Multiple mechanisms of cytochrome P450-catalyzed substrate hydroxylations

We have examined the 5-$\text{exo}$-hydroxylation of camphor by cytochrome P450 in [¹⁸O] water/buffer solution. In the $\text{NADH}\text{O}{}_2\text{-dependent}$ reaction of the reconstituted multienzyme system, no ¹⁸O-label is observed in the product alcohol. Similarly, in the m-chloroperbenzoic acid or cumene hydroperoxide supported reactions with ferric P450, solvent oxygen is not incorporated into hydroxycamphor. When the analagous reaction is carried out using iodosobenzene as the exogenous oxidant, however, the alcoholic oxygen of the product is derived entirely from the solvent. These results cannot be explained by equilibration of the iodosobenzene oxygen with solvent water before reacting with P450, and suggest a unique mechanism for iodosobenzene-supported P450 oxygenations. We propose two distinct mechanistic activities for cytochrome P450: a hydroxylase, and an oxene transferase, with the former encompassing the classic oxygenase as well as “peroxygenase” reactions.     

Induction of avian serum apolipoprotein II and vitellogenin by tamoxifen

Apo II and vitellogenin were detected in sera of nonestrogenized roosters by radioimmunoassay. Tamoxifen (30mg/kg) raised basal serum apo II more than 50 fold and vitellogenin more than 15 fold. Serum accumulation of apo II was biphasic in response to increasing doses of tamoxifen. This biphasic response was reflected in the relative rates of hepatic apo II synthesis. The tamoxifen metabolites, hydroxytamoxifen and desmethyltamoxifen, and the triphenylethylene antiestrogen, CI 628, also increased serum apo II. These studies show that the expression of vitellogenin and apo II genes occurs at a basal rate prior to exogenous estrogen treatment. These experiments also demonstrate that the triphenylethylene antiestrogens have agonist activity in the chicken when very sensitive techniques are used to measure estrogen-regulated proteins.     

Structure of cellulose II hydrate

A hydrate of cellulose II can be formed by swelling Fortisan fibers in hydrazine and then washing in water. The hydrate is stable at 93% relative humidity and has a monoclinic unit cell with dimensions a = 9.02 Å, b = 9.63 Å, c = 10.34 Å, and γ = 116.0°; the space group is P2₁. The unit cell contains disaccharide sections of two chains and approximately four water molecules. The structure was refined using the LALS method, based on 10 observed and 10 unobserved reflections. An antiparallel arrangement of adjacent chains was assumed, since this occurs in cellulose II (the starting material), and the hydrate also reverts to cellulose II on dehydration. Refinement of the positions and side-chain conformations of the chains shows that the chains are stacked in the same way as in cellulose II, and the hydrate is formed by insertion of water molecules between the stacks. However, all efforts to arrange the water molecules in crystallographically regular positions led to unsatisfactory agreement between the observed and calculated intensities. These results suggest an irregular arrangement of the water molecules, which was modeled using water-weighted atomic scattering factors. The analysis resulted in two refined models with relative chain staggers of ～ +c/4 and ～ -c/4, which are indistinguishable in terms of the x-ray agreement. Our preference is for the +c/4 model, for which the stacks of chains are analogous to those in cellulose II.     

Quantitative in situ hybridization reveals extent of sequence homology between related DNA sequences in Drosophila melanogaster

Cloned DNA from the larval serum protein one (LSP-1) genes was hybridized to polytene chromosomes of D. melanogaster. The ratio of grains deposited over any two of the three LSP-1 genes with any one LSP-1 subunit probe was constant. Varying the gene dose of any one LSP-1 subunit relative to the others by up to six fold gave a linear relationship of grain ratios to gene ratios. We show that these constant ratios closely reflect the extent of sequence homology between the genes as determined by heteroduplex mapping (Smith et al., 1981) and thermal denaturation studies. The results obtained demonstrate that the LSP-1 subunit genes are present in equal copies in the genome.     

