Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis

Summary In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the -amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33–120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.Correspondence to: S. Kakudo     

Inactivation of acrolein by sodium 2-mercaptoethanesulfonate using headspace-solid-phase microextraction gas chromatography and mass spectrometry

Acrolein, the metabolite of cyclophosphamide and ifosphamide, is an irritant of mucous membranes and seems to play an important role in hemorrhagic cystitis. Several methods are available to reduce the risk of hemorrhagic cystitis. Mesna is a regional detoxificant which inactivates acrolein. However, the interaction of mesna and acrolein has never been reported because no available method can detect acrolein. In this study, we measured acrolein to evaluate the effect of mesna in urine or phosphate-buffered saline using a headspace-solid-phase microextraction gas chromatography and mass spectrometry method which we had previously established. We also investigated the effect of mesna at different conditions of pH. Mesna was effective in a dose-dependent (10 microM to 20 mM) fashion in both urine and phosphate-buffered saline and completely inactivated acrolein at concentrations over 10 mM. Furthermore, mesna was more effective in alkaline conditions than in acid.     

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Variation in Storage α-Polyglucans of Red Algae: Amylose and Semi-Amylopectin Types in <Emphasis Type="Italic">Porphyridium</Emphasis> and Glycogen Type in <Emphasis Type="Italic">Cyanidium</Emphasis>

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.     

The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules resulted in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity caused loss of the leading process, whereas repression of JNK activity did not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. Our data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration.