Detection of DNA Sequence with Enhanced Sensitivity and Higher FRET Efficiency Using a Light-Emitting Polymer, Peptide Nucleic Acid Probe and Anionic Surfactant System.

An improved strategy has been developed for detection of DNA sequence by using water-soluble cationic conjugated polymer (PFP)/single-strand (ss) DNA and peptide nucleic acid labeled with fluorescent dye (PNAC*), where an anionic surfactant (sodium dodecyl sulphate, SDS) system has been used to improve the sensitivity of the sensor. The method of detection is simple to use, fast and cost-effective. This method uses the phenomenon of Forester Resonance Energy Transfer (FRET). The detection sensitivity of the biosensor has been improved by about ten times by using the anionic surfactant. It is observed that the effect of surfactant is to increase the photoluminescence (PL) intensity of the PNAC* when the sequence of the DNA is complementary (to that of PNA probe). On the other hand when the two sequences are non-complementary, the PL intensity of the PNAC* is further reduced as compared to the case when surfactant was absent.

Detection of known mutations for medical diagnostics by FRET spectroscopy.

A rapid, simple and low-cost method for the detection of known mutations in DNA oligonucleotide in a biothreat agent, Bacillus anthracis, has been reported. The technique is based on fluorescence resonance energy transfer (FRET), that utilizes a cationic conjugated polymer and a PNA probe labeled with Fluorescein dye (PNAC*). When the PNA probe is hybridized with a complementary target ssDNA and its mutated sequences separately, the energy transfer from polymer to PNAC*/ssDNA complex decreases with increasing number of mutations. It means that the efficiency of FRET or the degree of hybridization depends on the extent of mutations in the DNA sequence. The method is sensitive enough to detect upto 4 bases mismatch. We have, thus, explored a possible application of fluorescence-based technology for medical diagnostics.

Effect of oxazolidinone, RBx 7644 (ranbezolid), on inhibition of staphylococcal adherence to plastic surfaces.

Adhesion to biomaterial is assumed to be a crucial step in the pathogenesis of foreign body infection. Slime producing Staphylococcus epidermidis and Staphylococcus aureus have emerged as a preeminent cause of nosocomial bacteremia and infections of prosthetic medical devices. We evaluated the time-dependent anti-adhesive effect of RBx 7644 (ranbezolid), vancomycin, linezolid and quinupristin/ dalfopristin on two isolates each of S. epidermidis and S. aureus. Linezolid and quinupristin/ dalfopristin showed inhibition only at supra-inhibitory concentrations (2 and 4X MIC) following 2 and 4 h delayed treatment, whereas RBx 7644 demonstrated significant activity against adhesion of staphylococcal cells that had been treated with 2 to 6 h delay. When vancomycin treatment was delayed by 4 to 6 h, even concentrations above the MIC were unable to prevent adherence. This study indicates that RBx 7644 has anti-adhesion potential and may emerge as an important antibiotic for prevention and treatment of device-related infections caused by staphylococci.

Accidental gamma dose measurement using commercial glasses.

Commercial glasses have been investigated for their application in accidental gamma dose measurement using Thermoluminescent (TL) techniques. Some of the glasses have been found to be sensitive enough that they can be used as TL dating material in radiological accident situation for gamma dosimetry with lower detection limit 1 Gy (the dose significant for the onset of deterministic biological effects). The glasses behave linearly in the dose range 1-25 Gy with measurement uncertainty +/- 10%. The errors in accidental dose measurements using TL technique are estimated to be within +/- 25%. These glasses have shown TL fading in the range of 10-20% in 24 h after irradiation under room conditions; thereafter the fading becomes slower and reaches upto 50% in 15 d. TL fading of gamma-irradiated glasses follows exponential decay pattern, therefore dosimetry even after years is possible. These types of glasses can also be used as lethal dose indicator (3-4 Gy) using TL techniques, which can give valuable inputs to the medical professional for better management of radiation victims. The glasses are easy to use and do not require lengthy sample preparation before reading as in case of other building materials. TL measurement on glasses may give immediate estimation of the doses, which can help in medical triage of the radiation-exposed public.

Triple-FRET Technique for Energy Transfer Between Conjugated Polymer and TAMRA Dye with Possible Applications in Medical Diagnostics.

Three-component Förster resonance energy transfer (FRET) has been used to obtain efficient FRET between the cationic conjugated polymer (CCP) as donor and 5-carboxy tetramethylrhodamine (TAMRA) dye as acceptor, by using an intermediate donor, fluorescein. In spite of the fact that there is enough overlap between the emission spectra of CCP and absorption spectra of TAMRA, the efficiency of FRET between CCP and TAMRA is poor. The reason for this is that while the Förster critical distance is not very sensitive to the overlap, the FRET efficiency is extremely sensitive to it. However, it is observed that the FRET efficiency between CCP and TAMRA improves considerably when fluorescein is introduced in the solution. The triple FRET so obtained can be used for deoxyribonucleic acid sequence detection in medical diagnostics because the fluorescence emission from TAMRA is pH-insensitive.

Evaluation of dose distributions in gamma chamber using glass plate detector.

A commercial glass plate of thickness 1.75 mm has been utilized for evaluation of dose distributions inside the irradiation volume of gamma chamber using optical densitometry technique. The glass plate showed linear response in the dose range 0.10 Kilo Gray (kGy) to 10 kGy of cobalt-60 gamma radiation with optical sensitivity 0.04 Optical Density (OD) /kGy. The change in the optical density at each identified spatial dose matrix on the glass plate in relation to the position in the irradiation volume has been presented as dose distributions inside the gamma chamber. The optical density changes have been graphically plotted in the form of surface diagram of color washes for different percentage dose rate levels as isodose distributions in gamma chamber. The variation in dose distribution inside the gamma chamber unit, GC 900, BRIT India make, using this technique has been observed within +/- 15%. This technique can be used for routine quality assurances and dose distribution validation of any gamma chamber during commissioning and source replacement. The application of commercial glass plate for dose mapping in gamma chambers has been found very promising due to its wider dose linearity, quick measurement, and lesser expertise requirement in application of the technique.

Application of commercial glasses for high dose measurement using the thermoluminescent technique.

Commercial glasses under this study showed linear thermoluminescence (TL) response in gamma dose range 100 Gy to 10 kGy, glow peaks between 175 and 200 degrees C, fading under dark and room light 2.86-7.36% and 10.42-20.82%, respectively, in 24h and 34.86-70.80% under sunlight in 5h after exposure. The TL glass dosimetric results have been found to be reproducible within +/- 6.0%. Glasses have been observed as thermally unstable and its TL sensitivity reduces after annealing. The TL response of the glasses has been found to reduce by 7.40-51.49% after first annealing of the samples at 400 degrees C for 15 min. The trace element study suggests that presence of impurities has no role in TL sensitivity of glasses rather imperfections and dislocations in the lattice are the major contributor in the formation of TL centers. Commercial glasses can serve as good TL material for gamma irradiator and gamma chamber dosimetry. The various radiation parameters for glass TL dosimetry have been studied in detail and presented.

Anti-tumor effects of the bacterium Caulobacter crescentus in murine tumor models.

Caulobacter crescentus is a gram negative, non-pathogenic bacterium, common in aquatic and soil environments. One feature of note is a protein surface layer (S-layer) composed of a single protein, organized as a self-assembled crystalline array that coats the bacterium. In the course of efforts to express cancer-associated peptides as genetic insertions into the S-layer, we noted a tumor suppressive effect of the unmodified bacterium. C. crescentus was examined for anti-tumor activity against three transplantable tumor mouse models: Lewis lung carcinoma cells transfected with the MUC1 gene in C57BL/6, murine mammary carcinoma (EMT-6) in BALB/c (both in prophylactic and therapeutic mode) and murine leukemia cells (L1210) in DBA2. Mice were immunized three times i.p. with C. crescentus (2 x 10(7) cells/mouse). In prophylactic mode, the mice were challenged with tumor cells two weeks after the last immunization. Immunization with live C. crescentus resulted in anti-tumor activity in all three transplantable tumor models, as measured by prolonged survival, reduced tumor mass or reduced number of lung nodules, compared to saline control groups. In the Lewis lung and the EMT-6 mammary carcinoma murine models the number of lung nodules as well as the tumor weight was lower in mice treated with C. crescentus, compared to the control group; for EMT-6, this was observed in prophylactic and therapeutic modes. In the murine leukemia and Lewis lung carcinoma models prolonged survival was observed in the groups of mice immunized with Caulobacters. In most cases the live C. crescentus cells were markedly more efficacious than heat killed or formalin fixed cells, despite the fact that they do not grow or persist in mice. The results suggest that C. crescentus may be a safe, bacterial immunomodulator for the treatment of tumors.

Calcilytic compounds: potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone.

Despite the discovery of many ions and molecules that activate the Ca2+ receptor, there are no known ligands that block this receptor. Reported here are the pharmacodynamic properties of a small molecule, NPS 2143, which acts as an antagonist at the Ca2+ receptor. This compound blocked (IC50 of 43 nM) increases in cytoplasmic Ca2+ concentrations [Ca2+]i elicited by activating the Ca2+ receptor in HEK 293 cells expressing the human Ca2+ receptor. NPS 2143, even when tested at much higher concentrations (3 microM), did not affect the activity of a number of other G protein-coupled receptors, including those most structurally homologous to the Ca2+ receptor. NPS 2143 stimulated parathyroid hormone (PTH) secretion from bovine parathyroid cells (EC50 of 41 nM) over a range of extracellular Ca2+ concentrations and reversed the effects of the calcimimetic compound NPS R-467 on [Ca2+]i and on secretion of PTH. When infused intravenously in normal rats, NPS 2143 caused a rapid and large increase in plasma levels of PTH. Ca2+ receptor antagonists are termed calcilytics and NPS 2143 is the first substance (either atomic or molecular) shown to possess such activity. The pharmacodynamic properties of NPS 2143 together with the recently demonstrated effects of this compound on bone formation support the view that orally active calcilytic compounds might provide a novel anabolic therapy for osteoporosis.

Gamma-irradiated onions as a biological indicator of radiation dose.

Post-irradiation identification and dose estimation are required to assess the radiation-induced effects on living things in any nuclear emergency. In this study, radiation-induced morphological/cytological changes i.e., number of root formation and its length, shooting length, reduction in mitotic index, micronuclei formation and chromosomal aberrations in the root tip cells of gamma-irradiated onions at lower doses (50-2000 cGy) are reported. The capabilities of this biological species to store the radiation-induced information are also studied.

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

Novel peptidomimetic hematoregulatory compounds.

The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation.

Identification of unique truncated KC/GRO beta chemokines with potent hematopoietic and anti-infective activities.

SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GRO beta. In comparison to their full-length forms, truncated KC and truncated GRO beta were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GRO beta. To our knowledge, this represents the first example where any form of KC or GRO beta were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.

STUDY OF LOW BIRTH WEIGHT NEONATES.

Low birth weight neonates with 2000g or less birth weight constitute about 10% of live births with perinatal mortality as high as 32.4%. Perinatal morbidity is 19.3% with asphyxia neonatorum and neonatal jaundice heading the list. Epidemiological maternal factors include extremes of age and parity, lack of antenatal care, low socioeconomic status, illiteracy and underweight short women. Etiologic factors are obstetric complications, hypertensive disorders, systemic diseases or idiopathic. The scope of preventive measures include improvement of economic status and education about health and safe pregnancy. Proper antenatal care for early detection of high risk cases, adequate and timely management of complications and adequate facilities for neonatal care can reduce the perinatal morbidity and mortality.

Effect of amphotericin B lipid formulation on immune response in aspergillosis.

The immune response against Aspergillus fumigatus has been studied during infection and therapy in order to understand the mechanism of pathogenesis and the effect of treatment with amphotericin B. With this in view an animal model of aspergillosis was developed in Balb/c mice by intravenous injection of an optimized dose of 3. 6x10(6) A. fumigatus spores. Infection due to Aspergillus was well established by histopathological examination and fungal load in the animal. Lesions and eosinophil infiltration was observed in the infected tissues which indicated the involvement of a Type I hypersensitivity response. Evaluation of serological parameters indicated high levels of interleukin-4 (IL-4) and A. fumigatus specific IgG antibodies. The reduction in fungal load and modulation of immune response in the infected mice was studied following treatment with amphotericin B/cholesterol hemisuccinate vesicles (ABCV). The results clearly indicated significant reduction in the fungal load, disappearance of eosinophils and lesions with the appearance of macrophages and neutrophils in the infected lung tissue, a decrease in IL-4 (fourfold) and a concomitant increase of interferon-gamma (IFN-gamma; twofold) with an improvement in general condition of mice. In the non-treated mice, the rise of IL-4 level indicated the association of T(H)2 cell response with susceptibility to infection while the increase of IFN-gamma in the treated group suggested that T(H)1 cell response may be involved in resistance to Aspergillus infection.

Studies on the viability of metacercariae of Fasciola gigantica.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.

Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647 by fragment coupling.

Linear and convergent routes for the large-scale preparation of the hematoregulatory nonapeptide (Glp-Glu-Asp)2-DAS-(Lys)2 (2, SK&F 107647) were investigated. A convergent approach ('3 + 2'-route employing Boc-and benzyl ester protecting groups) was selected for the preparation of multihundred-gram quantities of 2. Key steps were the preparation and the coupling of tripeptide hydrochloride (HCl.H)2-DAS-(Lys(Z)-OBn)2 (6, DAS-2,7-L,L-diaminosuberic acid) and tripeptide Glp-Glu(OBn)-Asp(OBn)-OH (26). Several coupling reagents were investigated in order to reduce the amount of epimerization of this fragment coupling. TDBTU [O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl-1,1,3,3-tetrameth yluronium tetrafluoroborate] was identified as the condensation reagent of choice. Using this synthetic route > 97% pure final product in an overall yield of 35% calculated on di-Boc protected 2,7-L,L-diaminosuberic acid was prepared.