Bioluminescence — the biochemistry of photogens and photagogika

The present trend emphasizes the definitive chemistry of photogens, such as specific luciferins, and of photagogika (the actual light-emitters) rather than indirect evidence concerning components of bioluminescent systems as has been practices for almost a century, resulting in many misleading hypotheses. Recent advances indicate that, contrary to earlier conclusions, chemically identical components characterize divers types of bioluminescent organisms and that the r route to light emission probably always involves a hydroperoxide that, at least in some instances, leads to formation of a dioxytenone ring before decomposition.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.     

Circular Thin-layer Chromatography of Oligosaccharides

The physicochemical properties and chemical constituents of the blue protein from rice bran were investigated. The blue protein was a copper-containing glycoprotein, the molecular weight of which was found to be 18,300 Daltons by the sedimentation equilibrium method assuming the partial specific volume 0.72 cm³ g^?1. The hexose and pentose contents were 5.49 and 4.01 g per 100 g protein respectively. The copper content was 0.38% which corresponded to 1.09 atoms per one molecule of the protein. The electron spin resonance spectrum showed that the copper was in a cupric state. The standard oxidation-reduction potential of the copper was found to be +275 mV at 20°C and at pH 7.39. The visible and near infrared absorption maxima were found at 450, 600 and 890 mμ, and the 450 mμ band was optically active in the optical rotatory dispersion exhibiting a large Cotton effect.     

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.     

Synthesis of Prostaglandin-F1 Related Compounds

Synthesis of several prostaglandin-F₁ related compounds utilizing bicyclo(2,2,1) heptene derivatives as key intermediates were investigated.     

Inactivation of Buckwheat α-Glucosidase with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

The amino acid residue(s) involved in the activity of buckwheat α-glucosidase was modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester. The modification resulted in the decrease in the hydrolytic activity of the enzyme following pseudo-first order kinetics. Competitive inhibitors, such as Tris and turanose, protected the enzyme against the inactivation. Protection was provided also by alkali metal, alkaline-earth metal and ammonium ions, though these cations are non-essential for the activity of the enzyme. Turanose or K⁺ protected one carboxyl group per enzyme from the modification with carbodiimide and glycine ethyl ester. Free sulfhydryl group of the enzyme was also partially modified with carbodiimide, but the inactivation was considered to be mainly attributed to the modification of essential carboxyl group rather than to that of free sulfhydryl group.     

l-Alanine-α-Ketobutyrate Aminotransferase of Pseudomonas sp.

Modified fungal product 4-O-methylascochlorin (MAC) is an experimental agent affecting lipid and carbohydrate metabolism in mammals. The hypocholesterolemic properties of MAC were studied using rats fed on a standard laboratory diet. Because of the insolubility in water, reproducibility of the hypocholesterolemic activity had usually been poor for rats fed ad libitum. The difficulty was overcome by controlled reverse-phase feeding; MAC significantly lowered serum total cholesterol (s-TC) in rats only when given by gastric intubation soon after diet intake.

MAC increased fecal excretion of neutral and acidic sterols and also increased biliary flow accompanying increments in biliary cholesterol, bile acids and phospholipids. A much larger increase in neutral sterols was characteristic for MAC. However, intestinal absorption of cholesterol and cholic acid was unaffected by MAC. Three mechanisms therefore seemed to be working in hypocholesterolemic activity: (a) withdrawal of hepatic cholesterol into bile, (b) a larger fecal loss of sterols following increment of biliary sterols and (c) enhanced bile acid synthesis compensating the larger fecal loss. A negative sterol balance often leads to an increase in hepatic cholesterogenesis. However, cholesterogenesis, as judged from incorporation of the precursors, was unchanged by MAC.