Quantification of energy-converting protein complexes in plant thylakoid membranes
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I am a Professor of Biochemistry, Biophysics and Structural Biology and Plant and Microbial Biology at the University of California in Berkeley. I was born and raised in India, emigrated to the United States to attend university, earning a B.S. in Molecular Biology and a Ph.D. in Biochemistry at the University of Wisconsin in Madison. Following post-doctoral studies with Lawrence Bogorad at Harvard University where I became interested in genetic control of trace element quotas, I joined the department of Chemistry and Biochemistry at UCLA. One of the first to appreciate essential trace metals as potential regulators of gene expression, I articulated the details of the nutritional Cu regulon in Chlamydomonas. In parallel, I used genetic approaches to discover the genes governing missing steps in tetrapyrrole metabolism, including the attachment of heme to apocytochromes in the thylakoid lumen and the factors catalyzing the formation of ring V in chlorophyll. After biochemistry and classical genetics, I embraced genomics, taking a leadership role on the Joint Genome Institute’s efforts on the Chlamydomonas genome and more recently, contributing to high quality assemblies of several genomes in the green algal radiation, and large transcriptomic and proteomic datasets — focusing on the diel metabolic cycle in synchronized cultures and acclimation to key environmental and nutritional stressors — that are well-used and appreciated by the community. Finally, a new venture in Berkeley is the promotion of Auxenochlorella protothecoides as the true “green yeast” and as a platform for engineering algae to produce useful bioproducts.
Peppermint (Mentha x piperita L.) and Japanese catnip (Schizonepeta tenuifolia (Benth.) Briq.) accumulate p-menthane monoterpenoids with identical functionalization patterns but opposite stereochemistry. In the present study, we investigate the enantioselectivity of multiple enzymes involved in monoterpenoid biosynthesis in these species. Based on kinetic assays, mint limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, isopiperitenone reductase, and menthone reductase exhibited significant enantioselectivity toward intermediates of the pathway that proceeds through (-)-4S-limonene. Limonene synthase, isopiperitenol dehydrogenase and isopiperitenone reductase of Japanese catnip preferred intermediates of the pathway that involves (+)-4R-limonene, whereas limonene 3-hydroxylase was not enantioselective, and the activities of pulegone reductase and menthone reductase were too low to acquire meaningful kinetic data. Molecular modeling studies with docked ligands generally supported the experimental data obtained with peppermint enzymes, indicating that the preferred enantiomer was aligned well with the requisite cofactor and amino acid residues implicated in catalysis. A striking example for enantioselectivity was peppermint (-)-menthone reductase, which binds (-)-menthone with exquisite affinity but was predicted to bind (+)-menthone in a non-productive orientation that positions its carbonyl functional group at considerable distance to the NADPH cofactor. Here, the work presented here lays the groundwork for structure-function studies aimed at unraveling how enantioselectivity evolved in closely related species of the Lamiaceae and beyond.
Naphthalene is a ubiquitous combustion product and environmental contaminant with known human exposure. Chronic exposure to naphthalene vapor leads to respiratory tumor formation in rodents. Naphthalene forms DNA adducts (precursors to genotoxicity) in tissue explants but it is unclear if this occurs in vivo. Wild-type C57BL/6 mice were orally exposed to 50 mg/kg 14 C-naphthalene. Naphthalene-DNA adducts were detected by accelerator mass spectrometry at 2- to 72-h post-exposure in both lung and liver, with decreasing abundance over time. Adducts persisted even at 72-h after exposure, which indicates possible evasion of DNA repair and potential to contribute to mutagenesis.
From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.
Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.
Cardiovascular deconditioning, sometimes manifested in astronauts during standing postflight, may be related to the impairment of autonomic function and/or excessive production of endothelium-dependent relaxing factors. In the present study, we examined the cardiovascular responses to 7-day 30 degrees tail-suspension and a subsequent 6-h post-suspension period in conscious male Sprague-Dawley rats to determine the role of prostacyclin in the observed post-suspension reduction in mean arterial pressure (MAP). The specific prostacyclin synthase inhibitor U-51605 (0.3 mg/kg), or saline, was administered intravenously prior to release from suspension and at 2 and 4 h post-suspension. During 7 days of suspension, MAP did not change, however, there was a post-suspension reduction in MAP which was associated with significant increases in plasma prostacyclin and nitric oxide. U-51605 attenuated the observed post-suspension hypotension and reduced plasma prostacyclin levels, but not nitric oxide levels. The baroreflex sensitivity for heart rate was modified by U-51605: increased MAP threshold and effective MAP range. Thus, the post-suspension reduction in mean arterial pressure may be due to overproduction of prostacyclin and/or other endothelium-dependent relaxing factors and alteration in baroreflex activity.
Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.
A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA].
Recent studies indicate that hemodynamic forces such as cyclic strain and shear stress can increase prostacyclin (PGI2) secretion by endothelial cells (EC) but the effect of these forces on prostacyclin synthase (PGIS) gene expression remains unclear and is the focus of this study. Bovine aortic EC were seeded onto type I collagen coated flexible membranes and grown to confluence. The membranes and attached EC were subjected to 10% average strain at 60 cpm (0.5 sec deformation alternating with 0.5 sec relaxation) for up to 5 days. PGIS gene expression was determined by Northern blot analysis and protein level by Western blot analysis. The effect of cyclic strain on the PGIS promoter was determined by the transfection of a 1-kb human PGIS gene promoter construct coupled to a luciferase reporter gene into EC, followed by determination of luciferase activity. PGIS gene expression increased 1.7-fold in EC subjected to cyclic strain for 24 hr. Likewise, EC transfected with a pGL3B-PGIS (-1070/-10) construct showed an approximate 1.3-fold elevation in luciferase activity in EC subjected to cyclic strain for 3, 4, 8, and 12 hr. The weak stimulation of PGIS gene expression by cyclic strain was reflected in an inability to detect alterations in PGIS protein levels in EC subjected to cyclic strain for as long as 5 days. These data suggest that strain-induced stimulation of PGIS gene expression plays only a minor role in the ability of cyclic strain to stimulate PGI2 release in EC. These findings coupled with our earlier demonstration of a requisite addition of exogenous arachidonate in order to observe strain-induced PGI2 release, implicates a mechanism that more likely involves strain-induced stimulation of PGIS activity.