Physicochemical Properties and Dissolution Studies of Dexamethasone Acetate-β-Cyclodextrin Inclusion Complexes Produced by Different Methods

Inclusion complexes between dexamethasone acetate (DMA), a poorly water soluble drug, and β-cyclodextrin (βCD) were obtained to improve the solubility and dissolution rate of this drug. Phase-solubility profile indicated that the solubility of DMA was significantly increased in the presence of βCD (33-fold) and was classified as A_L-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by different methods (kneading, coevaporation, freeze drying) and physical mixture were characterized by differential scanning calorimetry, thermogravimetry, infrared absorption and optical microscopy. Preparation methods influenced the physicochemical properties of the products. The dissolution profiles of solid complexes were determined and compared with those DMA alone and their physical mixture, in three different mediums: simulated gastric fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and distilled water. The dissolution studies showed that in all mediums DMA presented an incomplete dissolution even in four hours. In contrast, the complexes formed presented a higher dissolution rate in simulated gastric fluid (SGF pH 1.2), which indicate that these have different ionization characteristics. According to the results, the freeze–dried and kneaded products exhibited higher dissolution rates than the drug alone, in all the mediums.     

Studies on the “Salvage” Synthesis of Ribonucleosides and Their 5′-Phosphates by Microorganisms

Studies on the “salvage” synthesis of ribonucleosides and their 5′-phosphates from nucleic acid bases by microorganisms were undertaken. After screening test of less than one hundred strains of type culture, it was found that inosine was produced from hypoxanthine by Arthrobacter ureafaciens, A. simplex, Flavobacterium aquatile and F. suaveolens.

In certain conditions, inosine was further oxidized and hydrolyzed into xanthosine, uric acid and etc.

As for the conditions of cultivation and reaction, the components of the medium and pH of the culture medium were important factors.

Using the standard method, the yield of inosine from hypoxanthine by F. suaveolens reached more than 60%, and the conversion was stoichiometric and any other by-products were not detected.

Inosine, xanthosine, guanosine and uridine were produced from adenine, xanthine, guanine and uracil, respectively, by F. suaveolens.     

Studies on the “Salvage” Synthesis of Ribonucleosides and their 5′-Phosphates by Microorganisms

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (ΔG) at 20°C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Conformational Studies of 3′-Spironucleosides (TSAO-T and Analogues) by NMR Spectroscopy

Abstract

The conformational properties in solution of the prototype compound TSAO-T (1) and its two analogues 2 and 3 have been determined by H and C NMR techniques. The three compounds showed a sugar ring conformation rare among HIV-inhibitory nucleosides, probably due to the presence, at the 3′-position of the spiro moiety.     

Assay of β-galactosidase activity by Flow Injection Analysis (FIA)

Summary A novel method to analyze -galactosidase by Flow Injection Analysis is presented with a linear working range extended to at least 2150 U/mL, being the detection limit 25 U/mL with 55 samples per hour frecuency and a BSD of 0.954% versus 2.4% obtained by manual assay. The method was tested with optimal results with samples from Escherichia coli cultures producing -galactosidase.     

Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma alpha(1)-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1-->4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2-->2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl-->N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas-liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.     

Studies of the Chinese Woodsiaceae (2) — Comparative Morphology and Anatomy of Mature Sporophytes

the comparative studies of morphology and anatomy of 19 species of Chinese Woodsiaceae are reported in the present paper. It includes anatomy of root, rhizome, rachis and joint on stipe; comparative morphology of the fronds and its parts, trichomes and indusia and an observation of the spores under SEM. The information is applied to discussing phylogeny and systematics briefly. The anatomical studies do provide taxonomic studies with evidence so that they should not be abandoned. For example, the stipe joints of Woodsia have two types, the stipe joint below the attachment point of the base pair of pinnae and the stipe joint just at the attachment point of that; the abscission layer of the former does not have pinna trace in it and the basic chromosome number is 39. However, the abscission layer of the latter does and the basic chromosome number is 41. It is obvious that the anatomical cha-racters can be used for infrageneric subdivision of Woodsia.     

