Methods: SERS and SEF: U-1000 Jobin-Ybon Spectrophotometer provided by an argon ion laser (514.5 nm line). Fluorescence: Perkin Elmer 50B with an excitation wavelength of 295 nm. CD: JASCO-710 spectropolarimeter between 200 and 240 nm. Stopped-flow: Biological SFM-3 system coupled to a transmittance detector. The change of transmittance at 490nm (maximum absorption for emodin-BSAf complexes)was monitored during the reaction time.
Results: From SERS and SEF we deduced that in complexes, the neutral and mono anionic drug species are predominant. From fluorescence and CD we calculate the binding constants. CD results indicate a change in the -helical contents of the protein when binding occurs. Stopped-flow experiments indicate the presence of two different mechanism of reaction for the binding.
Conclusions: 1) The primary interaction site of emodin is Sudlow’s site 2, where the bound drug presents a structure between neutral and mono anionic species due to the formation of some hydrogen bond. This interaction changes the -helical contents of the protein. This process occurs for [emodin]/[BSA]≤2.0 ratios, and it implies a fast reaction with a complex mechanism of reaction, where the observed rate constant, kobs, increases when [emodin]/[BSA] increases. After this interval, site 2 saturates.
2) The secondary interaction site of emodin to BSA occurs when [emodin]/[BSA]>2.0 and corresponds to Sudlow´s site 1 binding. Drug species binding to this site is not exactly the same than that binding to site 2, and it exhibits a form more displaced to the neutral one. This interaction does not change the -helical contents of BSA. This process implies a slow reaction with a different mechanism, where the observed rate constant, kobs, is invariable when [emodin]/[BSA] increases.
The impact of a mixture of doxycycline, an acid and a detergent on root canal débridement
SHABAHANG S1, TORABINEJAD M1
1Loma Linda University, Loma Linda, USA
Background: Pulp and periapical pathoses are microbial in nature. Removal of this flora is essential for healing. While débridement of the root canal system is achieved by mechanical instrumentation, supplemented with chemical disinfection, removal of the smear layer (created by the mechanical instrumentation) and the adherent bacteria are not consistently achieved. Here we have combined doxycycline with citric acid and a detergent (MTAD) that reduces surface tension thereby allowing enhanced penetration of the solution.
Methods: Part I (smear layer removal): Forty-eight extracted human teeth were prepared. Sterile water or 5.25% sodium hypochlorite (NaOCl) was used as intracanal irrigant. Canals were treated with 5ml of one of the following final rinses: sterile water, 5.25% NaOCl, 17% ethylenediaminetetracetic acid disodium (EDTA), or MTAD. Presence or absence of smear layer and erosion of the root canal walls were examined under a scanning electron microscope. Part II (disinfection): Eighty-five extracted human teeth were contaminated with Enterococcus faecalis. After biomechanical instrumentation using 1.3% or 5.25% NaOCl, the teeth were exposed to a 5-min application of MTAD, 1.3% NaOCl, 5.25% NaOCl or a 1-min application of EDTA followed by 5ml of 1.3% or 5.25% NaOCl. Teeth or dentin shavings were cultured for presence or absence of test bacteria.
Results: Part I (smear layer removal): MTAD was more effective in removing the smear layer and did not significantly change the structure of the dentinal tubules. Part II (disinfection): Fisher's exact test showed that combination of 1.3% NaOCl and MTAD was significantly more effective in disinfecting the root canal system than the other regimens. Chi2 test showed no difference among the other regimens.
Conclusions: Modification of doxycycline with citric acid and a detergent allows effective disinfection of the root canal system after short exposure. This contact disinfection might be partly due to acidity of the solution. Furthermore, the smear layer is removed without significant alteration to the dentin structure thereby enhancing the débridement process.
Authors’ disclosure statement: The authors acknowledge that, as an institution, Loma Linda University retains a financial interest in this product.
