Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl‐CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50‐ to 100‐fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30‐ to 50‐fold. More important, these cells had a 2‐fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using ¹³C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl‐CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.     

Neuroprotective molecular mechanisms of (−)-epigallocatechin-3-gallate: a reflective outcome of its antioxidant, iron chelating and neuritogenic properties

Tea, the major source of dietary flavonoids, particularly the epicatechins, signifies the second most frequently consumed beverage worldwide, which varies its status from a simple ancient cultural drink to a nutrient component, endowed possible beneficial neuro-pharmacological actions. Accumulating evidence suggests that oxidative stress, resulting in reactive oxygen species generation, plays a pivotal role in neurodegenerative diseases, supporting the implementation of radical scavengers and metal chelating agents, such as natural tea polyphenols, for therapy. Vast epidemiology data indicate a correlation between occurrence of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases, and green tea consumption. In particular, recent literature strengthens the perception that diverse molecular signaling pathways, participating in the neuroprotective activity of the major green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), renders this natural compound as potential agent to reduce the risk of various neurodegenerative diseases. In the current review, we discuss the studies concerning the mechanisms of action implicated in EGCG-induced neuroprotection and discuss the vision to translate these findings into a lifestyle arena.     

Induction of G2 arrest and binding to cyclophilin A are independent phenotypes of human immunodeficiency virus type 1 Vpr

Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl cis-trans isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and function (specifically, induction of G2 arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R (Vpr). In the present study, we examine the role of the Vpr-CypA interaction on Vpr-induced G2 arrest. We find that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35 of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA or its binding to Vpr is dispensable for the ability of Vpr to induce G2 arrest. Vpr expression in CypA-/- cells leads to induction of G2 arrest in a manner that is indistinguishable from that in CypA+ cells. CsA abolished CypA-Vpr binding but had no effect on induction of G2 arrest or Vpr steady-state levels. In view of these results, we propose that the interaction with CypA is independent of the ability of Vpr to induce cell cycle arrest. The interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on other functions of Vpr.     

Cellulosome-like entities inBacteroides cellulosolvens

Cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells ofBacteroides cellulosolvens. The extracellular fractions contained three to four major polypeptides and several minor polypeptide bands that were localized in two major gel filtration peaks indicating average molecular weights of about 700 kDa and >10 MDa. A relatively large molecular weight component (M_r 230 kDa) was found to contain carbohydrate, but no apparent enzymatic activity of its own could be detected. The cell sonicate displayed a more complicated polypeptide profile, and glycosylated polypeptides were larger (ca. 310 and 290 kDa) than that of the extracellular fraction. The 230-kDa extracellular component interacted strongly with the GSI isolectin fromGriffonia simplicifolia, exhibited immunochemical cross-reactivity with the S1 subunit of the cellulosome fromClostridium thermocellum, and displayed anomalous pH- and salt-dependent migratory behavior in SDS-PAGE. Taken together, this evidence strongly suggests a structural similarity between the glycoconjugates of these two distinct cellulolytic bacteria. A major 84-kDa polypeptide was identified as a xylanase, and a 50-kDa polypeptide displayed endoglucanase activity. Additional biochemical and cytochemical evidence indicated that cellulosome-like cellulolytic complexes are associated with the cell surface in this bacterium.     

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.     

The pleiotropic effects of 33258-Hoechst on the cell cycle in Chinese hamster cells in vitro

The fluorochrome 33258-Hoechst which binds to double-stranded DNA (dsDNA) has been previously shown to inhibit in several mammalian cell cultures the condensation of chromosomes in phase G2 and early mitosis. We have now found that this drug affects the cell cycle of Chinese hamster cells grown in vitro in several other ways. In cells treated with the drug, phase G2 is prolonged, the rate of DNA replication is drastically reduced and the cells are arrested most probably at very late S phase.     

3D treatment planning for heavy charged particles

Summary The comments herein describe, at a necessarily superficial level, a number of issues which must be addressed in developing plans for heavy charged particle therapy. Programs now exist which provide the needed capabilities. The challenge now is to make the planning process easier and faster- and possibly more efective. It seems likely that this will be achieved in the next few years.Invited paper given on the fourth workshop on Heavy Charged Particles in Biology and Medicine GSI, Darmstadt, FRG, September 23–25, 1991