Effect of Exogenous Glucose on Photosynthesis in the Diatom Thalassiosira weissflogii Depending on Nitrate Nitrogen Supply and Illumination

The rate of photosynthetic carbon fixation (P) in the diatom Thalassiosira weissflogii cultivated in the presence of exogenous glucose in the medium (0–10.56 g C/l) at different levels of illumination—25, 50, and 100 E/(m² s)—was studied as a function of nitrate nitrogen supply. In the diatoms limited in nitrogen and assimilating exogenous glucose, P was found to decrease or increase depending on the light intensity, glucose concentration, and the duration of exposure. In the diatoms assimilating both nitrate nitrogen and glucose, compared to those supplied with nitrates alone, P was higher at the medium and high light intensities and lower at the low light intensity. The interrelation of the processes of carbon and nitrogen metabolism in mixotrophic algae and the ecological role of glucose uptake by phytoplankton are discussed.     

Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii

Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.     

Tet-system for the regulation of gene expression during embryonic development

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTA ^CMV (M1) and rTA ^CMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and -galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTA ^CMV (M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTA ^CMV -3/NZL-2 embryos at E13.5. Doxycycline abolished -gal expression in tTA ^CMV (M1)/NZL-2 but induced it in rTA ^CMV -3/NZL-2 embryos including late stages of embryogenesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that double reporter animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.     

Hydrogen production by cyanobacteria in an automated outdoor photobioreactor under aerobic conditions

The possibility of hydrogen production by a hydrogenase impaired mutant strain of Anabaena variabilis in outdoor culture was studied. A computer-controlled rooftop (outdoor) tubular photobioreactor (4.35 L) was assembled. H(2) production rates by A. variabilis PK84 grown in CO(2) + air in the photobioreactor were measured together with other parameters such as temperature, irradiance, pH, dry biomass weight, and pO(2), and Chl a concentrations during summer months of 1998 and 1999. Efficiencies of light energy bioconversion to H(2) energy and energy accumulated in biomass were calculated. The influence of irradiance, temperature, and mode of cultivation on H(2) production and efficiency of light energy bioconversion were evaluated. The culture produced up to 1.1 L H(2) day(-1) PhBR(-1). The efficiency of light energy to H(2) energy bioconversion on some days was 0.094%. However, the conditions for maximum H(2) photoproduction and for maximum efficiency of light energy to H(2) energy bioconversion were not the same. A. variabilis PK84 could produce hydrogen for prolonged periods (up to 40 days) without injection of fresh inoculum. During this period photobioreactor produced 24.5 L of H(2). Possibilities for increasing the efficiency of light energy conversion are discussed.     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.     

Neurons of the human basal ganglia (striatum and basolateral amygdala) expressing the enzyme NADPH-d

In human striatum and basolateral amygdala NADPH-d+ neurons were revealed (after Vincent et al., 1983); and in striatum strio-cortical neurons were also revealed using DiI marker (after Dahtstrom and Belichenko, 1995). The NADPH-d+ neurons were numerous in both formations. Staining of NADPH-d+ neurons with their processes, and our previous study of striatal and amygdalar human neurons by Golgi method made it possible to identify the species of neurons with their assessment as sparsely or densely branched. The main efferent neurons of striatum and basolateral amygdala (densely branched medium spiny and bushy spiny, respectively) and their densely branched interneurons were not marked. Efferent NADPH-d+ neurons included the most numerous ones in both formations. A projection of reticular striatal neurons to cortex was also shown. The NADPH-d+ interneurons belonged to sparsely branched forms. In striatum they included slender-dendritic and long-dendritic bipolars (numerous), ordinary bipolars, twisted and large poor-dendritic cells; in amygdala--the same bipolars and radial cells. Thus, the NADPH-d positive cells in the formations under study were represented by more "ancient" or less structurally complex cell forms.