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Drug Cell Interaction at Molecular Level
SHAD FK
Panjwani Center for Molecular Medicine and Drug Research
International Center for Chemical Sciences, University of Karachi, Karachi-75270
ftmshad@yahoo.com
Background: We are using Patch clamp technique to measure the ionic currents involved in neuromuscular disorders and are looking at the drug cell interaction at the molecular level taking muscular dystrophy as an example. Many forms of muscular dystrophy are associated with a structural fragility of the muscle membrane, whereby membrane damage exceeds the ability of muscle to repair itself, resulting in the progressive degeneration of muscle fibers. Clinically and genetically, they are a heterogeneous group of inherited diseases. Mutations in several individual genes are now known to underlie the pathogenesis of different types of muscular dystrophy. Some of these gene mutations involve proteins expressed throughout the body, yet appear to preferentially affect skeletal muscle.
We are interested to detect association of acetylcholine receptor protein with voltage gated sodium currents in biopsied cultured cells.
Methods: Whole cell and single channel configurations of patch clamp technique were used to measure the ionic currents gated by nicotinic acetylcholine receptors using acetylcholine and non depolarizing neuromuscular agent tubocurarine along with sodium channel agonist veratridine.
Results: Patient 1, a 6-year-old boy, had severe myasthenic symptoms since infancy. Patient 2 was a 44-year-old man. Both used wheelchairs and had a 30-50% EMG decrement on 2-Hz stimulation. Evoked quantal release was reduced to approximately 25% of normal in both. In Patient 2, the synaptic response to acetylcholine was further compromised by degeneration of the junctional folds with concomitant loss of the acetylcholine receptor (AChR). Patch clamp study of the muscle biopsy cultured cells confirmed the depletion of nicotinic receptor channel activities and sodium channel agonist veratridine increases the acetylcholine gated currents.
Conclusions: Combined clinical and in vitro electrophysiological findings define two types’ congenital myasthenic syndromes gated by acetylcholine that can be regulated by sodium channel agonist veratridine.
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Bacterial Drug Efflux Pumps: Significance for Antibiotic Resistance and Pathogenicity
SHAFER WM1,2, WARNER D3, ZALUCKI Y 1,2, JOHNSON P1,2, JERSE AE3
1VA Medical Center, Decatur, Georgia, U.S.A., 2Emory University School of Medicine, Atlanta, Georgia, U.S.A., 3Uniformed Services University of the Health Sciences, Bethesda, Maryland, U.S.A
Background: The development and use of chemicals with antibacterial properties revolutionized the treatment of infectious diseases, but mechanisms of bacterial resistance to many antibacterials have evolved and threaten public health. Interestingly, the acquisition of resistance genes often decreases bacterial fitness in vitro, but little definitive information is available regarding their impact on bacterial fitness during infection. We examined this problem with the human pathogen Neisseria gonorrhoeae and an experimental murine infection model and tested if regulation of a multi-drug efflux pump can impact bacterial fitness.
Methods: This study employed a murine vaginal infection and strains of N. gonorrhoeae bearing defined mutations in the mtrR or mtrA genes; mtrR encodes the MtrR repressor and mtrA encodes the transcriptional activator of the mtrCDE operon. Fitness differences were determined by co-infection of mice with wild type and mutant strains. Promoter mapping was performed by primer extension analysis and mutations were identified by DNA sequencing.
Results: We determined that transcriptional regulatory mutations that de-repressed efflux pump gene expression increased levels of gonococcal resistance to antimicrobials and fitness in vivo but not in vitro. These mutations mapped to the transcriptional repressor gene mtrR, a 13 bp inverted repeat sequence within the mtrR promoter and at position -120 upstream of the mtrCDE operon; the latter gives evidence of generating a new promoter element. In contrast to mutations that de-repressed mtrCDE gene transcription, a null mutation in the mtrA gene decreased gonococcal fitness in vivo, but this could be counteracted by second site mutations that de-repressed mtrCDE expression.
Conclusions: The MtrC-MtrD-MtrE efflux pump produced by gonococci is similar to other efflux pumps possessed by Gram-negative bacteria. We propose that this pump is required for gonococcal proliferation at mucosal sites and levels of its expression can significantly impact both antibiotic resistance and in vivo fitness.
