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We have reported the time resolved pulsed Photoacoustic (PA) spectrum of formaldehyde molecules (CH2O) We have introduced newly designed PA resonance (L2=6.5cm, R2=0.75 cm) coupled with special filter. The 4th harmonic i.e. 266nm (UV) pulses obtained from Q-switched Nd: YAG laser having 7 ns pulse width is used to excite the resonant modes of the cavity. We have recorded the Photoacoustic spectrum of formaldehyde in time and frequency domains. In addition, we have studied other important factors such as minimum detection limit, incident laser energy, effect of sample pressure, Acquiescing time and the efficiency of PA cells with different excited modes.

We have successfully demonstrated, the detection of THz signal in plasma formed in ambient air using Photoacoustic closed window cell filled with methanol vapour. The type-I SHG cut beta-barium borate (BBO) crystal is used to generate Terahertz by focusing a 40 fs, 800 nm laser pulse in the air to create a superposition of the fundamental and the second harmonic optical fields in the plasma filament. In addition, we have studied the effect of and rotation of BBO crystal on the intensity of acoustic signal associated with the signature of THz radiation. We have also characterized the plasma filamentation using external microphone which was placed close to the focusing point. The variation of the PA signal with incident laser power reflected the dependency of generated THz on the incident laser power. لقد أثبتنا بنجاح ، اكتشاف إشارة THz في البلازما المتكونة في الهواء المحيط باستخدام خلية ضوء-صوتية مغلقة مملوءة ببخار الميثانول. تُستخدم بلورة بورات-بيتا الباريوم (BBO) من النوع I-SHG لتوليد تيراهيرتز من خلال تركيز نبضة ليزر تبلغ 40 fs و 800 نانومتر في الهواء لإنشاء تراكب للحقول الضوئية الأساسية والثانية في خيوط البلازما. بالإضافة إلى ذلك ، قمنا بدراسة تأثير دوران بلورة BBO على شدة الإشارة الصوتية المرتبطة بإشعاع التيراهيرتز. كما قمنا بدراسة خصائص فتيل البلازما باستخدام ميكروفون خارجي تم وضعه بالقرب من نقطة التركيز. اخيرا قمنا بدراسة تباين الموجات الضوء-صوتية مع طاقة الليزر المسلطة والتي تعتمد عليها أشعة التيرا هيرتز المتولدة

A simple, rapid and rugged RP-HPLC method was developed for evaluation and quantification of impurities present in Pirfenidone (PFD) drug substance. Impurities were separated and determined on a Zorbax RX-C18 column (250 mm length, 4.6 mm inner diameter and 5.0 μm particle size, octadecylsilane chemically bonded to porous silica) with 0.02 M KH2PO4 buffer and acetonitrile as mobile phase using a simple gradientelution program. The column flow rate of 1.0 mL per minute was used for the separation. The detection wave length was fixed at 220 nm. The method was substantiated with respect to specificity, precision, linearity, range, accuracy, ruggedness, limit of detection and quantitation. The impurities were identified as 2-hydroxy-5-methylpyridine and Iodobenzene. The linearity range obtained was 0.017 to 0.380 μg/mL for 2-hydroxy-5-methylpyridine, 0.047 to 0.382 μg/mL for Pirfenidone and 0.030 to 0.99 μg/mL for Iodobenzene with the retention times of 3.248 min, 10.608 min and 24.241 min for 2-hydroxy-5-methylpyridine, Pirfenidone and Iodobenzene, respectively. The percentage recoveries of 2-hydroxy-5-methylpyridine and Iodobenzene were in the range of 94.08% - 104.12%. The LOD and LOQ values were found 0.000005 mg/mL, 0.000017 mg/mL for 2-hydroxy-5-methylpyridine and 0.009 μg/mL, 0.030 μg/mL for Iodobenzene, respectively. The method is found to be suitable for the quantitation of impurities along with Pirfenidone drug substance. The method was validated as per the International Conference on Harmonization (ICH) guidelines.