Nucleotide sequence variants of Rattus norvegicus mitochondrial DNA

The mitochondrial DNA (mtDNA) molecules of different albino, domesticated rats (Rattus norvegicus) of the SASCO colony are of two kinds (SASCO-1 and SASCO-2) in regard to their sensitivity at certain sites to a number of restriction enzymes. MtDNA molecules from Utah wild R. norvegicus (Wild-UT) have sensitivities to restriction enzymes which differ at some sites from either SASCO-1 or SASCO-2 mtDNA molecules. Four single nucleotide differences were found among the HindIII F fragments (169 nucleotides) of SASCO-1, SASCO-2, and Wild-UT mtDNAs. Arguments are presented in favor of the interpretation that each variant nucleotide is the third nucleotide of the codon containing it, and that none of the four differences would result in a difference in the respective amino acid translated.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Erasure of the memory response in MEL cells

When MEL cells are reexposed to DMSO after an interruption in inducer treatment, they can initiate commitment to differentiation without the lag period observed after the primary exposure to inducer. This property is known as memory. Here we have employed metabolic inhibitors to analyze the basis of the memory response. Treatment of cells with cycloheximide or cordycepin during the inducer withdrawal period causes memory erasure. Cells must recapitulate an entire lag period upon reexposure to DMSO. The memory response is maintained, however, if cells are treated with metabolic inhibitors in the presence of DMSO. Our results suggest that the capacity of MEL cells for memory requires the synthesis of cell components which are normally stable in the absence of DMSO. Experiments involving reciprocal shifts between two different inhibitors have been performed. Evidence is presented that the process leading to the initiation of commitment is composed of at least three components acting in sequence.     

Involvement of smooth and coated vesicles in the function of the contractile vacuole complex of some cryptophycean flagellates

The contractile vacuole complex of cryptophycean flagellates comprises the contractile vacuole, a pore and a vesicular spongiome. A minority of spongiome vesicles bear a 15-nm coat on the cytoplasmic surface of the membrane. The coat superficially resembles a clathrin coat. The majority of vesicles are smooth surfaced. Both types of vesicles are found at the same time. Smooth vesicles can be seen in profile suggesting vesicle-vesicle and vesicle-vacuole fusion. It is suggested that smooth vesicles are involved in the segregation of fluid from the cytoplasm and in filling the vacuole. Coated elements exist only as independent vesicles and as coated pits in the contractile vacuole membrane. There is no evidence of fusion of coated vesicles. It is suggested that coated vesicles function to retrieve specific membrane components from the contractile vacuole.     

Genetic and Developmental Analysis of a Temperature-Sensitive Minute Mutation of DROSOPHILA MELANOGASTER

A temperature-sensitive (ts) third chromosome Minute (M) mutation, designated Q-III, has been recovered and characterized. Q-III heterozygotes raised at 29° exhibit all of the dominant traits of M mutants including small bristles, rough eyes, prolonged development, reduced viability and interactions with several unrelated mutations. Q-III homozygotes raised at 29° are lethal; death occurs primarily during the first larval instar. When raised at 22°, Q-III heterozygotes are phenotypically normal and Q-III homozygotes display moderate M traits. In addition, Q-III elicits ts sterility and maternal-effect lethality. As it true of M lesions, the dominant traits of Q-III are not expressed in triploid females raised at 29°. Complementation tests suggest that Q-III is a ts allele of M(3)LS4, which is located in 3L near the centromere.—Reciprocal temperature-shift experiments revealed that the temperature-sensitive period (TSP) of Q-III lethality is polyphasic, extending from the first instar to the latter half of pupation. Heat-pulse experiments further resolved this into two post-embryonic TSPs: one occurring during the latter half of the second larval instar, and the other extending from the larval/pupal boundary to the second half of pupation. In addition, heat pulses elicited a large number of striking adult phenotypes in Q-III individuals. These included pattern alterations such as deficiencies and duplications and other morphological defects in structures produced by the eye-antennal, leg, wing and genital imaginal discs and the abdominal histoblasts. Each defect or pattern alteration is associated with a specific TSP during development.—We favor the interpretation that most of the major Q-III defects, particularly the structural duplications and deficiencies, result from temperature-induced cell death in mitotically active imaginal anlagen, while the small macrochaete phene probably results from the direct effects of Q-III on bristle synthesis. The hypothesis that the Q-III locus specifices a component required for protein synthesis is discussed, and it is concluded that this hypothesis can account for the pleiotropy of Q-III, and that perhaps it can be extended to M loci in general.