/kg/day, LH levels were significantly decreased. Initial transitory rise in FSH was seen with both doses. Both doses of tamoxifen decreased testosterone levels. Changes in the circulating estradiol levels were inconsistent, and no apparent relationship between dose and days of treatment was observed. Thus, this study supports our thesis of tamoxifen as a potential male contraceptive agent.

Animals↗
PubMed1998

Regulation of in vitro and in vivo T cell activation by CD43.

Accessory molecule interactions can be critical in determining the outcome of a T cell's encounter with antigen. Cell adhesion proteins may augment T cell responses by facilitating TCR engagement of the antigen-MHC complex, while co-stimulatory molecules may deliver distinct signals that modulate T cell responsiveness. CD43 (leukosialin, sialophorin) has been suggested to influence cell activation by steric hindrance based upon the large size and glycosylation of the protein, as well as the relative abundance of the protein on the cell surface. In this paper we examine both in vitro and in vivo T cell-dependent responses in CD43-deficient mice. We demonstrate that T cells from CD43-deficient mice are hyper-responsive following both in vivo and in vitro activation, and that this is observed in response to not only TCR-CD3-mediated stimulation, but also following receptor-independent activation. This data suggests that mechanisms other than non-specific steric hindrance are important in the regulation of T cell activation by CD43.

Animals↗
PubMed1998

Crack propagation directions in unfilled resins.

Posterior composite restorative materials undergo accelerated wear in the occlusal contact area, primarily through a fatigue mechanism. To facilitate the timely development of new and improved materials, a predictive wear model is desirable. The objective of this study was to develop a finite element model enabling investigators to predict crack propagation directions in resins used as the matrix material in composites, and to verify these predictions by observing cracks formed during the pin-on-disc wear of a 60:40 BISGMA:TEGDMA resin and an EBPADMA resin. Laser confocal scanning microscopy was used to measure crack locations. Finite element studies were done by means of ABAQUS software, modeling a cylinder sliding on a material with pre-existing surface-breaking cracks. Variables included modulus, cylinder/material friction coefficient, crack face friction, and yield behavior. Experimental results were surprising, since most crack directions were opposite previously published observations. The majority of surface cracks, though initially orthogonal to the surface, changed direction to run 20 to 30 degrees from the horizontal in the direction of indenter movement. Finite element modeling established the importance of subsurface shear stresses, since calculations provided evidence that cracks propagate in the direction of maximum K(II)(theta), in the same direction as the motion of the indenter, and at an angle of approximately 20 degrees. These findings provide the foundation for a predictive model of sliding wear in unfilled glassy resins.

Composite Resins↗
PubMed1998

A sensate liner for biomedical monitoring applications.

This program develops and demonstrates technologies useful for implementing a manageable cost effective systems approach to monitoring the medical condition of personnel by way of an instrumented uniform hereafter referred to as a Sensate Liner (SL). The SL consists of a form fitting garment which contains and interconnects sensing elements and devices to an electronics pack containing a processor and transmitter. The SL prototype requires fiber, textile, garment and sensor development. The SL textile consists of a mesh of electrically and optically conductive fibers integrated into the normal structure (woven or knitted) of fibers and yarns selected for comfort and durability. A suite of SL garment compatible embedded biological and physical sensors are then integrated into the SL. The initial SL sensor suite is selected to improve triage for combat casualties. Additional SL sensor concepts for medical monitoring will be discussed.

Clothing↗