The heme environment of recombinant human indoleamine 2,3-dioxygenase. Structural properties and substrate-ligand interactions

Indoleamine 2,3-dioxygenase is a heme enzyme that catalyzes the oxidative degradation of L-Trp and other indoleamines. We have used resonance Raman spectroscopy to characterize the heme environment of purified recombinant human indoleamine 2,3-dioxygenase (hIDO). In the absence of L-Trp, the spectrum of the Fe(3+) form displayed six-coordinate, mixed high and low spin character. Addition of L-Trp triggered a transition to predominantly low spin with two Fe-OH(-) stretching modes identified at 546 and 496 cm(-1), suggesting H-bonding between the NH group of the pyrrole ring of L-Trp and heme-bound OH(-). The distal pocket of Fe(3+) hIDO was explored further by an exogenous heme ligand, CN(-); again, binding of L-Trp introduced strong H-bonding and/or steric interactions to the heme-bound CN(-). On the other hand, the spectrum of Fe(2+) hIDO revealed a five-coordinate and high spin heme with or without L-Trp bound. The proximal Fe-His stretching mode, identified at 236 cm(-1), did not shift upon L-Trp addition, indicating that the proximal Fe-His bond strength is not affected by binding of the substrate. The high Fe-His stretching frequency suggests that Fe(2+) hIDO has a strong "peroxidase-like" Fe-His bond. Using CO as a structural probe for the distal environment of Fe(2+) hIDO revealed that binding of L-Trp in the distal pocket converted IDO to a peroxidase-like enzyme. Binding of L-Trp also caused conformational changes to the heme vinyl groups, which were independent of changes of the spin and coordination state of the heme iron. Together these data indicate that the strong proximal Fe-His bond and the strong H-bonding and/or steric interactions between l-Trp and dioxygen in the distal pocket are likely crucial for the enzymatic activity of hIDO.     

Biosynthesis of HNK-1 glycans on O-linked oligosaccharides attached to the neural cell adhesion molecule (NCAM): the requirement for core 2 beta 1,6-N-acetylglucosaminyltransferase and the muscle-specific domain in NCAM

The HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of beta1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding beta1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 beta1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.     

Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coli XL1-blue cells after isopropyl beta-D-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5 degrees C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.     

ER transport signals and trafficking of potassium channels and receptors

Channels and receptors on the cell surface mediate neuronal signaling. It is therefore important to understand how their surface density is controlled. Recent studies on the trafficking of potassium channels and neurotransmitter receptors have revealed unexpected complexity in the regulation of transport from the endoplasmic reticulum to the Golgi apparatus, raising the possibility that the surface composition of channels and receptors may be adjusted by controlling their export from the endoplasmic reticulum.     

The new generation dihydropyridine type calcium blockers, bearing 4-phenyl oxypropanolamine, display alpha-/beta-adrenoceptor antagonist and long-acting antihypertensive activities

