Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.     

Design and synthesis of metabolically stable atrial natriuretic factor analogs. Amino- and carboxy-terminal stabilization

Two analogs of rat atrial natriuretic factor, rANF7-28-NH2 and [Mpr7,Ala20,D-Arg27]rANF7-27-NH2, were prepared by the solid-phase method. These peptides had 2-fold and 7-fold less affinity, respectively, than rANF1-28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5-fold less potent than rANF1-28 in relaxing serotonin-contracted rabbit aortic rings. rANF7-28-NH2 was rapidly degraded by rat kidney homogenates but [Mpr7,Ala20,D-Arg27]rANF7-27-NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.     

Hydroperoxide-dependent epoxidation of 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene by ram seminal vesicle microsomes and by hematin

Addition of arachidonic acid to ram seminal vesicle microsomes oxidizes 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene (BA-3,4-diol) to five more polar products. Four of the products are identified by chromatographic and spectroscopic analysis as tetrahydrotetraols, which are solvolysis products of dihydrodiolepoxides. The fifth product is a 10-methyl ether formed by methanolysis of the anti-diolepoxide. Quantitation of the individual products indicates that anti-diolepoxides predominate over syn-diolepoxides by approximately 2:1. Identical product profiles are detected from the reaction of BA-3,4-diol with hematin and 13-hydroperoxy-octadecadienoic acid in the presence of Tween 20. No other products are detected in either system, which indicates that peroxyl radicals oxidize BA-3,4-diol exclusively by epoxidation of the 1,2-double bond. The stereochemical and regiochemical differences between oxidation of BA-3,4-diol by peroxyl radicals and cytochrome P-450 are dramatic and suggest that BA-3,4-diol is uniquely suited as a probe to quantitate peroxyl radical-dependent epoxidation in vitro and in vivo.     

Genetic control of the mitochondrial form of superoxide dismutase in hexaploid wheat

Extracts of mature grains of a large number of aneuploid derivatives of Triticum aestivum cv. Chinese Spring and of the members of five wheat-alien chromosome addition series were subjected to isoelectric focusing in polyacrylamide gels in order to study the genetic control of superoxide dismutase (SOD). Evidence was obtained that homologous structural genes for the mitochondrial form of SOD are located in the long arms of the homoeologous group 2 chromosomes of Chinese Spring and in chromosome 2R of Secale cereale cv. Imperial. The SOD gene loci located in chromosomes 2A, 2B, 2D, and 2R were designated Sod-A1, Sod-B1, Sod-D1, and Sod-R1, respectively. Chromosome-arm pairing data indicate that 2DL is not homoeologous to either 2AS or 2BL. The results of this study suggest, however, that 2BL is partially homoeologous to both 2AL and 2DL.Technical article No. 21074 of the Texas Agricultural Experiment Station. This work was supported by USDA Grant 83-CRCR-1-1322 to GEH.     

1H-NMR study of the OR1 operon in bacteriophage lambda. I. Assignment of signals from imino protons and adenine C2 protons

An oligodeoxyribonucleotide composed of 17 residues, d(TATCACCGCCAGAGGTA), and a complementary chain were synthesized. Their duplex was identical with the operator OR1, the binding site for bacteriophage lambda cro and c1 repressors. The 1H NMR spectra (500 MHz) of the duplex imino and aromatic protons were studied at 10, 20 and 25 degrees C. Signals from the imino protons of complementary base pairs and from the C2 protons of adenine (with the exception of the duplex terminal nucleotides) were assigned using the NOE technique and the known characteristics of short DNA fragment melting. No signals from the imino protons of the terminal base pairs were detected even at 10 degrees C due to fraying which increased as the temperature was raised. The assignment of signals can be used to identify centers of interaction between the operator OR1 and repressors, as well as to study possible local changes in DNA geometry.     

Mechanisms of depletion of substance P by capsaicin

Capsaicin is a neurotoxin that can deplete sensory nerves of their content of substance P and interfere with certain sensory functions, such as responses of animals to noxious heat stimuli. In adult guinea pigs, a species that is susceptible to the effects of capsaicin on both substance P content and sensory function, capsaicin induces selective depletion of substance P from dorsal root ganglia and the dorsal spinal cord, sites of the cell bodies and central terminals of primary afferent neurons, respectively. As the onset of thermal analgesia in guinea pigs precedes depletion of substance P, direct neural actions of capsaicin probably account for its effects on sensory function. Capsaicin interferes with the retrograde transport of nerve growth factor (NGF) to the cell bodies of sensory nerves. Decreased availability of NGF at the site of neural protein synthesis leads to decreased synthesis of substance P. After failure of synthesis of substance P, the content of the peptide in sensory nerves gradually decreases until depletion occurs.     

Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Class I and class II restriction pattern polymorphisms associated with independently derived RT1 haplotypes in inbred rats

This communication reports the DNA level identification of class I and class II sequences associated with 20 RT1 haplotypes which have been assigned previously to eight RT1 groups. Sixteen to 22 bands in genomic blots hybridized with the mouse pH-2III class I cDNA probe. Only the three RT1 ^khaplotypes associated with identical class I restriction fragment patterns. Differences in restriction bands between putatively identical RT1 haplotypes were either less than or equal to 6%, or greater than 50%, suggesting a relatively high level of recombination between serologically identified RT1.A genes and the majority of class I sequences. Restriction fragment patterns associated with three RT1 ^uhaplotypes differed by less than 6%. However, intra-RT1 ^a,intra-RT1 ^b,and intra-RT1 ^lrestriction fragment differences were between 50 and 64%. In specific cases, different RT1 haplotypes associated with identical class I restriction patterns, e.g., RT1 ^m(MNR) and RT1 ^d(MR); higher resolution confirmed the difference (two bands) between RT1 ^mand RT1 ^d.Results of hybridization with the human DC₁ probe confirmed that the AVN RT1 ^aand NSD RT1 ^bhaplotypes were generated by recombinations within the vicinity of the RT1.B : RT1.D regions. These results demonstrate that a previous classification of RT1 haplotypes was incomplete and did not include the majority of class I and class II sequences which distinguish RT1 haplotypes.