The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.     

Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin-C

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8-16 weeks gestation obtained following prostaglandin-induced pregnancy terminations. They were maintained on collagen-coated tissue culture dishes in selective arginine-free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H-thymidine. The labeling index of hepatocytes was consistently enhanced by 25-250 ng/ml human placental lactogen (HPL), 25-250 ng/ml human growth hormone (HGH), 10-50 ng/ml insulin-like growth factor I/somatomedin-C (IGF I/Sm-C), and 10% dialyzed fetal calf serum, reaching a maximum of three- to four-fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF I/Sm-C. Although this proportion did not change following treatment with HGH and HPL, IGF I/Sm-C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF I/Sm-C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF I/Sm-C.     

Formation of organic products in self-radiolyzed calcium carbonate

Summary Calcium carbonate labeled with carbon-14 was self-irradiated by means of the -decay of its carbon-14. A number of products containing one or two carbon atoms were identified by high performance liquid chromatography. Formic and oxalic acids were produced in relatively high yields, while glyoxylic, glycolic, and acetic acids, as well as formaldehyde and methanol, were formed in lower yields. These results support the suggestion that carbonates subjected to ionizing radiation could have been a source of carbon for organic synthesis on the primitive earth.     

Pliocene hominid partial mandible from Tabarin, Baringo, Kenya

Sediments in the Tugen Hills, west of Lake Baringo, Kenya, form one of the best fossiliferous successions known in Africa spanning the period from 14 my to less than 4 my. Hominoid fossils have previously been recovered from a number of localities in the region. We describe here a new hominid mandible (KNM-TH 13150) from the site of Tabarin, in the Chemeron Formation. Isotopic determinations on a tuff below the fossiliferous horizon gives dates of 4.96 my and 5.25 my. The associated fauna is consistent with these results and independently suggests a minimum age for the specimen of 4.15 my. Although fragmentary, the preserved morphology of the Tabarin mandible is consistent with the diagnosis of the Pliocene hominid Australopithecus afarensis. It can be distinguished from all other currently recognized hominoid taxa.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Development of High-Frequency Delivery System for Transposon Tn919 in Lactic Streptococci: Random Insertion in Streptococcus lactis subsp. diacetylactis 18-16

The conjugative transposon Tn919, originally isolated in Streptococcus sanguis FC1, is capable of low-frequency transfer (10⁻⁷ and 10⁻⁸ per recipient) on membrane filters to a wide number of streptococcal recipients including the industrially important lactic streptococci. The introduction of pMG600 (Lac⁺ Lax⁻; a lactose plasmid capable of conjugative transfer at high frequencies and which, in certain hosts, confers an unusual clumping phenotype) into a Streptococcus lactis CH919 donor, generating S. lactis CH001, resulted in a significant improvement in the transfer frequency of Tn919 to S. lactis CK50 (1.25 × 10⁻⁴ per recipient). In addition, these matings could be performed on agar surfaces, allowing the recovery of a greater number of recipients than with filter matings. Tn919 also transferred at high frequency to S. lactis subsp. diacetylactis 18-16S but not to Streptococcus cremoris strains. Insertion in 18-16S transconjugants generated from filter matings with an S. lactis CH919 donor was random, occurring at different sites on the chromosome and also in plasmid DNA. Thus, the conditions necessary for the practical exploitation of Tn919 in the targeting and cloning of genes from a member of the lactic streptococci, namely, high-frequency delivery and random insertion in host DNA, were achieved.     

The kinetics of milk coagulation: IV. The kinetics of the gel-firming process

A mechanistic kinetic model of gel firmness development during milk gel formation is presented. The model correctly accounts for the influence of enzymatic kappa-casein hydrolysis on the rate of firmness development in renneted milk gels. The model used is based on two first-order reactions occurring in series. The first reaction is enzymatically controlled and corresponds to the formation of gel crosslink sites by kappa-casein hydrolysis. The second reaction is nonenzymatic and corresponds to the process of crosslink formation and depletion of active sites. The model successfully predicts gel firmness development in the temperature range 31-45 degrees C for a variety of initial enzyme concentrations.     

Maintenance of innate feeding preferences by a polyphagous insect despite ingestion of applied deleterious chemicals

The question of whether the appearance of a novel deleterious chemical in a preferred host plant could alter the relative degree of preference by a polyphagous insect during its lifetime was considered. The behavioral preference of fall armyworm, Spodoptera frugiperda (J. E. Smith), for corn (Zea mays L.) over red kidney bean Phaseolus spp. was quantified in a two-way choice test, and followed by a no-choice test in which the same larvae were fed either treated or untreated corn. Each treatment consisted of one of 15 compounds, including 13 naturally occurring allelochemicals and two synthetic insecticides. None of the test chemicals had any inherently deterrent effect at the rates tested. Deleterious effects on the larvae during exposure included reduced growth, reduced size, irritancy, and in some cases death. After this exposure the two way choice test was repeated. All test compounds failed to elicit a change in relative preference during the final two-way choice test. However, larvae exposed to gossypol and chrysanthemum monocarboxylic acid ester fed less than expected during the latter stages of no-choice conditions. The degree of aversion learning by each larva was related to the degree of prior sickness it had experienced.

---

Résumé Nous avons examiné si l'introduction d'une substance chimique nocive dans la plante-hôte préférée pouvait modifier la hiérarchie des choix durant le reste de la vie d'un insecte phytophage. Le comportement de préférence de Spodoptera frugiperda pour le maïs (Zea mays) vis à vis des Phaseolus, a été quantifié dans un dispositif à choix binaire, ont suivi des expériences au cours desquelles les chenilles devaient s'alimenter, soit sur maïs sain, soit sur maïs traité. Chaque essai concernait un composé chimique (15 au total), 13 étant des substances allélochimiques naturelles, 2 des insecticides de synthèse. Aux concentrations étudiées, aucun des produits n'a eu d'effet dissuasif. Les effets nocifs pendant l'exposition sur les chenilles comprenaient une croissance et une taille réduites, une irritation et, dans quelques cas, la mort. Aucune des expériences de choix binaire, avec des chenilles ayant subi cette exposition, n'a modifié les préférences. Cependant les chenilles, qui avaient été exposées au gossypol ou à l'acide monocarboxylique éthyl ester de chrysanthemum, ont consommé moins pendant les stades ultérieurs que ce que l'on pouvait attendre en absence de choix. Le degré d'aversion acquis par chaque chenille dépendait de l'importance des maux qu'elle avait subis précédemment.