The Expression of Tumor Necrosis Factor-α (TNF-α) by the Intrathecal Injection of Lipopolysaccharide in the Rat Spinal Cord

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor α (TNF-α) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-α synthesis is still a matter of controversy. Therefore, we focused our study on TNF-α protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-α reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-α exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-α might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation. Aiguo Shen and Dan Zhou contributed equally to this work.     

Studies on the inhibition of AmpC and other β-lactamases by cyclic boronates

BackgroundThe β-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of β-lactamases, which collectively are able to hydrolyse all classes of β-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) β-lactamase families.MethodsUsing biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important β-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (n = 132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem.ResultsCrystallographic analysis of a bicyclic boronate in complex with AmpC from Pseudomonas aeruginosa reveals it binds to form a tetrahedral boronate species. Microbiological studies on the clinical coverage (in combination with meropenem) and induction of β-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum β-lactamase inhibitors.ConclusionsTogether with reported studies on the structural basis of their inhibition of class A, B and D β-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis.General significanceBicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.     

Kinetic Studies on the Inhibition of GABA-T by γ-Vinyl GABA and Taurine

γ-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of γ-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: γ-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by γ-vinyl GABA and taurine at concentrations of 51.0 and 78.5?mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (K_i) values for γ-vinyl GABA and taurine were found to be 26±3?mM and 68±7?mM respectively. The transamination process of α-ketoglutarate was not affected by the presence of γ-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when α-ketoglutarate was the substrate. The inhibition dissociation constant (K_ii) for this system was found to be 96±10?mM. The Michaelis-Menten constant (K_m) in the absence of inhibition, was found to be 0.79±0.11?mM, and 0.47±0.10?mM for GABA and α-ketoglutarate respectively.     

Sensory cells of the “rod-” and “cone-type” in the pineal organ of Rana esculenta,as revealed by immunoreaction against opsin and by the presence of an oil (lipid) droplet

Summary The pineal organ of the frog, Rana esculenta, was studied by use of light- and electron-microscopic methods including immunoreaction against opsin. Most of the morphologically classified cone-type outer segments of the pineal photoreceptors reacted with antisera against opsin of the bovine retina that is dominated by rods. Some of the outer segments of pineal photoreceptor cells remained unstained in accord with the reference tissue, the frog retina, where generally the rods were opsin-positive and most of the cones opsin-negative.The opsin-negative outer segments of pineal photoreceptors were found in continuity with inner segments each containing a large oil (lipid) droplet. These oil droplets stained intensely with osmic acid, Sudan III, Sudan Black B or Scharlach R in cryostat sections, and were soluble in lipid solvents. In ultrathin sections of osmicated material, the oil droplets were homogeneous and of varying electron density. Approximately one tenth of the pineal photoreceptors contained oil droplets and at the same time possessed opsin-immunonegative outer segments.Since in the retina oil droplets and a negative immunoreaction against bovine opsin are characteristic of cones, we suggest that in the pineal organ they also mark conetype photoreceptors scattered among rod-type photo-receptors, the latter displaying a positive immunoreaction with the antisera used.Support from the Deutsche Forschungsgemeinschaft (Ok 1/25-3) is gratefully acknowledged     

Quantitative Study of Anomeric Forms of Glucose Produced by α-Glucosidases and Glucoamylases

A gas-liquid chromatographic method was applied to the determination of anomeric forms of sugar produced by carbohydrases. Anomeric forms of glucose released from maltotriose, phenyl α-maltoside and phenyl α-glucoside were determined quantitatively. Thirteen α-glucosidases tested, including α-glucosidase from honey bee and acid α-glucosidase from pig′s liver, were found to produce α-glucose exclusively, and two kinds of glucoamylases, only β-glucose. This method proved very useful for the determination of the anomeric forms of sugar produced. It was confirmed that mammalian acid α-glucosidase does not belong to the category of exo-1,4-α-glucosidase (EC 3.2.1.3).