Objective: The main objective of present study was to Isolate, characterize and validate a reverse phase high performance liquid chromatographic method was validated for quantification of bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine in Olanzapine drug substance; it decreases the mental disorders in human body. The method is specific, rapid, precise and accurate for the separation and determination of bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine in Olanzapine drug substance form.Methods: The bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine of Olanzapine was resolved on a Zorbax RX-C 8, 250 mm X 4.6 mm, 5 micron column (L-1) using a mobile phase system containing 0.03 M sodium dodecyl sulphate in water pH 2.5 with 1 N sodium hydroxide solution and acetonitrile in the ratio of (Mobile phase A-52:48 v/v) and (Mobile phase B-buffer and Acetonitrile 30:70 v/v) by using the gradient program. The mobile phase was set at a flow rate of 1.5 ml/min and the volume injected was 20μl for every injection. The detection wavelength was set at 220 nm and the column temperature was set at 35 °C.Results: The proposed method was productively applied for the quantitative determination of bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo [f]azulene)]-1,4-piperazine in Olanzapine drug substance form. The linear regression analysis data for calibration plots showed a good linear relationship over a concentration range of 0.025to 0.903 µg/ml for bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine, 0.081-0.608 µg/ml for Olanzapine. The mean values of the correlation coefficient were 0.999 and 0.999 for bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine and Olanzapine. The method was validated as per the ICH guidelines. The detection limit (LOD) was about 0.007 µg/ml, 0.024 µg/ml and quantitation limit (LOQ) was about 0.024 µg/ml, 0.081 µg/ml for bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine and Olanzapine. The relative standard deviation was found to be 1.64 % and 2.18 % for bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine and Olanzapine.Conclusion: The validated HPLC method and the statistical analysis showed that the method is repeatable and selective for the estimation of the bis-[10-(2-methyl-4H-3-thia-4,9-diazabenzo[f]azulene)]-1,4-piperazine of the Olanzapine drug substance.

Objective: The main objective of present study was to develop and validate a reverse phase enantioselective chiral high performance liquid chromatographic method was developed for enantiomeric resolution of Tenofovir disoproxil fumarate; it decreases the HIV infection in human body. The method is specific, rapid, precise and accurate for the separation and determination of (S)-isomer in tenofovir disoproxil fumarate drug substance form.Methods: The S-Isomer of Tenofovir disoproxil fumarate was resolved on a Chiral AGP (150 × 4.0 mm, 5 µm) column (L-41) using a mobile phase system containing 0.1 M ammonium acetate in water pH 6.8 with ammonia solution and methanol in the ratio of (85:15 v/v). The mobile phase was set at a flow rate of 0.8 ml/min and the volume injected was 10μl for every injection. The detection wavelength was set at 260 nm and the column temperature was set at 15 °C.Results: The proposed method was productively applied for the quantitative determination of (S)-isomer in Tenofovir disoproxil fumarate drug substance form. The linear regression analysis data for calibration plots showed a good linear relationship over a concentration range of 0.125 to 3.75 µg/ml for (S)-isomer, 0.125-3.75 µg/ml for Tenofovir disoproxil fumarate. The mean values of the correlation coefficient were 0.999 and 0.999 for (S)-isomer and Tenofovir disoproxil fumarate. The method was validated as per the ICH guidelines. The detection limit (LOD) was about 0.05 µg/ml and quantitation limit (LOQ) was about 0.125 µg/ml for (S)-isomer and Tenofovir disoproxil fumarate. The relative standard deviation was found to be 0.78 % for (S)-isomer in Tenofovir disoproxil fumarate.Conclusion: The developed and validated HPLC method and the statistical analysis showed that the method is repeatable and selective for the estimation of the (S)-isomer of the Tenofovir disoproxil fumarate drug substance.

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

Hb S is a naturally occurring mutation of human tetrameric hemoglobin in which the β subunits have a hydrophobic Val in place of a negatively charged Glu at the β6 position. The consequence of this mutation is that solubility of Hb S decreases when oxy Hb S loses oxygen. When deoxy Hb S becomes oversaturated deoxy Hb S assembles into long, multi-stranded fibers under physiological conditions (1, 2). Fiber formation is characterized by a delay time prior to polymerization, which is explained by homogeneous and heterogeneous nucleation. During oversaturated Hb S conditions deoxy Hb S monomers form very small polymers by homogeneous nucleation, and these polymers grow by the end addition of hemoglobin molecules in solution. The surface of these growing fibers also can serve as heterogeneous nucleation sites for further growth of additional polymers (1, 3). The polymer then assembles into 14-stranded fibers, which finally form a viscous gel. Intracellular polymers or fibers cause reduction in red blood cell deformability (sickling), leading to obstruction of flow in the microcirculation, thus creating vasooclusion and a wide array of physiological problems including episodes of painful crises (1, 4). Computational refinements of x-ray-determined crystal structures clarified the details of many of the axial and lateral contacts in Hb S polymers (5). These results and properties of the βS chain and of βS peptides (6, 7) show a slight hinge-like motion of the A helix in the βS-globin subunits that makes intermolecular contact with the adjacent Hb S tetramer. Crystal structural analysis of Hb S in the deoxy form also showed not only β6Val in a largely hydrophobic acceptor pocket, but other contact sites involving a hydrogen bond between β4Thr and β73Asp in Hb S which play a critical role in protein interactions. The β4 and β73 positions are located near β6Val and the EF helix acceptor sites in the β subunit, respectively, which are critical lateral contact regions in Hb S polymerization (1, 5, 8). In addition, recent advances in understanding the molecular and cellular pathophysiology of sickle cell disease, coupled with new insights into the developmental regulation of human globin-gene expression as well as characterization of Hb S polymerization, have provided the scientific impetus and clinical rationale to attempt augmentation of the production of Hb F (4). Furthermore, rational drug design (also known as structure-based drug design) through the use of computer modeling and results of x-ray diffraction is an emerging approach that is revolutionizing the practice of drug discovery. The first anti-sickling molecules designed from receptor-based molecular modeling, the substituted benzaldehyde BW12C (now referred to as 12C79), were described by Beddell et al (9). This drug has reached clinical trials, but analysis of the covalent complex of 12C79 and Hb A showed that this agent binds to the N-terminal amino group rather than the direct interaction sites of Hb S polymers including the β6Val donor site, β85Phe and β88Leu hydrophobic acceptor sites or the β4Thr-β73Asp hydrogen bond. Our mutational studies on Hb S polymerization showed that the Hb S β73His variant (β6Val and β73His) promoted polymerization compared to deoxy Hb S, while Hb S β73Leu (β6Val and β73Leu) inhibited polymerization like naturally occurring deoxy Hb C-Harlem (β6Val and β73Asn) (10). These results suggest that the β73 position (Asp in Hb S) serves as a unique site to promote or inhibit polymerization by amino acid replacement (10). Kinetics of polymerization, solubility and minimum concentration required for polymerization of the Hb S β73 variants were affected by β73 amino acid (inhibition of polymerization: His <

A molecular understanding of volatile general anesthetic mechanisms of action will ultimately require high-resolution structural descriptions of anesthetic–protein complexes. Structural changes in proteins following anesthetic binding have been technically difficult to detect, but presumably underlie many of the resulting reversible alterations in protein function. Using three different spectroscopic approaches, evidence is presented that binding of a modern general anesthetic to the four-α-helix bundle (Aα 2 -L1M/L38M) 2 results in conformational changes in the target. Comparable structural changes in in vivo central nervous system protein targets may underlie some, or all, of the behavioral effects of these widely used clinical agents.

A 68 nucleotide segment of the light neurofilament (NF-L) mRNA, spanning the translation termination signal, participates in regulating the stability of the transcriptin vivo. Aldolases A and C, but not B, interact specifically with this segment of the transcriptin vitro. Aldolases A and C are glycolytic enzymes expressed in neural cells, and their mRNA binding activity represents a novel function of these isozymes. This unsuspected new activity was first uncovered by Northwestern blotting of a brainstem/spinal cord cDNA library. It was confirmed by two-dimensional fractionation of mouse brain cytosol followed by Northwestern hybridization and protein sequencing. Both neuronal aldolases interact specifically with the NF-L but not the heavy neurofilament mRNA, and their binding to the transcript excludes the poly(A)-binding protein (PABP) from the complex. Constitutive ectopic expression of aldolases A and C accelerates the decay of a neurofilament transgene (NF-L) driven by a tetracycline inducible system. In contrast, mutant transgenes lacking mRNA sequence for aldolase binding are stabilized. Our findings strongly suggest that aldolases A and C are regulatory components of a light neurofilament mRNA complex that modulates the stability of NF-L mRNA. This modulation likely involves endonucleolytic cleavage and a competing interaction with the PABP. Interactions of aldolases A and C in NF-L expression may be linked to regulatory pathways that maintain the highly asymmetrical form and function of large neurons.

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-R-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four- R-helix bundle (AR2-L1M/L38M)2. The R-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-R-helix bundle (AR2-L38M)2 reported earlier (Johansson, J. S.; Scharf, D.; Davies, L. A.; Reddy, K. S.; Eckenhoff, R. G. Biophys. J. 2000, 78, 982). The affinity for binding halothane is somewhat improved with a Kd ) 120 ( 20 IM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-R-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.