Changes in body composition associated with smoltification and premature transfer to seawater in Coho salmon (Oncorhynchus kisutch) and King salmon (O. tschawytscha)

Serum protein, glucose and fat levels were determined for Coho salmon parrs, freshwater smolts, seawater smolts, seawater stunts and freshwater desmolts. Liver and muscle glycogen, fat, protein and water concentrations were also calculated. Serum protein, glucose and fat levels were significantly lower in the freshwater smolts than in the parrs. Furthermore, both liver and muscle total fat levels were markedly decreased in the smolts, suggesting that smoltification is associated with increased catabolism. Smolts that failed to reach seawater by late summer reverted to a parr-like appearance and also regained the biochemical characteristics of parrs (high body fat and glycogen). Premature transfer of Coho presmolts into seawater caused impaired growth (stunting). Stunted Coho had higher muscle protein levels than normal smolts and parrs. Tissue water levels were not significantly different between stunts and normal seawater smolts, suggesting that the stunting phenomenon may not be caused primarily by osmoregulatory failure but may be due to shifts in metabolic patterns. In contrast to the pronounced changes seen in Coho salmon, transfer of King salmon to sea water did not result in increased catabolism of body reserves or in the production of stunts.     

Biosynthetic relationship between tetrahydroanthracene and anthraquinone in Aloe saponaria

The incorporation of Me¹⁴COONa into aloesaponol I, laccaic acid D methyl ester and aloesaponarin I was demonstrated. The biosynthetic relation between aloesaponol I and aloesaponarin I was established, but incorporation of aloesaponol I into laccaic acid D methyl ester, or vice versa was not demonstrated and this result was confirmed by an investigation using labelled laccaic acid D methyl (¹⁴CH₃) ester. It was possible to show that aloesaponol I and laccaic acid D methyl ester were biosynthesized in parallel in Aloe saponaria.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.     

The alkyl-connected 2-amino-6-vinylpurine (AVP) crosslinking agent for improved selectivity to the cytosine base in RNA

We have previously reported that the 2-amino-6-vinylpurine (AVP) nucleoside exhibits a highly efficient and selective crosslinking reaction toward cytosine and displayed an improved antisense inhibition in cultured cells. In this study, we further investigated the alkyl-connected AVP nucleoside analogs for more efficient crosslinking to the cytosine base (rC) of the target RNA. We synthesized three AVP analogs which connect the 2-amino-6-vinylpurine unit to the 2′-deoxyribose through a methylene, an ethylene, or a butylene linker. The ODN incorporating the AVP analog with the methylene or the butylene linker showed a slightly higher crosslinking to the target rC of RNA than the original AVP with no linker. In contrast, the AVP with the ethylene linker formed a selective and efficient crosslink to the rC of the target RNA.     

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Recombinant l-aminoacylase (PhoACY) from a hyperthermophilic archeon, Pyrococcus horikoshii, is a zinc-containing metalloenzyme. When the zinc was substituted by Mn²⁺ or Ni²⁺, its specific activity was significantly increased with acetyl-l-methionine as a substrate. The thermostability of PhoACY was improved when it was incubated with 1 mM Zn²⁺, Mn²⁺ or Ni²⁺. The enzyme with external Zn²⁺ addition had no significant loss of the activity when held at 90°C for up to 12 h and moreover had more than a 10-fold longer half-life even at 100°C, compared to the enzyme without Zn²⁺ addition. A thermostable structure of the enzyme associated with zinc binding is described based on differential scanning calorimetry.     

Anaerobic Nitrogen-Fixing Consortia Consisting of Clostridia Isolated from Gramineous Plants

We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N₂-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N₂ fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N₂ fixation arises in association with nondiazotrophic endophytes.     

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Lotus japonicus Infested with Herbivorous Mites Emits Volatile Compounds That Attract Predatory Mites

Lotus Japonicus has an indirect defense mechanism against spider mites, Tetranychus urticae, we investigated the responses of predatory mites, Phytoseiulus persimilis, to volatile compounds released from T. urticae-infested L. japonicus in a Y-tube olfactometer. Plants infested with spider mites attracted more P. persimilis than did clean air. Uninfested plants and artificially damaged plants did not attract P. persimilis. When infested by spider mites, L. japonicus plants started emitting (Z)-3-hexenyl acetate, (E)-4,8-dimethyl-1,3,7-nonatriene, germacrene d, 1-octen-3-ol and methyl salicylate (MeSA). These compounds were considered to be T. urticae-induced plant volatile compounds. When three L. japonicus mutants deficient in nodule organogenesis were infested by the spider mites, they all attracted P. persimilis. However, two of the infested mutants emitted blends of induced volatile compounds that were qualitatively different from those emitted from infested wild type L. japonicus. Received 8 August 2000/ Accepted in revised form 12 October 2000     

X-ray irradiation altered chemosensitivity of a p53-null non-small cell lung cancer cell line

Radiotherapy is an effective approach to treating many types of cancer. Recent progress in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates. However, the emergence of tolerant cells during or after radiotherapy remains problematic. In the present study, we first established a cell population from H1299, the p53-null non-small cell lung cancer cell line, by 10 Gy irradiation using 6 MV X-rays. The radio- and chemosensitivity of this cell population (referred to as H1299-IR) was determined using colony formation analyses and MTS assays. Compared with the parental cell line, the radiosensitivity of H1299-IR was apparently the same. H1299 and H1299-IR were both more radio tolerant than the A549 cell line. However, H1299-IR became significantly more sensitive to cisplatin, an antitumor agent. After exposure to 25 mug/ml cisplatin for 2 h, parental cells steadily grew during the MTS assay, whereas the sensitivity of H1299-IR cells doubled both at 24 and 48 h. Microarray analysis of over 30,000 H1299-IR genes (Agilent Technology) revealed that 12 and 15 genes were up- (> 2.0) and down- (< 2.0) regulated, respectively. Rad51d (homologous recombination repair protein) gene was down-regulated 2.8-fold, whereas matrix metalloproteinase 1 (collagenase-1) gene was up-regulated 4.4-fold. These results indicated that some p53-null non-small cell lung cancers could be successfully treated when X-ray radiotherapy was administered with subsequent or concurrent cisplatin chemotherapy.