Production, purification and characterization of recombinant human interferon gamma.

An essentially three-step chromatographic purification procedure, i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-gamma (rhIFN-gamma) from the inclusion bodies produced in genetically transformed Escherichia coli cells. Batchwise adsorption of the cloudy solution of renatured rhIFN-gamma obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing of E. coli cells grown in a 30-l fermentor gave about 70 mg of a homogeneous preparation of rhIFN-gamma. The specific biological activity of purified rhIFN-gamma is ca. 3.4 x 10(7) I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI greater than pH 9) with a monomer relative molecular mass of 15,000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-gamma. However, its amino acid composition and its relative molecular mass (15,067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.     

Empirical entropic contributions in computational docking: evaluation in APS reductase complexes

The results from reiterated docking experiments may be used to evaluate an empirical vibrational entropy of binding in ligand-protein complexes. We have tested several methods for evaluating the vibrational contribution to binding of 22 nucleotide analogues to the enzyme APS reductase. These include two cluster size methods that measure the probability of finding a particular conformation, a method that estimates the extent of the local energetic well by looking at the scatter of conformations within clustered results, and an RMSD-based method that uses the overall scatter and clustering of all conformations. We have also directly characterized the local energy landscape by randomly sampling around docked conformations. The simple cluster size method shows the best performance, improving the identification of correct conformations in multiple docking experiments.     

Photochromic and fluorescence properties of coumarin fulgimides

Four new fulgimides possessing a fluorescent coumarin unit were synthesized from the corresponding fulgides, and their photochromic as well as fluorescence properties were investigated. The open-ring forms of coumarin fulgimides were found to exhibit fluorescence in the visible region. Upon exposure to UV light, the fulgimides were transformed into the nonfluorescent closed-ring forms, which can be reverted to the initial fluorescent open-ring forms on exposure to visible light. The efficiency of quenching of fluorescence was as high as 95% at the photostationary state of UV irradiation.     

Electrophilic substitution of diazonium salts in 2-amino-4-(alkyl or aryl)oxazoles

Aryl diazonium fluoborates react readily with 2-amino-4-alkyloxazoles and 2-amino-4-aryloxazoles under mild conditions. The products are the corresponding 5-arylazo derivatives formed, in 44-98% yield free from side products.     

Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides

The adsorption characteristics of a variety of synthetic peptide hormones and di-, tri- and tetrapeptides on Cu(II) immobilized on two commercially available high-performance chelating gels run under various experimental conditions are described. Methods for determining the concentration of immobilized Cu(II) in situ are also described. The Cu(II)-charged columns exhibit a net negative charge as judged from the significantly higher retention of some basic peptides in the absence of NaCl in the equilibration and elution buffers. At higher NaCl concentrations (2-4 M), aromatic interactions seem to be superimposed on the metal ion affinity characteristics of the peptides. The relationship between resolution of peptides and the concentration of immobilized Cu(II) ions has also been established for the Chelating Superose gel where 40 mumol Cu(II) ml-1 gel apparently gives the optimum resolution. The nature of the gel matrix also plays a role in the resolution of some peptides, the extent of which is difficult to predict. The results obtained also suggest that peptides containing aromatic and hydroxy amino acids are retarded more than those which lack them. Moreover, these same amino acids apparently strengthen the existing strong binding of peptides containing His, Trp or Cys to a Chelating Superose-Cu(II) column. Dipeptides with C-terminal His (i.e., X-His) are neither bound nor retarded on a column of Chelating Superose-Cu(II) whereas those having the structure His-X are strongly bound. Some tri- and tetrapeptides containing His were also found not to bind to the column. The underlying cause of this anomalous adsorption behaviour is discussed and is ascribed to "metal ion transfer" arising from the relatively higher affinity of such peptides towards immobilized Cu(II) ions than the chelator groups (iminodiacetate) which are covalently bound to the gel matrix.     