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Cytogenetic Response of Imatinib Mesylate in Chronic Phase Chronic Myeloid Leukemia
SHARMA KD, HA CB, KARKI P
BP Koirala Institute of Health Sciences, Dharan, Nepal
Background: The chronic myeloid leukemia (CML) is characterized by presence of Philadelphia Chromosome (Ph) in more than 90 percent of cases. The reciprocal translocation between chromosomes 9 & 22 results in the formation of Ph chromosome and a chimeric BCR-ABL fusion gene that causes CML.The disease follows a triphasic course – initial chronic phase (CP), accelerated phase (AP) and terminal blast crisis phase (BP). Imatinib mesylate (Glivec®, GleevecTM, formerly STI571), a selective tyrosine kinase inhibitor, decreases the number of BCR-ABL positive colony formation in CML patients. Aims: 1) To perform chromosomal analysis in CML patients. 2) To determine the cytogenetic response of Imatinib in Ph positive CML patients in chronic phase (CP).
Materials and Methods: This study was carried out in the Cytogenetic Laboratory at BP Koirala Institute of Health Sciences, Dharan, Nepal. Fifty clinically diagnosed and hematologically proven CML patients in chronic phase were selected for bone marrow aspiration at the time of diagnosis for chromosomal analysis. Those who were Ph positive received Imatinib mesylate 400mg orally daily. The follow up chromosomal analysis was done after six to twelve months of Imatinib therapy to monitor the size and progression of the Ph positive clone. Cytogenetic studies were performed by GTG banding techniques. Karyotypes were interpreted according to the 1995 International System of Human Cytogenetic Nomenclature. Cytogenetic responses (CRs) were determined by the percentage of metaphase cells that were negative for the Ph chromosome.
Results: Out of 50 CML-CP patients 48 (96%) were Ph positive and 2 (4%) were Ph negative at the time of diagnosis. Out of 48 Ph positive patients who were under Imatinib treatment, 25 patients were available for follow up. After a median follow up of 12 months, 16 (64%) patients had major cytogenetic response (Ph+ve cells less than 35%)[partial CR (Ph+ve cells more than 0% to 35%) in 15 (60%) and complete CR (Ph+ve cells 0%) in 1(4%)], 7 (28%) patients had minor cytogenetic response (Ph+ve cells 35% to 65%) and 2 (8%) patients showed Imatinib resistance.
Conclusion: Imatinib reduced Ph chromosome in 23 (92%) out of 25 newly diagnosed Ph positive CML patients in chronic phase.
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Intragraft Administration Of Abciximab And Verapamil Combined With Direct Stenting Prevents Slow-Flow And No-Reflow Phenomenon In Saphenous Vein Graft Percutaneous Coronary Intervention
SHARMA S, LARDIZABAL JA, ANTONESCU A, BHAMBI B, NYITRAY W, DESAI K, ISHIMORI T
Cardiology, Bakersfield Heart Hospital/Central Cardiology, 2901 Sillect Avenue,Ste # 100, Bakersfield, CA 93308, USA
Background. Slow flow/ no-reflow phenomenon (SF-NR) complicates up to 15% cases of percutaneous coronary intervention (PCI) in saphenous vein grafts (SVG). We hypothesized that a strategy of prophylactic intragraft administration of abciximab and verapamil into the SVG, combined with immediate direct stenting of the graft lesion without pre-dilatation, would reduce the risk of platelet activation, microvascular vasospasm and distal plaque embolization respectively and cause a reduction in the incidence of SF-NR.
Methods. Data from 130 consecutive patients who underwent PCI of SVG lesions in a single center over a 7-year period were reviewed. Patients who underwent conventional PCI technique (balloon pre-dilatation of the target lesion prior to stent deployment; optional use of intragraft verapamil or intravenous abciximab) were assigned to the control group (n=72). The patients who received prophylactic intragraft administration of abciximab (0.25 mg/kg) and verapamil (100-300 mcg, depending upon blood pressure and heart rate) through the guiding catheter followed by direct stenting were assigned to the novel strategy group (n=58). The primary outcome was the occurrence of SF-NR Clinical endpoints included death, MI, target vessel revascularization (TVR), and MACE during the hospitalization period, 30 days and at 1 year.
