Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion [Lederer, F., & Mathews, F.S. (1987) in Flavins and Flavoproteins, Proceedings of the Ninth International Symposium, Atlanta, GA, 1987 (Edmondson, D.E., & McCormick, D.B., Eds.) pp 133-142, Walter de Gruyter, Berlin]. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Do neural crest cells in the pancreas differentiate into somatostatin-containing cells?

Summary The possibility that the somatostatin cells are derived from the neurectoderm has been questioned in avian embryos. Isotopic and isochronic transplantations of the neural primordium from quail into chick embryos were made at the vagal level (somites 1 to 7). Quail and chick cells can be distinguished by the structure of their nucleus. The somatostatin cells were characterized immunocytochemically. In no case did quail cells showing the immunological reaction originate from the neural crest.     

Ultrastructural evidence of oestradiol receptor by immunochemistry

Antiserum against calf uterus oestradiol receptor has been used for detecting oestradiol receptor in rat pituitary cells at the ultrastructural level after immunochemical reaction according to Sternberger. The gonadotropic, lactotropic and somatotropic cells were positive, but not the thyrotropic and corticotropic cells. In peripubertal and adult rats, both cytoplasmic and nuclear receptors were seen, but in a long-term castrated rat, the receptor was found only in the cytoplasm. After oestradiol administration to 21-day-old animals, the cytoplasmic receptor decreased and the nuclear receptor increased in gonadotropic cells, supporting the concept of hormone-receptor complex translocation. Antibodies against α1-foetoprotein demonstrated the presence of this oestrogen-binding plasma protein in all pituitary cells, but only in the cytoplasmic area. These results and the immunological controls related to antibody specificity give the first evidence of steroid receptor at the ultrastructural level.     

Effect of phenobarbital administration to rats on the level of the in vitro synthesis of cytochrome P-450 directed by total rat liver RNA.

Total liver RNA has been isolated from male rats at different time points subsequent to a single injection of phenobarbital, and the level of cytochrome P-450 synthesis directed by these RNA preparations in a cell-free translation system has been determined. It is observed that the maximum $\text{in vitro}$ synthesis of cytochrome P-450 occurs at 16 hours (3-fold above uninduced level) which is approximately 30 hours prior to the maximum induction of spectrophotometrically detectable cytochrome P-450 measured in liver homogenates. Thus, while cytochrome P-450 mRNA is involved in the induction process, its synthesis does not appear to be rate limiting. In addition, phenobarbital induced cytochrome P-450 is not synthesized $\text{in vitro}$ in a form larger than that isolated from endoplasmic reticulum, but rather is also found to have a molecular weight of 50,000.     

Gastrointestinal somatostatin cells in the human fetus

Summary The stomach, duodenum, jejunum and ileum from 6 to 24 week-old human fetuses or deceased premature infants and from one totally anencephalic fetus were stained with antisomatostatin serum. The somatostatin containing cells appear in different segments of the gastrointestinal tract from the tenth week of gestation. The somatostatin-containing cells are lead haematoxylin positive. Somatostatin was also detected in the duodenum of the anencephalic fetus. The study of inhibition of the immunofluorescent reaction by homologous and heterologous antigens confirmed the specificity of antiserum.These data demonstrate that somatostatin can be synthetized elsewhere than in the hypothalamus.We are indebted to Professor P. Magnin, Hôpital Edouard Herriot, Professor M. Dumont, Hôpital de la Croix-Rousse, Professor Notter and Professor Garmier, Hôtel Dieu, Professor M. Bethenod, Hôpital Debrousse, Lyon, whose kind cooperation allowed us to carry out the rapid removal of gastrointestinal tracts for these studies. We thank Professor Assan, Hôtel Dieu, Paris and Professor Lambert U. 45, Inserm, Lyon, who graciously provided us with antisera. We also wish to express our gratitude to Professor Roger Guillemin, The Salk Institute, La Jolla, California, whose interest and cooperation made these studies possible.This work was supported by a grant of the Institut National de la Santé et de la Recherche Médicale.     

Anthocyanin inheritance in petals of flax, Linum usitatissimum

Twelve anthocyanins have been isolated from flax: the 3-glucosylrutinosides of pelargonidin, cyanidin and delphinidin; the 3-triglucosides of delphinid     

The effect of microbial mycolytic agents on Trichophyton rubrum

Chitinolytic microorganisms isolated from forest soil and from healthy gypsy moth larvae (Porthetria dispar (L.) were screened for their ability to lyse Trichophyton rubrum mycelia. A few of these isolates were mycolytic on both autoclaved and on actively growing, intact, T. rubrum mycelia. Supernatants from these isolates, utilizing live T. rubrum as the sole carbon source, showed the same mycolytic ability. Assays of the supernatants for enzymatic activity revealed exocellular, stable enzymes that releases reducing substances including N-acetylglucosamine from the mycelia.     

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil‐borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo‐gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.