Friedel-crafts reactions of 2-amino-4-(alkyl or aryl)oxazoles with acid chlorides and acid anhydrides: Synthesis of 5-acyl-2-amino-4-alkyloxazoles

Acid chlorides and anhydrides react with 2-amino-4-alkyloxazoles in the presence of aluminum chloride to produce 5-acyl substituted 2-amino-4-alkyloxazoles in modest yields. However, in the absence of the Lewis acid reaction occurs at the amino group to give the corresponding amides. This provides a viable entry for functionalising and making carbon-carbon bond at C-5 of this heterocyclic system.     

14CO2 labeling : a reliable technique for rapid measurement of total root exudation capacity and vascular sap flow in crop plants

Root release of organic compounds and rate of the vascular sap flow are important for understanding the nutrient and the source-sink dynamics in plants, however, their determination is procedurally cumbersome and time consuming. We report here a simple method involving ¹⁴C labeling for rapid and reliable measurement of root exudates and vascular sap flow rate in a variable groundnut population developed through seed gamma irradiation using a cobalt source (⁶⁰Co). An experimental hypothesis that a higher ¹⁴C level in the vascular sap would indicate a higher root release of carbon by the roots into the rhizosphere was verified.     

Novel BODIPY-bridged cyclotriphosphazenes

Three new 2-component unsubstituted ( 4P ), diiodo- ( 5P ), and dibromo- ( 6P ) distyryl-BODIPY-bridged cyclotriphosphazene dimers were designed and synthesized. The newly synthesized BODIPY-cyclotriphosphazene systems were characterized by ¹ H, ¹³ C, and ³¹ P NMR spectroscopy. The photophysical properties of the distryl-BODIPYs (4–6) and BODIPY-cyclotriphosphazene dyads ( 4P – 6P ) were studied by UV-Vis absorption and fluorescence emission spectroscopy. In these derivatives, the bino-type cyclotriphosphazene derivative bearing unsubstituted BODIPY unit 4P exhibited high fluorescence and no singlet oxygen generation due to the lack of spin converter. The attachment of heavy atoms (iodine and bromine) enabled the production of singlet oxygen. The bino-type BODIPY-cyclotriphosphazenes ( 5P and 6P ) were also used as triplet photosensitizers in the photooxidation of 1,3-diphenylisobenzofuran to endoperoxide via generation of the singlet oxygen in dichloromethane. The singlet oxygen production of these compounds was also investigated via a direct method and produced a singlet oxygen phosphorescence peak at 1270 nm.     

Low loaded Pt-Co catalyst surfaces optimized by magnetron sputtering sequential deposition technique for PEM fuel cell applications: physical and electrochemical analysis on carbon paper support

A series of thin Pt-Co films with different metal ratios were deposited by using the sequential cosputtering directly on a commercial hydrophobic carbon paper substrate at room temperature and in ultra-high vacuum (UHV) conditions. Their electrocatalytic properties toward the oxygen reduction reaction were investigated in 0.5 M H₂SO₄ solution by means of cyclic voltammetry (CV) and linear sweep voltammetry (LSV) on rotating disc electrode (RDE). The results showed that Pt particles, deposited by dc-magnetron gun, surround the large Co-clusters deposited by rf-magnetron gun. In addition, the increase of Co content led to an increase in the electrochemical active surface area (EASA) from 23.75 m²/gPt to 47.54 m²/gPt for pure Pt and Pt:Co (1:3), respectively, which corresponded the improvement of the utilization of Pt by a factor of 1.91. This improvement indicated that the sequential magnetron cosputtering was one of the essential technique to deposit homogeneous metal clusters with desirable size on the gas diffusion layer by adjustment plasma parameters.     

Imaging Lipid Metabolism in Live Caenorhabditis elegans Using Fingerprint Vibrations

Quantitation of lipid storage, unsaturation, and oxidation in live C. elegans has been a long‐standing obstacle. The combination of hyperspectral stimulated Raman scattering imaging and multivariate analysis in the fingerprint vibration region represents a platform that allows the quantitative mapping of fat distribution, degree of fat unsaturation, lipid oxidation, and cholesterol storage in vivo in the whole worm. Our results reveal for the first time that lysosome‐related organelles in intestinal cells are sites for storage of cholesterol in C. elegans.