Results: SF-NR occurred more frequently in the control group compared to the novel strategy group (11% vs. 2%, P=0.04). One patient in the control group died after developing persistent SF-NR and acute MI post-PCI. No death was reported in the novel strategy group. Three patients, all in the control group, developed post-PCI MI during the index hospitalization. The difference in 30-day MI and TVR rates did not reach statistical significance. There was a non-statistically significant trend towards higher 1-year MI rate in the control group (8% vs. 2%; P=0.13). The control had significantly higher rates of MACE (25% vs. 7%, P=0.01) and TVR (22% vs. 7%, P=0.03) at 1 year as compared to the novel strategy group.
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Substance Use and Its Functions, Dysfunctions, and Alternative Functions in Contemporary Society
SHAW VN
California State University-Northridge, Northridge, USA
Background: People use and produce substances with some purposes; substance use exists and persists for some reasons; and substance users fit into society and fulfill some functions for it. Aims: 1) To identify various functions substance use fulfills in contemporary society. 2) To identify various dysfunctions substance use has in contemporary society. 3) To identify various alternative functions substance use serves in contemporary society.
Methods: This study is based on general literatures in the humanities and social sciences. Major research methods used range from classification, categorization, correlation, comparison, and logical reasoning to theoretical argumentation. For example, contrasting categorization distinguishes manifest, material, short-term, and peripheral from latent, moral, long-term, and core functions or dysfunctions.
Results: Substance use has both functions and dysfunctions. Specifically, functions or positive effects include pain alleviation, symptom management, stress control, socializing, exchange, trade activities, service provision, and job creation. Dysfunctions or negative consequences involve dependency, withdrawal syndrome, social vice, crime, black market, waste of social resources, and drain on the taxpayers’ money.
Conclusions: 1) Substance improves human adaptation to nature. 2) Substance use and abuse act as a substitute for more serious deviance and even crime in a society. 3) Substance users serve their group, culture, and historical era as messengers of critical issues or innovators of alternative lifstyles.
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POSTIRRADIATION activation of LINE DNA Retroposition in realization of the adaptive response
SHERLINA S, VLADIMIROV V
(Scientific Research Institute of Military Medicine, S. Petersburg, Russia)
mobile LINE, resistance, retroposition
There is a set of concepts of an important role of the mobile genetic elements in particular LINE (long interspersed) DNA in adaptation to the variable environment. However, small molecular mechanisms of resistance are not clear. So an issue of biological significance and origin of nucleic acids of blood plasma as an indicator of extreme states arises.
To study the role of LINE retroposition, a rat model of the radiation stress was used. The dependence of dynamics of the contents of 5’- and 3'-end fragments of LINE and high-molecular (hm) and low-molecular (lm) DNA on the irradiation dose in blood plasma and application of radio protector (WR-2721) was studied. Dynamics of the contents of LINE 5'-end fragments and [3H]-thymidine, included in DNA of the liver, spleen and thymus cells of irradiated animals was also examined. The methods of PCR and PAAG electrophoresis were used.
Unequal reactions of 5’- and 3'-end fragments of LINE to the radiation dose were revealed, pointing to the postirradiation activation of retroposition process. The degree of this activation nonlinearly depends on the dose. At the sublethal doses, LINE retroposition was growing with increasing in irradiation dose, whereas at minimum absolute lethal dose it was partially inhibiting. Dynamics of the separate fractions of extra cellular DNA 2 - 5 h after irradiation was defined by decreasing amount of hmDNA and increasing amount of lmDNA.
Influence of radio protector on the irradiated animals was expressed by increasing in the speed of clearance of hmDNA, not accompanied by accelerating the accumulation of lm DNA of blood plasma. This result shows the role of hmDNA capture by the cells. The changes of LINE retroposition parameters were similar to that of hmDNA in these conditions. It indicates the involvement of the horizontal transfer of genes in mechanisms of radio protective effect.
The revealed drift of 5'-end fragments of LINE and [3H]-thymidine included in DNA of the liver, spleen and thymus cells suggests the involvement of the mechanism of LINE retroposition in the coupling of functions of immunocompetent tissues, important for the adaptation.
In conclusion, the dynamics of LINE retroposition parameters in the blood plasma (5'- / 3'-end fragments ratio) reflects the efficiency of adaptive systems and injury severity of organism in extreme states.
