In vivo incorporation of acetate into the acyl moieties of polar lipids from etiolated leek seedlings

Seven-day-old leek seedlings actively synthesize lipids in vivo from [1-¹⁴C]acetate, both in the light and in the dark. In the dark, phospholipid synthesis is more effective than galactolipid synthesis. Whatever the time of acetate incorporation by the etiolated seedlings, very long chain fatty acids having from 20 to 26 carbon atoms are found in all the polar lipids, including the acyl-CoAs. All of the labelled very long chain fatty acids incorporated into the polar lipids are saturated. On the other hand, the labelled C₁₈-fatty acids are unsaturated in phospholipids and galactolipids and almost no label is found in the saturated or unsaturated C₁₈-fatty acids of the acyl-CoAs.     

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

Covalently cyclized agonist and antagonist analogues of bombesin and related peptides.

During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V-G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures.     

Expression of the human papillomavirus E7 oncogene during cell transformation is sufficient to induce susceptibility to lysis by activated macrophages.

Human papillomaviruses (HPV), and in particular HPV type 16, are etiologic agents in the development of cervical cancer, which is the second most common form of cancer in women worldwide. Mammalian cells are susceptible to transformation in vitro by the E6 and E7 oncogenes derived from the HPV-16 genome. NIH-3T3 cells transfected with the HPV-16 E7 oncogene were found to exhibit cytolytic susceptibility to murine-activated macrophages. In comparison, E6 oncogene-expressing cells were not susceptible to lysis by activated macrophages. The E7 oncoprotein is multifunctional, being capable of complexing with the retinoblastoma tumor suppressor gene (anti-oncogene) product, stimulating DNA synthesis, and causing cell transformation in vitro. Macrophage killing assays performed on cell lines expressing E7 mutants revealed that the ability to complex the retinoblastoma tumor suppressor gene product and stimulate DNA synthesis did not induce susceptibility to activated macrophages, whereas the ability of E7 to cause transformation was required to induce susceptibility to activated macrophages. These data suggest that cell transformation is a more important prerequisite for inducing susceptibility to activated macrophages than is the loss of tumor suppressor gene function. This study also provides an initial link between HPV-16 oncogene expression and the ability of activated macrophages to selectively recognize and destroy HPV-16-associated neoplastic cells.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.     

Inhibition of rat tissue kallikrein gene family members by rat kallikrein-binding protein and alpha 1-proteinase inhibitor.

The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue-kallikrein family members isolated from the rat submandibular gland, with rat kallikrein-binding protein (rKBP) and alpha 1-proteinase inhibitor (alpha 1-PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK10 with association rate constants in the 10(3)-10(4) M-1.s-1 range, whereas only 'true' tissue kallikrein rK1 was not susceptible to alpha 1-PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the only member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.     

Correlation between Pairing Initiation Sites, Recombination Nodules and Meiotic Recombination in Sordaria Macrospora

The decrease of meiotic exchanges (crossing over and conversion) in two mutants of Sordaria macrospora correlated strongly with a reduction of chiasmata and of both types of "recombination nodules." Serial section reconstruction electron microscopy was used to compare the synapsis pattern of meiotic prophase I in wild type and mutants. First, synapsis occurred but the number of synaptonemal complex initiation sites was reduced in both mutants. Second, this reduction was accompanied by, or resulted in, modifications of the pattern of synapsis. Genetic and synaptonemal complex maps were compared in three regions along one chromosome arm divided into well marked intervals. Reciprocal exchange frequencies and number of recombination nodules correlated in wild type in the three analyzed intervals, but disparity was found between the location of recombination nodules and exchanges in the mutants. Despite the twofold exchange decrease, sections of the genome such as the short arm of chromosome 2 and telomere regions were sheltered from nodule decrease and from pairing modifications. This indicated a certain amount of diversity in the control of these features and suggested that exchange frequency was dependent not only on the amount of effective pairing but also on the localization of the pairing sites, as revealed by the synaptonemal complex progression in the mutants.     

Pharmacological Characterization and Autoradiographic Localization of Histamine H2 Receptors in Human Brain Identified with [125I]Iodoaminopotentidine

125I-Aminopotentidine (125I-APT), a reversible probe of high specific radioactivity and high affinity and selectivity for the H2 receptor, was used to characterize and localize this histamine receptor subtype in human brain samples obtained at autopsy. On membranes of human caudate nucleus, specific 125I-APT binding at equilibrium revealed a single component, with a dissociation constant of 0.3 nM and maximal capacity of about 100 fmol/mg of protein. At 0.2 nM, 125I-APT specific binding, as defined with tiotidine, an H2-receptor antagonist chemically unrelated to iodoaminopotentidine, represented 40-50% of the total. Specific 125I-APT binding was inhibited by a series of typical H2-receptor antagonists that displayed apparent dissociation constants closely similar to corresponding values at the reference biological system, i.e., guinea pig atrium. This indicates that the pharmacology of the H2 receptor is the same in the human brain as on this reference system. However, histamine was about 10-fold more potent in inhibiting 125I-APT binding to membranes of human brain than of guinea pig brain. 125I-APT binding was also inhibited by amitriptyline and mianserin, two antidepressant drugs, in micromolar concentrations corresponding to effective plasma concentrations of treated patients. The distribution of H2 receptors was established autoradiographically with 125I-APT on a series of coronal sections of human brain after assessing the pharmacological specificity of the labeling. The highest density of 125I-APT sites was found in the basal ganglia, various parts of the limbic system, e.g., hippocampus or amygdaloid complex, and the cerebral cortex. H2 receptors displayed a laminar distribution in cerebral cortex and hippocampal formation. A low density of sites was found in cerebellum as well as in hypothalamus, the brain area where all the perikarya and the largest number of axons of histaminergic neurons are found. The widespread distribution of H2 receptors in the human brain is consistent with the alleged modulatory role of histamine mediated by this subtype of receptor.     

K+/H+ exchange in yeast mitochondria: sensitivity to inhibitors, solubilization and reconstitution of the activity in proteoliposomes.

The K+/H+ exchange activity of the inner mitochondrial membrane was investigated in the yeast Saccharomyces cerevisiae. Swelling experiments in potassium acetate indicated that the K+/H+ exchange was active without any additional treatment after the mitochondria isolation, such as a Mg2+ depletion. As in mammalian mitochondria, the activity of yeast mitochondria was stimulated by increasing pH and was inhibited by the amphiphilic amines quinine and propranolol and by the carboxyl reagent dicyclohexylcarbodiimide. However, the activity was poorly inhibited by Mg2+ and consequently was only slightly stimulated by the Mg2+/H+ exchanger A23187. On the other hand, Zn2+ was very efficient for inhibiting the exchange and consequently the activity was strongly stimulated by the permeant metal-chelator o-phenanthroline. The [86Rb]Rb+ accumulation in mitochondria and mitoplasts was only partially inhibited by quinine and propranolol suggesting that part of the accumulation monitored under these conditions was due to cation leak through the inner membrane together with adsorption on the membrane. The DCCD-sensitive activity could be reconstituted from mitochondria and from mitoplasts solubilized with Triton X-100; this activity, measured by [86Rb]Rb+ accumulation, was quinine- and propranolol-sensitive. A spectrophotometric method, based on the capacity of negatively charged proteoliposomes to swell, was then developed in order to continuously follow the reconstituted activity.