Phospholipases. I. Effect ofn-alkanols on the rate of enzymatic hydrolysis of egg phosphatidylcholine

Summary The rate of hydrolysis of unsonicated liposomes of egg lecithin by phospholipase A (from bee venom and Russell viper venom) and phospholipase C (fromBacillus cereus andClostridium welchii) is markedly dependent on the nature and concentration of a variety of added alcohols. Typical plots of rate against alcohol concentration are bell-shaped. The maximum rate and the alcohol concentration at which it is achieved are alcohol-specific. In a homologous series ofn-alkanols, the maximal rates increase and the optimal concentrations decrease as the chain length is increased from C₄ to C₈. For longer alcohols (C₉ to C₁₂), progressively higher concentrations are required to elicit maximal activation. The optimal activating concentrationsC for C₄ to C₈ n-alkanols obey the relationshipp C=a logP _octanol+constant [cf. Hansch & Dunn,J. Pharm. Sci. 61:1 (1972)], suggesting that the alcohol-activating effect is a consequence of their incorporation into the liposomes with resultant modification of liposomal structure.     

STUDIES OF SPERMIOGENESIS IN A NEMATODE, NIPPOSTRONGYLUS BRASILIENSIS

The fine structure of the developing spermatids and the mature sperm of Nippostrongylus brasiliensis was investigated. Immature spermatids are found at one end of the tubelike testis, and the mature sperm at the other. The spermatid has a prominent nucleus, with the chromatin clumped at the margin. It also contains a pair of centrioles, located near the nucleus. The cytoplasm is filled with ribosomal clusters, but it lacks an organized Golgi area or endoplasmic reticulum. Besides the normal mitochondria, the spermatid has specialized mitochondrionlike inclusions with dense matrix, few broad cristae, and a crystalloid structure always facing the nucleus. As spermiogenesis proceeds, the nucleus elongates, comes to lie at one end, and later evaginates to form a separate head structure, leaving the mitochondria and other cytoplasmic organelles in a broad cytoplasmic region. The nuclear material becomes filamentous and spiral, and the centrioles come to lie at one end near the junction of the head and the cytoplasmic portion of the sperm. Microtubules are found in the cytoplasmic region extending from the tubelike nucleus. The specialized mitochondria are about eighteen in number, and are arranged in rows in staggered groups of three around the microtubules in the cytoplasmic region. The mature sperm is aflagellate and lacks an acrosome. No movement of the sperm was ever observed.     

Methods for Isolation of Auxotrophic Mutants of Methanobacterium ivanovii and Initial Characterization of Acetate Auxotrophs

To develop a biochemical genetic approach to understanding cell carbon synthesis or metabolic pathways in methanogens, Methanobacterium ivanovii was selected as a model organism for genetic manipulation studies. The organism displayed a colony size of 3 to 6 mm in less than 2 weeks and had a plating efficiency of about 90%, which made it suitable for replica plating. Mutagenesis and selection techniques were developed for selection of acetate auxotrophs. Chemical mutagenesis with ethyl methanesulfonate, followed by enrichment with bacitracin as a selective agent, resulted in stable acetate auxotrophs. M. ivanovii was very sensitive to UV, but UV-induced acetate auxotrophs were unstable and reverted within two to four transfers. The acetate auxotrophs were analyzed in relation to wild type for carbon monoxide dehydrogenase enzyme activity.     

Lateral interaction of cholesterol in diacylphosphatidylcholesterol bilayers

Thermotropic phase-transition properties of the aqueous dispersions of several diacylphosphatidylcholesterol (DRCh) analogs are examined. The aqueous dispersions of their calcium salts exhibit characteristic endothermic thermotropic transitions due to a change in the conformation of acyl chains. These dispersions consist of osmotically intact liposomes that trap ions, and at the transition temperature there is anomalous increase in the ion leakage. Wide-angle electron diffraction studies of DPCh · Ca monolayers also exhibit a transition from a sharp 4.25 Å band to a broad one centering at 4.7 Å, reflecting an order-disorder transition in the acyl chains. The long-range order in the organization of acyl chains of DRCh molecules could arise from intermolecular interactions between the cholesterol moleties to form a functional dimer, and such dimers are apparently cross-linked by Ca²⁺ to form a long-range interacting lattice of acyl chains. Evidence for this model is adduced from the fluorescence properties of the dispersions of dimyristoylphosphatidylcholesta-5,7,9-trienol. The phase-transition properties of DRCh are an ideal illustration of calcium-induced isothermal phase transition.     

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

Effect of salinity,alkalinity and iron application on the availability of iron,manganese, phosphorus and sodium in pea (Pisum sativum L.) crop

Summary The effect of the salinity, alkalinity and Fe application on the dry matter yield and availability of Fe, Mn, P and Na were studied in the greenhouse on pea (Pisum sativum L.) crop. The highest dry matter yield was recorded in normal soil which decreased with the increase in the salinity and alkalinity, minimum being at 40 ESP. Alkalinity was more harmful to pea crop than salinity.Fe at 10 ppm increased the dry matter yield significantly. Highest Fe concentration (408.12 ppm) was recorded in 40 ESP soil followed by 20 ESP (395.2 ppm). Salinity alongwith marginal or higher alkalinity reduced harmful effect of alkalinity. The uptake of Fe was the highest in normal soil due to the high dry matter yield. All the three sources increased the concentration of Fe and its uptake than the control in all the soils but did not show much distinction among themselves.The concentration of Mn decreased more with the increase in alkalinity than salinity but salinity with alkalinity improved Mn concentration. Similarly uptake of Mn also decreased sharply with the increase in salinity and alkalinity. The application of Fe sources decreased Mn concentration but increased the uptake. The highest decrease was caused with FeSO₄ and lowest with Fe rayplex.Like Mn the concentration and uptake of P decreased with the increased levels of salinity and alkalinity. The addition of Fe decreased the concentration of P, highest depression being with Fe KE-MIN.Increase in ESP increased the concentration and the uptake of Na greatly. Addition of Fe through all the sources increased Na concentration and uptake significantly but sources did not differ much in their effect on Na.     

Naringenin Ameliorates Chronic Sleep Deprivation‐Induced Pain via Sirtuin1 Inhibition

Neurochemical Research - Growing experimental evidences have suggested the reciprocal correlation between sleep deprivation and pain. Inflammation and oxidative stress are among the key pathways...     

Activation of phospholipase A2 by freshly added lysophospholipids

Reaction progress curves for the hydrolysis of dimyristoylphosphatidylcholine by pig pancreatic phospholipase A₂ exhibits a latency phase. Addition of 1-palmitoyllysophosphatidylcholine to the preformed vesicles reduces the latency phase and enhances the binding of phospholipase A₂ to the vesicles. In contrast, the binary codispersions prepared from diacylphospholipids premixed with lysophosphatidylcholine do not exhibit such enhanced susceptibility to the phospholipase. This effect appears to be due to organizational defects created by asymmetrical incorporation of lysophospholipid molecules into the outer monolayer of the vesicles, and the action of phospholipase is not observed when the additive is equilibrated in both the monolayers of the vesicles.     

Estrogen receptor β and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain

The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.