A new series of dihydropyridine derivatives, bearing oxypropanolamine moiety on phenyl ring at the 4-position of the dihydropyridine base, were prepared. Oxypropanolamine was synthesized by replacing the phenolic OH of vanillin or other compounds, having a phenyl aldehyde group, with epichlorohydrin, followed by cleavaging the obtained epoxide compounds with tert-butylamine, n-butylamine or 2-methoxy-1-oxyethylamino benzene (guaiacoxyethylamine), respectively. Obtained various oxypropanolamine compounds, still remaining a phenyl aldehyde moiety, were then performed by Hantzsch condensation reaction with methylacetoacetate or ethylacetoacetate, respectively, to give our new series of dihydropyridine linked with the 4-phenyl ring. These compounds were evaluated for inotropic, chronotropic, and aorta contractility that associated with calcium channel and adrenoceptor antagonist activities. Dihydropyridine derivatives that with oxypropanolamine side chain on their 4-phenyl ring associated alpha-/beta-adrenoceptor blocking activities created a new family of calcium entry and the third generation beta-adrenoceptor blockers. Optimizing this research to obtain more potent alpha-/beta-adrenoceptor blocking and long-acting antihypertensive oxypropanolamine on the 4-phenyl ring of dihydropyridine series compounds was thus accomplished and classified as third generation dihydropyridine type calcium channel blockers, in comparison with previous short-acting type nifedipine and long-acting type amlodipine. We concluded that compounds 1a, 1b and 1g showed not only markedly high calcium-antagonistic activity but also the highest antihypertensive effect; compounds 1b, 1c, 1f, 1g, 1i and 1j induced sustained antihypertensive effects are major and attributed to their calcium entry and alpha-adrenoceptor blocking activities in the blood vessel due to their introduction of 2-methoxy, 1-oxyethylamino benzene moiety in the side chain on the 4-phenyl ring of dihydropyridine. Bradycardiac effects of all the compounds 1a-1j resulted from calcium entry and beta-adrenoceptor blocking, which attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 1b as candidate compound for further pharmacological and pre-clinical evaluation studies.     

Cloning,expression, and genomic organization of mouse mp29 gene

The convulsions of approximately 25% of epileptics are inadequately controlled by currently available medication; therefore the preparation of new antiepileptic drugs is of great interest. Aryl semicarbazones can be considered a new class of compounds presenting anticonvulsant activity. In addition, they can be orally administered and are more active as anticonvulsants than mephenytoin or phenobarbital. However, one disadvantage of these compounds is their low water solubility. As a strategy to circumvent this problem, a 1:1 inclusion compound of benzaldehyde semicarbazone (BS) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was prepared and characterized. The anticonvulsant activities of the free semicarbazone and of the inclusion compound were evaluated in rats using the maximum electroshock and audiogenic seizures screenings. In both tests the minimum dose of compound necessary to produce activity decreases from 100mg/kg for the free semicarbazone to 35 mg/kg for the inclusion compound, indicating a significant increase in the bio-availability of the drug.     

Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation

Osteogenic Protein-1 (OP-1, BMP-7), a member of the bone morphogenetic protein family, stimulates synthesis of biochemical markers characteristic of the osteoblastic and chondrocytic phenotypes and induces new bone formation. Interleukin-6 (IL-6), a cytokine produced by a wide variety of cells, appears to interact with other factors producing different biological effects. In the present study, we showed that OP-1 action in fetal rat calvaria (FRC) cells was enhanced by the combination of IL-6 and the soluble receptor IL-6sR. OP-1 alone induced alkaline phosphatase (AP) activity by 4- to 5-fold above the control. Exogenous IL-6 soluble receptor (IL-6sR) synergistically stimulated the OP-1-induced AP activity and mineralized bone nodule formation by an additional 3-fold. The stimulation was IL-6sR concentration-dependent. The combination of IL-6 and IL-6sR synergistically stimulated OP-1 action by an additional 6- to 7-fold. BMPR-II receptor mRNA expression in FRC cells treated with OP-1 and IL-6 plus IL-6sR was stimulated further, while BMPR-IA, -IB, and ActR-I expressions were not affected. The intracellular signaling molecules Smad2 and Smad5 mRNA expressions were not changed under these conditions. The expression of selected BMP family members (BMP-3, -4, and -6) was altered in FRC cells treated with OP-1 in combination with IL-6 and IL-6sR. The combination of IL-6 and IL-6sR reduced the OP-1-stimulated BMP-3 mRNA levels and enhanced the suppressive effect of OP-1 on BMP-4 and -6 mRNA expressions. In conclusion, the present results demonstrate that exogenous IL-6 and IL-6sR synergistically stimulate OP-1 action in primary cultures of rat osteoblastic cells. One possible mechanism of synergy involves differential regulation of the effects of OP-1 on the expression of the type II BMP receptor and several other BMPs.