Photocatalyzed Anaerobic Oxidation of Nicotinamide Coenzyme Dimers to NAD+ by Adriamycin

The nicotinamide adenine dinucleotide dimers (NAD)₂ obtained by electrochemical reduction of NAD⁺ are oxidized by adriamycin in anaerobic photocatalyzed reaction yielding NAD⁺ and 7-deoxyadriamyci-none. Under the same conditions NADH is not oxidized.     

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

The composition and the structure of the product from the known electrochemical dimerization of the NAD⁺ have been conclusively demonstrated. A detailed analysis of the ¹H and ¹³C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H₂O₂ is the reduction product. NAD⁺ was identified as the oxidation product by an enzymatic method.     

Conjugation of metallothionein to a murine monoclonal antibody

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.     

Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) damages dopaminergic neurons as seen in Parkinson disease. Here we show that after administration of MPTP to mice, there was a robust gliosis in the substantia nigra pars compacta associated with significant upregulation of inducible nitric oxide synthase (iNOS). These changes preceded or paralleled MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking the iNOS gene were significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that iNOS is important in the MPTP neurotoxic process and indicates that inhibitors of iNOS may provide protective benefit in the treatment of Parkinson disease.     

Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐solids loadings

Effective and efficient breakdown of lignocellulosic biomass remains a primary barrier for its use as a feedstock for renewable transportation fuels. A more detailed understanding of the material properties of biomass slurries during conversion is needed to design cost‐effective conversion processes. A series of enzymatic saccharification experiments were performed with dilute acid pretreated corn stover at initial insoluble solids loadings of 20% by mass, during which the concentration of particulate solids and the rheological property yield stress (τ_y) of the slurries were measured. The saccharified stover liquefies to the point of being pourable (τ_y ≤ 10 Pa) at a total biomass conversion of about 40%, after roughly 2 days of saccharification for a moderate loading of enzyme. Mass balance and semi‐empirical relationships are developed to connect the progress of enzymatic hydrolysis with particle concentration and yield stress. The experimental data show good agreement with the proposed relationships. The predictive models developed here are based on established physical principles and should be applicable to the saccharification of other biomass systems. The concepts presented, especially the ability to predict yield stress from extent of conversion, will be helpful in the design and optimization of enzymatic hydrolysis processes that operate at high‐solids loadings. Biotechnol. Bioeng. 2009; 104: 290–300 © 2009 Wiley Periodicals, Inc.     

Thermal stress response in a dinoflagellate-bearing nudibranch and the octocoral on which it feeds

In this study, we examined two non-scleractinian taxa, the rare nudibranch Phyllodesmium lizardensis and Bayerxenia sp., the octocoral on which the nudibranch lives and feeds, to investigate the effect of experimental heat stress on their symbioses with Symbiodinium. Bleaching has not been studied in nudibranchs. Bayerxenia sp. belongs to the alcyonacea family Xeniidae, members of which are known to be heat sensitive, but the genus has never been subject to heat stress experiments or bleaching observations. While qPCR did not reveal any changes to the symbiont community composition, the two host species responded differently to increased temperature. There were changes in the relative proportion of tissue types in Bayerxenia sp., but these were not attributable to the temperature treatment. Bayerxenia sp. exhibited no changes in cellular structure (apoptosis or cell necrosis), or symbiont functioning, cell size, density, or cladal community structure. On the other hand, the host, P. lizardensis, experienced tissue loss and symbiont densities decreased significantly with the majority of the remaining symbiont cells significantly degenerated after the heat stress. This decrease did not influence symbiont community composition, symbiont cell size, or photosynthetic efficiency. While the bleaching process in nudibranchs was demonstrated for the first time, the physiological and molecular pathways leading to this response still require attention.     

Site-directed chemical modification and cross-linking of a monoclonal antibody using equilibrium transfer alkylating cross-link reagents

A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include alpha,alpha-bis[(p-chlorophenyl)methyl]- and alpha,alpha-bis[(p-tolylsulfonyl)methyl]acetophenones substituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an 125I-labeled ETAC reagent and a 111In-labeled DTPA (diethylenetriaminepentaacetic acid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the cross-links on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the 125I-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with pH whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was pH 5.0; (7) the 111In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the 111In bis cyclic anhydride DTPA labeled antibody.     

Photocatalyzed oxidation of NAD dimers

A mixture of dimers of nicotinamide adenine dinucleotide, largely 4,4?-linked, obtained by electrochemical reduction of NAD⁺, can be photooxidized back to NAD⁺ in the presence of oxygen. Oxygen is consumed during the photooxidation process with the production of hydrogen peroxide. The oxidation is almost pH independent and is stimulated by light whose wavelength exceeds 300 nm. Lactate dehydrogenase and alcohol dehydrogenase added to the solutions under irradiation increased the oxygen uptake by the NAD dimers in a concentration-dependent way. These observations suggest that light induces the homolytic cleavage of NAD dimers to NAD radicals which in turn are oxidized to NAD⁺ by oxygen.