NMR observation of the interaction of small oligopeptides with phospholipid vesicles

Using proton spin-lattice relaxation times, the interaction of small oligopeptides with sonicated vesicles of synthetic β-γ-dimyristoyl L-α-lecithin has been monitored at 29°C in D₂O. The measured relaxation times for the lecithin choline methyl, alkyl chain, and terminal methyl protons were observed to shorten markedly with increasing concentration of peptide, the relaxation remaining exponential. Noticeable resonance broadening was observed at the highest peptide concentration studied. The data reported are for the effect of the pharmacologically active pentapeptide methionine-enkephalin. Similar results have been observed for the effect of tetraglycine. The relaxation of the observable resonances of the added peptide appear to be unaffected. The results are discussed in terms of peptide-vesicle interactions.     

Purification and properties of 3-hexulosephosphate synthase from Methylomonas M 15.

3-Hexulosephosphate synthase, the first enzyme of the ribulose monophosphate cycle, was purified 15-fold from methanol-grown Methylomonas M 15. The purification procedure involved chromatography on DEAE-cellulose, Sephadex G-75, and DEAE-Sephadex A-50. The purified enzyme was more than 95% pure as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was calculated to be 43000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a molecular weight of 22000. The enzyme catalyzes specifically the condensation formaldehyde with ribulose 5-phosphate to yield D-arabino-3-hexulose 6-phosphate. The Km values were found to be 1.1 mM for formaldehyde and 1.6 mM for ribulose 5-phosphate. A bivalent cation is essential for activity and stability of the enzyme, Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5--8.0.     

Kinetic studies of Bacillus polymyxa nitrogenase.

Nitrogenase from the facultative anaerobe Bacillus polymxa was separated into its component proteins, which were recombined in the ratio that produced optimal specific activity (125 to 175 nmol of C2H2 reduced/min per mg of total protein). The apparent Michaelis constants (Km)for the magnesium adenosine triphosphate complex, reducible substrates azide, acetylene, and N2 and the nonphysiological electron donor hydrosulfite (S2O42-) were determined to be 0.7, 0.7, 0.2, 0.06, and 0.03 MM, respectively. These apparent Km values are in reasonable agreement with those reported for the nitrogenases of Azotobacter vinelandii and Klebsiella pneumoniae. Either a total lack of cooperativity between binding sites or a single binding site for reducible substrates is indicated by analysis of Hill plots. Hill plot slopes of approximately 1.7 suggest that multiple binding sites exist for both ATP and S2O42-.     

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV Plum Pox Virus - CP coat protein - CaMV Cauliflower Mosaic Virus - CP+ coat protein expressing plant - CP– control plant = non coat protein expressing plant - TMV Tobacco Mosaic Virus - NPTII neomycin phosphotransferaseII - IBA indole-3-butyric acid - BAP 6-benzylaminopurine; - MS Murashige Skoog - ELISA enzyme linked immunosorbent assay     

In vitro plant regeneration from leaf explants of C3 and C3-C4-intermediate Flaveria species

Summary A procedure is described for the invitro regeneration of whole plants of Flaveria cronquistii (C₃ species) F. pubescens and F. chloraefolia (both C₃-C₄ intermediate species) using different concentrations of 6-benzylaminopurine and alpha-napnthalenic acid.Abbreviations BAP 6-benzylaminopurine - NAA alpha-naphthalenic acid - MS medium Murashige-Skoog-medium     

Identification of rabbit microsomal cytochrome P-450 isozyme,form 1, as a hepatic progesterone 21-hydroxylase

Six highly purified forms of rabbit microsomal cytochrome P-450, isolated from hepatic microsomes, exhibit differences in the regiospecific metabolism of progesterone. Only one of the isozymes studied, form 1, catalyzes the formation of deoxycorticosterone from progesterone at an appreciable rate. This cytochrome P-450 isozyme may participate in the conversion of progesterone to deoxycorticosterone during pregnancy. All six forms of cytochrome P-450 catalyze 6β- and 16α-hydroxylation at the two concentrations of progesterone tested. Form 3b exhibits a lower apparent Km for 6β-hydroxylation than the other five.     

