Apparatus and mechanism of photosynthetic oxygen evolution: a personal perspective*

This historical minireview describes basic lines of progress in our understanding of the functional pattern of photosynthetic water oxidation and the structure of the Photosystem II core complex. After a short introduction into the state of the art about 35 years ago, results are reviewed that led to identification of the essential cofactors of this process and the kinetics of their reactions. Special emphasis is paid on the flash induced oxygen measurements performed by Pierre Joliot (in Paris, France) and Bessel Kok (Baltimore, MD) and their coworkers that led to the scheme, known as the Kok-cycle. These findings not only unraveled the reaction pattern of oxidation steps leading from water to molecular oxygen but also provided the essential fingerprint as prerequisite for studying individual redox reactions. Starting with the S. Singer and G. Nicolson model of membrane organization, attempts were made to gain information on the structure of the Photsystem II complex that eventually led to the current stage of knowledge based on the recently published X-ray crystal structure of 3.8 A resolution in Berlin (Germany).With respect to the mechanism of water oxidation, the impact of Gerald T. Babcock's hydrogen abstractor model and all the considerations of electron/proton transfer coupling are outlined. According to my own model cosiderations, the protein matrix is not only a 'cofactor holder' but actively participates by fine tuning via hydrogen bond networks, playing most likely an essential role in water substrate coordination and in oxygen-oxygen bond formation as the key step of the overall process.     

Evidence of cytochrome P450-catalyzed cleavage of the ether bond of phenoxybutyrate herbicides in Rhodococcus erythropolis K2-3

Bacterial strain Rhodococcus erythropolis K2-3 can cleave theether bond of the phenoxybutyrate herbicides, i.e., 4-(2,4-dichlorophenoxy)butyrate(2,4-DB) and 4-(4-chloro-2-methylphenoxy)butyrate (MCPB), by anenzyme system that is constitutively expressed. The enzyme(s) involved were investigated in this study. The rate ofdisappearance of 2,4-DB determined in a whole cell assay amounted to0.6 mmol/h ¶ g_{dry mass}.Carbon monoxide difference spectra of dithionite-reduced wholecells and crude cell extracts suggested that strain K2-3 contains a soluble cytochrome P450(P450), named P450_PB-1. The addition of various phenoxybutyrate substrates to crude cell extracts resulted in typical difference spectra following the type I pattern ofsubstrate binding with P450. The rate of 2,4-DB cleavage was reduced by inhibitors of P450: 5 mM metyrapone and carbon monoxide at a CO/O₂ ratio of 10 reduced the activity by about 20%, and 70%, respectively. The ether cleaving activity completely disappearedafter disruption of the cells and could not be detected in crude extracts. To elucidate theenzymatic basis of this reaction, P450 was partially purified. With the resulting enzyme preparation,2,4-DB cleavage activity was re-established, becoming measurable after the addition of eitherphenazine methosulfate or ferredoxin and ferredoxin/NADP oxidoreductase from spinach. We detected no activities attributable to -ketoglutarate-dependent dioxygenase orNAD(P)H-dependent monooxygenase. These results collectively indicatethat cleavage of the ether bond of phenoxybutyrate herbicides is catalyzed by P450-mediated activityin this strain. One of the products derived from this reaction is dichlorophenol, and comparativechromatographic analyses suggest that the other product is a C4-carbonicacid, most likely succinic semialdehyde/succinate.     

Nuclear apoptosis induced by isolated mitochondria

We isolated and purified mitochondria from mouse livers and spinach leaves.When added into egg extracts of Xenopus laevis,they caused nuclei of mouse liver to undergo apoptotic changes.Chromatin condensation,margination and DNA ladder were observed.After incubating isolated mitochondria in some hypotonic solutions,and centrifuging these mixtures at mgh speed,we got mitochondrial supernatants.It was found that in the absence of cytosolic factor,the supernatant alone was able to induce apoptotic changes in nuclei.The effective components were partly of protein.DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC-YVAD-CHO.Meanwhile,caspase inhibitors fully blocked chromatin condensation.Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted.It was found that this endonuclease is different from endonuclease G,cytochrome c-induced nuclease,or Ca^2 -activated endonuclease.     

L-Propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia–reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H₂O₂ UV-photolysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Unequal cleavage in the early Tubifex embryo

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non-cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin-based, cell contact-dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4-cell stage.     

Cleaved Slit directs embryonic muscles

The formation of functional musculoskeletal system relies on proper connectivity between muscles and their corresponding tendon cells. In Drosophila, larval muscles are born during early embryonic stages, and elongate toward tendons that are embedded within the ectoderm in later. The Slit/Robo signaling pathway had been implicated in the process of muscle elongation toward tendons. Here we discuss our recent findings regarding the critical contribution of Slit cleavage for immobilization and stabilization of the Slit signal on the tendon cells. Slit cleavage produces 2 polypeptides, the N-terminal Slit-N, which is extremely stable, undergoes oligomerization, and associates with the tendon cell surfaces, and the C-terminal Slit-C, which rapidly degrades. Slit cleavage leads to immobilization of Slit signaling on tendons, leading to a short-range repulsion, which eventually arrest further muscle elongation. Robo2, which is co-expressed with Slit by the tendon cells facilitates Slit cleavage. This activity does not require the cytoplasmic signaling domain of Robo2. We suggest that Robo2-dependent Slit cleavage, and the formation of Slit-N oligomers on the tendon cell surfaces direct muscle elongation, and provide a stop signal for the approaching muscle, through binding to Robo and Robo3 receptors expressed by the muscles.     

Effect and mechanism of the Ang-(1-7) on human mesangial cells injury induced by low density lipoprotein

Hyperlipidemia is an independent risk factor for renal disease, and lipid deposition is associated with glomerulosclerosis. The angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis (ACE2-Ang-(1-7)-Mas axis) has been reported to participate in lipid metabolic regulation but its mechanism remains unclear. We hypothesized Ang-(1-7) would reduce lipid uptake in human mesangial cells (HMCs) by regulating the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr–SREBP2–SCAP) negative feedback system, and improve glomerulosclerosis by regulating the transforming growth factor-β1 (TGF-β1). In this study we found that ACE2 was undetected in HMCs. The administration of LDL caused normal LDLr–SREBPs–SCAP negative feedback effect. Exogenous Ang-(1-7) enhanced this negative feedback effect via down-regulating LDLr, SREBP2, and SCAP expression, and effectively inhibited LDL-induced lipid deposition and cholesterol increases. This enhanced inhibitory effect was reversed by the Mas receptor antagonist A-779. Meanwhile, Ang-(1-7) significantly decreased the high LDL-induced production of TGF-β1, an effect blocked by A-779. Interestingly, HMCs treated with Ang-(1-7) alone activated the TGF-β1 expression. Our results suggested that Ang-(1-7) inhibits LDL accumulation and decreases cholesterol levels via modulating the LDLr–SREBPs–SCAP negative feedback system through the Mas receptor. Moreover, Ang-(1-7) exhibits a dual regulatory effect on TGF-β1 in HMCs.     

Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris

We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2–10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF‐β proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010