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Screening Phage Genomes to Identify Novel Antimicrobial Targets in Mycobacteria and Related Organisms
SHIBAYAMA Y, DABBS ER
Univ. of the Witwatersrand, Johannesburg, South Africa
Background: Abundance of phages in nature and enormous diversity in their genetic makeup suggest the presence of a huge number of unknown bactericidal mechanisms and can be exploited to identify potential susceptibility targets in drug-resistant bacteria. Aim: 1) To discover genes from novel phages whose products inhibit Mycobacteria and related organisms. 2) To elucidate their mechanisms of action.
Methods: Phages plaquing on Rhodococcus were isolated from soil and purified by CsCl gradient centrifugation. Their morphologies were determined by transmission electron microscopy and genome sizes by pulsed-field electrophoresis. Genomic libraries were constructed using an E. coli – Rhodococcus shuttle vector pDA71. Library clones were individually screened by transformation into the host and Mycobacterium. Inhibitory DNA fragments were subcloned to determine minimum necessary DNA which were then sequenced.
Results: Four phages plaquing on Rhodococcus were isolated. All belonged to the Siphoviridae family and possessed genomes of 39kb to 140kb. Out of 80 randomly selected clones from their genomic libraries, 21 were inhibitory to R. erythropolis, as they had transformation efficiencies of < 102/µg DNA compared to 2 × 104/µg DNA for the vector-only control. Of 9 inserts selected for subcloning, 5 also inhibited M. smegmatis. Inhibitory DNA from the subclones revealed 18 open reading frames, 13 of which had no sequence similarity in databases. ORFs with sequence similarities were to thymidilate synthase complementing protein, HNH endonuclease, capsid protein, tail-related protein, and α subunit of DNA polymerase III.
Conclusion: Isolated phages were shown to provide a large pool of novel antibacterial genes. ORFs with sequence similarities have shown the promise of this approach, as they suggested potential interference with the biosynthesis of dTMP, DNA and protein, and the integrity of DNA and plasma membrane.
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Treatment strategies for multiple liver metastases from colorectal cancer
SHIMADA H1)2) TANAKA K1), ENDO I1)
Dept. of Gastroenterological Surgery, Yokohama City Univ. Graduate School of Medicine, Yokohama, Japan
Harue General Hospital, Fukui, Japan
Background: As extended hepatectomy (Hx) and chemotherapy including targeted agent has developed during the last several years, the treatment outcome for multiple liver metastases from colorectal cancer has implored.
Aim: To clarify the current status of Hx with chemotherapy.
Method&Results: Extended Hx 87 patients who underwent Hx for multiple (>4) and bilobar liver metastases from colorectal cancer from 1992 to 2006 were enrolled in this study.
Results: The mean resected volume of straightforward Hx in 51 patients, Hx after portal embolization (PE) in 13 and two-stage Hx with or without PE in 23 was 401.9g, 654.5 and 879.9 individually. There was no mortality nor significant difference of morbidity among them. The hypertrophy ratio of the future remaining liver volume (before/ after the first procedure) in Hx after PE, two-stage Hx and two-stage Hx with PE was 1.25, 1.25 and 1.6 individually. The 5-year overall survival rate and 5-year disease-free survival rate after Hx were 38.0% and 11.7% individually. From these results, when the remaining cancer-free liver volume is only 200gm, two-stage Hx with PE should be employed. Perioperative chemotherapy
Methods: The treatment consists of systemic chemotherapy with 5FU+FA+CDDP or currently FOLFOX4 and hepatic artery infusion (HAI) with 5FU+FA+CDDP.
Results: 5-year survival rate of adjuvant systemic chemotherapy (49.9%) or adjuvant HAI (54.8%) was significantly higher than that of the absence or less than 5g of total injected 5FU through the HAI. There was a significant difference in survival of patients with 6 or more tumors and CEA > 11ng/ml between patients with and without neoadjuvant chemotherapy. In the 5-year overall survival rate of the responders consisting CR, PR and ST to the chemotherapy (48.8%) was significantly higher than that of the non responders (22.6%). Downstaged chemotherapy provided 5-year survival rate of 34.7% after Hx in 19 out of 138 patients (13.8%) with initially irresectable liver metastases.
Conclusion: Alternation therapy of potent chemotherapy and extended Hx enabled patients with multiple bilobar liver metastatis to survive a long period, specifically for patients of which tumor showed responsiveness to chemotherapy.
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