Physiological and Biochemical Studies of Chilling Injury in Bananas

The physiological changes in green bananas (cv. Sin-zun), which are very sensitive to chilling injury, were studied during and after exposure to low temperatures (4±1°C, 6±0.5°C) for various periods. While the fruits injured by chilling did not fail to produce CO₂ and ethylene, the pattern of both CO_2- and ethylene production in these chilled fruits (9 and 15 days at 6°C) after transfer to 20°C was not normal. The contents of acetaldehyde and ethanol in chilled fruits, both in peels and pulps, increased with the advance of chilling, injury. There was an accumulation of α-keto acids in the peels of chilled fruits. Only half the conversion of ¹⁴C (fed as succinic acid-1, 4-¹⁴C) to citric acid and isocitric acid was observed in chilled tissues as compared with healthy ones; the activity of citrate synthase in banana peels appears therefore to be inhibited by chilling injury. A histological study of the tissues showed that the browning substances (polyphenols) present in chilled fruits accumulate around the vascular tissues.     

Inter- and intramolecular stacking interaction between indole and adeninium rings

Crystals of 1,9-dimethyladeninium-indole-3-acetate (1:1) complex (I) and 9-(3-indol-3-ylpropyl)-1-methyladeninium iodide (II), an inter- or intramolecular model for the stacking interaction between the tryptophanyl residue and the methylated (or protonated) adenine base, were subjected to X-ray analyses. Nearly parallel stacking and interplanar spacing near to 3.4 A were observed between the indole and adeninium rings of both crystals. In particular, one of the two stacking pairs formed in I showed the existence of a partial charge-transfer interaction in their ground states. On the basis of the molecular orbital consideration, the mutual orientation between these stacked aromatic rings is considerably governed by the orbital interaction between the highest occupied molecular orbital of the indole ring and the lowest unoccupied one of the adeninium ring. The ring stacking observed in II was stabilized by the strong coupled dipole-dipole interaction. Absorption, fluorescence, and proton nuclear magnetic resonance spectra indicated the existence of a stacking interaction in the aqueous solutions of I and II, as well as in their crystalline states. The biological implication for the observed stacking interactions has been discussed.     

Inulavosin and its benzo‐derivatives,melanogenesis inhibitors,target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo‐derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper‐binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo‐derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo‐tyrosinase that has a conformational defect.     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.     

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

Apolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.     

Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.     

Intraspecific variation in the status of ant symbiosis on a myrmecophyte,<Emphasis Type="Italic"> Macaranga bancana,</Emphasis> between primary and secondary forests in Borneo

A tree species, Macaranga bancana , distributed in South East Asian tropics has a mutualistic relationship with specific symbiotic ant species, which defend the plant from herbivores. To examine the intraspecific variation in the status of the ant-plant symbiosis among microhabitats of different light conditions, we investigated the species composition of nesting ants and the herbivory damage on M. bancana saplings by field observations and sampling in primary and secondary forests in Sarawak. In addition, the effectiveness of non-ant (physical and chemical) defenses were estimated by feeding the larvae of a polyphagous lepidopteran with M. bancana leaves from saplings in the two types of forests. All saplings in the primary forest were colonized by two Crematogaster ant species that had been known to be the obligate symbionts of M. bancana, while in the secondary forest, about half of the saplings were occupied by several ant species that were not obligate symbionts. There was little herbivory damage on saplings colonized by the two Crematogaster symbiont ants in both forest types, while the saplings colonized by the other ant species suffered a 10–60% loss of leaf area. Larval mortality of the polyphagous lepidopteran Spodoptera litura was significantly higher when larvae fed on leaves of M. bancana saplings in the secondary forest than when fed on leaves of M. bancana saplings in the primary forest. These results suggest that the symbiosis between ants and M. bancana is looser and the non-ant-defenses are stronger in secondary forests, where light is more intense, than in primary forests.