Thermo-optically Induced Reorganizations in the Main Light Harvesting Antenna of Plants. I. Non-Arrhenius Type of Temperature Dependence and Linear Light-intensity Dependencies

Thermo-optically induced structural reorganizations have earlier been identified in isolated LHCII, the main chlorophyll a/b light harvesting complexes of Photosystem II, and in granal thylakoid membranes [Cseh et al. (2000) Biochemistry 39: 15250–15257; Garab et al. (2002) Biochemistry 41: 15121–15129]. According to the thermo-optic mechanism, structural changes can be induced by fast, local thermal transients due to the dissipation of excess excitation energy. In this paper, we analyze the temperature and light-intensity dependencies of thermo-optically induced reversible and irreversible reorganizations in the chiral macrodomains of lamellar aggregates of isolated LHCII and of granal thylakoid membranes. We show that these structural changes exhibit non-Arrhenius type of temperature dependencies, which originate from the ‘combination’ of the ambient temperature and the local thermal transient. The experimental data can satisfactorily be simulated with the aid of a simple mathematical model based on the thermo-optic effect. The model also predicts, in good accordance with experimental data published earlier and presented in this paper, that the reorganizations depend linearly on the intensity of the excess light, a unique property that is probably important in light adaptation and photoprotection of plants.     

Barley leaf unrolling. The proline connection

Unrolling of 1 cm sections, taken between 3 and 4 cm from the apex, of 6-day-old, etiolated barley leaves, was promoted by blue (426 nm) and red (658 nm) light. Accompanying such unrolling was a reduction in the level of the free proline of the tissue. When leaf unrolling was prevented by irradiation with far-red (728 nm) light, or treatment with abscisic acid (ABA) following red light irradiation, the level of proline remained more or less unchanged, at the level of the untreated, dark controls. The proline analogue, azetidine carboxylic acid (AZC) powerfully inhibited the light induced leaf opening, emphasizing the significance of proline-containing, structural and functional proteins in barley leaf unrolling. The inhibition imposed by AZC is partially reversible by added proline.     

Papain Does Not Cleave Operator-Bound Lambda Repressor: Structural Characterization of the Carboxy Terminal Domain and the Hinge

Abstract

The circular dichroism spectra of three different purified carboxy terminal fragments 93–236, 112–236 and 132–236 of the bacteriophage γ cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1–92. All three carboxy terminal fragments contain mostly β-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93–131 region previously called a hinge is structured. Fourier transformed infrared spectra also showed that fragment 93–236 contains α-helices, β-sheets and turns but fragment 132–236 contains no detectable α-helix, only β-sheets and turns. Papain is known to cleave the γ repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer. For the 132–236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 µM protein concentration, with a KD of 13.2 µM and 19.1 µM respectively. The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92–93; however, the subsequent cleavages are faster for SN228. The three Cys residues in the 132–236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain. Denaturation of the 132–236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.     

Comparative chlorine and temperature tolerance of the oyster crassostrea madrasensis: Implications for cooling system fouling

Crassostrea madrasensis is an important fouling oyster in tropical industrial cooling water systems. C. madrasensis individuals attach to surfaces by cementing one of their two valves to the substratum. Therefore, oyster fouling creates more problems than mussel fouling in the cooling conduits of power stations, because unlike the latter, the shell of the former remains attached to the substratum even after the death of the animal. However, there are no published reports on the tolerance of this species to chlorination and heat treatment. The mortality pattern and physiological behaviour (oxygen consumption and filtration rate) of three size groups (13 mm, 44 mm and 64 mm mean shell length) of C. madrasensis were studied at different residual chlorine concentrations (0.25, 0.5, 0.75, 1, 2, 3 to 5 mg l^{m 1}) and temperatures (30°C to 45°C). The effect of shell size (=age) on C. madrasensis mortality in the presence of chlorine and taking into account temperature was significant, with the largest size group oysters showing highest resistance. At 1 mg l^{m 1} residual chlorine, the 13 mm and 64 mm size group oysters took 504 h (21 d) and 744 h (31 d), respectively to reach 100% mortality. At 39°C, the 13 mm size group oysters took 218 min to reach 100% mortality, whereas the 64 mm size group oysters took 325 min. The oxygen consumption and filtration rate of C. madrasensis showed progressive reduction with increasing residual chlorine concentrations. However, the filtration rate and oxygen consumption responses of C. madrasensis were not significantly different between 30°C (control) and 37.5°C. There was a sharp decrease in the filtration rate and oxygen consumption at 38.5°C. A comparison of the present mortality data with previous reports on other bivalves suggests that the chlorine tolerance of C. madrasensis lies in between that of Perna viridis and Perna perna, while its temperature tolerance is significantly higher than that of the other two bivalve species. However, in power station heat exchangers, where simultaneous chlorine and thermal stresses are existent, C. madrasensis may have an edge over other common foulants, because of its high temperature tolerance.     

Phosphorylation on histidine is accompanied by localized structural changes in the phosphocarrier protein, HPr from Bacillus subtilis.

The histidine-containing protein (HPr) of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) serves a central role in a series of phosphotransfer reactions used for the translocation of sugars across cell membranes. These studies report the high-definition solution structures of both the unphosphorylated and histidine phosphorylated (P-His) forms of HPr from Bacillus subtilis. Consistent with previous NMR studies, local conformational adjustments occur upon phosphorylation of His 15, which positions the phosphate group to serve as a hydrogen bond acceptor for the amide protons of Ala 16 and Arg 17 and to interact favorably with the alpha-helix macrodipole. However, the positively charged side chain of the highly conserved Arg 17 does not appear to interact directly with phospho-His 15, suggesting that Arg 17 plays a role in the recognition of other PTS enzymes or in phosphotransfer reactions directly. Unlike the results reported for Escherichia coli P-His HPr (Van Nuland NA, Boelens R, Scheek RM, Robillard GT, 1995, J Mol Biol 246:180-193), our data indicate that phosphorylation of His 15 is not accompanied by adoption of unfavorable backbone conformations for active site residues in B. subtilis P-Ser HPr.     

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.