Cytochrome P450cam-catalyzed oxidation of a hypersensitive radical probe.

trans-1-Phenyl-2-vinylcyclopropane, a hypersensitive radical probe, is oxidized by cytochrome P450cam (CYP101) to a diastereomeric mixture of the corresponding epoxide (81%), (trans-2-phenylcyclopropyl)acetaldehyde (6%), and trans-5-phenyl-2-penten-1,5-diol (13%). trans-5-Phenyl-2-penten-1-ol and (trans-2-phenylcyclopropyl)ethane-1,2-diol are not detectably formed. Authentic standards of all the products have been synthesized and used to establish the identities (or the absence) of the metabolites. Studies with [18O]H2O demonstrate that the oxygens at positions 1 and 5 in the rearranged diol derive from molecular oxygen and water, respectively. Catalytic turnover of the enzyme is required for product formation from the olefin, but incubation of the epoxide metabolite with the enzyme, or with buffer alone, yields both the aldehyde and the rearranged diol products. The absence of trans-5-phenyl-2-penten-1-ol implies that the lifetime of the putative radical intermediate is so short that its existence as a discrete entity is questionable. A cationic intermediate is unlikely but cannot be excluded because the same metabolites are formed in a secondary reaction, even at pH 8.0, from the epoxide. The results provide no evidence for the involvement of radicals or cations in the epoxidation reaction, in agreement with results on the oxidation of olefins in organic solvents by metalloporphyrin catalysts.     

Substrate oxidation by the heme edge of fungal peroxidases. Reaction of Coprinus macrorhizus peroxidase with hydrazines and sodium azide

The peroxidase from Coprinus macrorhizus is inactivated by phenylhydrazine or sodium azide in the presence of H2O2. Inactivation by phenylhydrazine results in formation of the delta-meso-phenyl and 8-hydroxymethyl derivatives of the prosthetic heme group and covalent binding of the phenyl moiety to the protein but not in the detectable formation of Fe-phenyl- or N-phenylheme adducts. Alkylhydrazines are catalytically oxidized but do not inactivate the enzyme. Catalytic oxidation of sodium azide produces the azidyl radical and results in its addition to the delta-meso position of the prosthetic heme group. Comparison of the heme adducts obtained with C. macrorhizus peroxidase with those generated by horseradish peroxidase shows that the regiochemistry of the addition reactions is the same in both cases. The results suggest that substrates interact primarily or exclusively with the heme edge rather than the ferryl oxygen of C. macrorhizus peroxidase and indicate that the interaction occurs with the same sector of the heme edge as in horseradish peroxidase. The active-site topologies of this pair of plant and fungal peroxidases thus appear to be similar, although the observation that alkylhydrazines add to the heme edge of horseradish but not C. macrorhizus peroxidase clearly shows that there are significant differences in the two active sites.     

Fertilized and unfertilized ova are transported at different rates by the hamster oviduct

To determine if the egg provides any clues for the regulation of ovum transport in the hamster, oocyte and embryo transport were compared. On the evening preceding ovulation, the animals were randomly assigned to one of five groups. They were caged overnight with a male of proven fertility (Group 1) or they were isolated (Group 2). Other females were artificially inseminated in both uterine horns at 2200 h either with fertile epididymal spermatozoa (Group 3), spermatozoa rendered infertile by freezing and thawing (Group 4), or with fertile spermatozoa in one uterine horn and infertile spermatozoa in the contralateral horn (Group 5). The number, condition, and distribution of ova in the genital tract were assessed at various intervals during the next 4 days. The rate of fertilization and normal development in females or sides inseminated with fertile or infertile spermatozoa was over 90% and 0% respectively. Embryos in Groups 1 and 3 reached the uterus 1 day earlier than unfertilized oocytes in Groups 2 and 4. In group 5, the transport of embryos resulting from insemination with fertile spermatozoa followed a pattern similar to those in Groups 1 and 3; the oocytes in the contralateral tract resembled those of Groups 2 and 4. The different transport rates of embryos and oocytes were not associated with the reproductive state of the female but with the condition of the ova. Moreover, the different transport rates were observed in animals transporting the two types of eggs simultaneously on different sides indicating that there is a local recognition of some unidentified factor unequally present in fertilized and unfertilized eggs.     

Features of the cell-free translation of a spider fibroin mRNA

The massive production of fibroin by the large ampullate glands of the spider, Nephila clavipes, serves as a model system in which to study the synthesis and control of a large secretory protein. Their tissue-specific product, fibroin, produced during the entire adult life of the female, is approximately 320 kilodaltons, and rich in glycine and alanine. Highly purified fibroin mRNA from the glands has been translated in a rabbit reticulocyte cell-free system with variable supplements. The translational products analyzed by SDS-PAGE display two features, tRNA modulation and discontinuous pauses during elongation. tRNA complements exert their effects both in the translational efficiency and in the size of the peptides generated. The pauses observed during translation generate subsets of smaller discrete peptides, visualized in the gels as ladders of variable relative intensities, appearing exclusively and concomitantly with the fibroin. Definitive linkage between the discrete peptides and fibroin synthesis process has been established by their selective labeling with specific radioactive amino acids.     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.