Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC3.1.4.4 [EC] ) from soybean (Glycine max L.) suspension-cultured cellwas purified around 1,200-fold to homogeneity by acetone precipitation,Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4Baffinity chromatography. The purified enzyme released 1,600µmol of choline per min per mg of protein. The enzymeis monomeric with a molecular mass of 92 kDa, as estimated bySDS-PAGE. One of the most interesting characteristics of thepurified soybean phospholipase D was the dependence of the pHoptimum on the Ca²⁺ ion concentration in the assay. With 10mM, 20 mM and 40 mM Ca²⁺ ions, the optima were at pH 7.5, 6and 5.5, respectively. The specific adsorption of phospholipaseD onto octyl-Sepharose gel suggests that the molecule becomesmore hydrophobic in the presence of Ca²⁺ ions. The amino acidsequence of the first 18 N-terminal residues of soybean phospholipaseD revealed a high degree of homology with those previously publishedfor cabbage leaf and castor bean endosperm enzymes. Westernblots of the soybean phospholipase D showed an immunoreactivitywith antibodies raised against a synthetic peptide correspondingto the 15 N-terminal aminoacid residues of phospholipase D fromcabbage leaves. (Received March 13, 1995; Accepted May 29, 1995)     

Nest building by lowland gorillas in the Lopé Reserve,Gabon: Environmental influences and implications for censusing

We analyzed data from 373 fresh nest-sites (containing 2435 nests) of lowland gorillas (Gorilla g. gorilla)during a 4-year period in the Lopé Reserve, Gabon, to determine whether the observed variability in nest building was due to environmental influences. We recognized and defined seven types of nest in terms of the degree of construction and the raw materials used. Overall, nests built on the ground from herbaceous plants are the most common type (40%), followed by tree nests (35%). Frequencies of the different nest-types vary significantly between eight habitat-types. In habitat-types with high densities of understory herbs, ground nests predominated, but when herbs were rare, the majority of nests were in trees. A general preference for sleeping in herbaceous ground nests is indicated since trees are abundant in all habitat-types, except savanna. The frequency of nesting in trees shows a significant positive correlation with rainfall, but effects of climate are confounded by seasonal variation in use of different habitat-types. When elephants were attracted to the same localized food sources as gorillas, many tree nests were built even when herbs were available. We conclude that different nest-types reflect a variety of solutions to maximize comfort, depending on available raw materials and the probability of rainfall or disturbance by elephants or both factors. Nests are a powerful tool for population censuses and demographic studies of great apes, but problems exist in interpreting data on lowland gorilla nests. Results from this analysis show that only a third of nest-sites accurately reflects group size (of weaned individuals) and that 26% of all gorilla nest-sites could be mistaken for those of chimpanzees, as all nests, or all those visible from a transect, were in trees. Gorilla nests at Lopé were nonrandomly distributed with respect to habitat-types, and nest construction varied seasonally, thereby introducing sources of bias to transect nest counts. We discuss these problems and ones related to assessing the decay rate of nest-sites and make recommendations relevant to census work.     

The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential.

We report the cloning and primary structure of the Drosophila insulin receptor gene (inr), functional expression of the predicted polypeptide, and the isolation of mutations in the inr locus. Our data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The INR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The INR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension which contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature INR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of INR have different signaling potentials. Finally, we show that loss of function mutations in the inr gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of inr appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are affected by inr mutations.     

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.     

Incidence of heavy metals in the mangrove flora and sediments in Kerala, India

The mangroves of Kerala on the south-west coast of India are fast disappearing due to land reclamation and other anthropogenic disturbances. There are very few ecosystem level studies made in these much threatened biotopes in Kerala. The present study involves the measurement of heavy metals in the mangrove flora and sediments of three mangrove habitats along the Kerala coast. Sampling was carried out for a period of one year at bi-monthly intervals, with concentrations of Fe, Mn, Cu,Zn,Pb and Co analysed using atomic absorption spectrophotometry. An appreciable variation was observed in metal concentrations indifferent mangrove species. Cu, Zn and Pb were found to be in higher concentrations in Avicennia officinalis whereas higher levels of Fe, Mn and Co were observed in the species Barringtonia racemosa. The analysis of heavy metals indicated a high level of metal pollutants such as Fe, Cu, Zn and Pb in the mangrove habitats of Quilon and Veli compared to the relatively uncontaminated areas of Kumarakom. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Influence of central command and ergoreceptors on the splanchnic circulation during isometric exercise

The splanchnic circulation can make a major contribution to blood flow changes. However, the role of the splanchnic circulation in the reflex adjustments to the blood pressure increase during isometric exercise is not well documented. The central command and the muscle chemoreflex are the two major mechanisms involved in the blood pressure response to isometric exercise. This study aimed to examine the behaviour of the superior mesenteric artery during isometric handgrip (IHG) at 30% maximal voluntary contraction (MVC). The pulsatility index (PI) of the blood velocity waveform of the superior mesenteric artery was taken as the study parameter. A total of ten healthy subjects [mean age, 21.1 (SEM 0.3) years] performed an IHG at 30% MVC for 90 s. At 5 s prior to the end of the exercise, muscle circulation was arrested for 90 s to study the effect of the muscle chemoreflex (post exercise arterial occlusion, PEAO). The IHG at 30% MVC caused a decrease in superior mesenteric artery PI, from 4.84 (SEM 1.57) at control level to 3.90 (SEM 1.07) (P = 0.015). The PI further decreased to 3.17 (SEM 0.70) (P = 0.01) during PEAO. Our results indicated that ergoreceptors may be involved in the superior mesenteric artery vasodilatation during isometric exercise.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.