Presence and disappearance of nerve growth factor receptors on sensory neurons in culture

The age-dependent presence of nerve growth factor (NGF) receptors on neurites of sensory neurons of dorsal root ganglia removed from chicks of different embryonic ages and subsequently kept in culture with NGF and/or brain extract has been investigated by autoradiography. Most of the neurons removed at embryonic Day 10 (E10), (82–95%) can be labeled with iodinated NGF, irrespective of whether they are selected for survival by means of NGF, brain extract, or both. However, when neurons are isolated from E16 chicks and maintained in culture with brain extract, only about 28% of the neurons have NGF receptors at reduced density. This percentage is higher than that expected if the number of neurons surviving with NGF would be exactly correlated with the number of neurons displaying NGF receptors: at E16 only about 5% of the neurons survive with NGF alone. In order to determine if the decrease in the number of neurons displaying NGF receptors also occurs in vitro, E10 neurons were cultured for various periods of time either with NGF or brain extract. Most of the neurons grown with NGF do not lose their NGF receptors. In contrast, the majority of the neurons grown in the presence of brain extract lose their receptors: after 6 days in culture, only about 25% of the neurons can be labeled with NGF. It is concluded that in vitro, a maturation with regard to the NGF receptor occurs in the presence of brain extract similar to that observed in vivo.     

The Topographical Distribution of Alanine, Aspartate, γ-Aminobutyric Acid, Glutamate, Glutamine, and Glycine in the Pigeon Optic Tectum and the Effect of Retinal Ablation

Abstract: The concentrations of alanine, aspartate, γ-aminobutyric acid, glutamine, glutamate, and glycine were measured in the pigeon optic nerve and in the individual tectal layers. Characteristic topographical distribution patterns were observed for the different amino acids. After unilateral retinal ablation, the concentration of aspartate and glutamate was decreased in the nerve and contralateral tectum. The reduction was restricted to the superficial part of the tectum, which receives a direct retinal input. The maximal loss was measured in the first two layers, where aspartate was reduced by 51% and glutamate by 75% in comparison with the ipsilateral side 4 weeks after ablation. The results favor a special role for aspartate and glutamate in pigeon retino-tectal afferents.     

Thiocyanate catalyzes myeloperoxidase-initiated lipid oxidation in LDL

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase-hydrogen peroxide (MPO/H2O2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN-) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN*. As relatively abundant levels of SCN- are found in plasma of smokers--a well-known risk group for cardiovascular disease--the ability of SCN- to act as a catalyst of LDL atherogenic modification by MPO/H2O2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H2O2 revealed that SCN- catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN- on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite--recently found to be a substrate for MPO--showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN- -catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN- catalyzed the cell-mediated LDL oxidation. The MPO/H2O2/SCN- system may have the potential to play a significant role in the oxidative modification of LDL--an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.     

Cadherin-catenin complex: Protein interactions and their implications for cadherin function

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. In epithelial cells, E-cadherin represents a key molecule in the establishment and stabilization of cellular junctions. On the cellular level, E-cadherin is concentrated at the adherens junction and interacts homophilically with E-cadherin molecules of adjacent cells. Significant progress has been made in understanding the extra- and intracellular interactions of E-cadherin. Recent success in solving the three-dimensional structure of an extracellular cadherin domain provides a structural basis for understanding the homophilic interaction mechanism and the calcium requirement of cadherins. According to the crystal structure, individual cadherin molecules cooperate to form a linear cell adhesion zipper. The intracellular anchorage of cadherins is regulated by the dynamic association with cytoplasmic proteins, termed catenins. The cytoplasmic domain of E-cadherin is complexed with either β-catenin or plakoglobin (γ-catenin). β-catenin and plakoglobin bind directly to α-catenin, giving rise to two distinct cadherin-catenin complexes (CCC). α-catenin is thought to link both CCC's to actin filaments. The anchorage of cadherins to the cytoskeleton appears to be regulated by tyrosine phosphorylation. Phosphorylation-induced junctional disassembly targets the catenins, indicating that catenins are components of signal transduction pathways. The unexpected association of catenins with the product of the tumor suppressor gene APC has led to the discovery of a second, cadherin-independent catenin complex. Two separate catenin complexes are therefore involved in the cross-talk between cell adhesion and signal transduction. In this review we focus on protein interactions regulating the molecular architecture and function of the CCC. In the light of a fundamental role of the CCC during mammalian development and tissue morphogenesis, we also discuss the phenotypes of embryos lacking E-cadherin or β-catenin. © 1996 Wiley-Liss, Inc.