Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.     

The MHC class II-associated chicken invariant chain shares functional properties with its mammalian homologs

The nucleotide sequence of chicken invariant chain (Ii) was determined, and the amino acid sequence similarity with human Ii is 61%. Certain regions important for the biological function of human Ii are highly conserved between chicken and mammals. The cytoplasmic tail of chicken Ii fused to the plasma membrane reporter molecule neuraminidase relocated the protein to endosomes. Moreover, like the mammalian orthologs, the cytoplasmic tail was found to contain two independent leucine-based endosomal sorting signals. Chicken Ii was found to interact with human Ii and crosslinking studies also indicate that chicken Ii assembles as a trimer. The chicken Ii can furthermore bind the human MHC class II (HLA-DR1). Many of the functional properties between the chicken Ii and its mammalian orthologs are thus maintained in spite of their sequence differences.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.     

A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties

A sample set of registered perennial ryegrass varieties was used to compare how morphological characterisation and AFLP^® (AFLP^® is a registered trademark of Keygene N.V.) and STS molecular markers described variety relationships. All the varieties were confirmed as morphologically distinct, and both the STS and AFLP markers exposed sufficient genetic diversity to differentiate these registered ryegrass varieties. Distances obtained by each of the approaches were compared, with special attention given to the coincidences and divergences between the methods. When correlations between morphological, AFLP and STS distances were calculated and the corresponding scatter-plots constructed, the variety relationships appeared to be rather inconsistent across the methods, especially between morphology and the molecular markers. However, some consistencies were found for closely related material. An implication could be that these molecular-marker techniques, while not yet suited to certain operations in the traditional registration of new varieties, could be suitable methods for investigating disputable distinctness situations or possible EDV (EDV= essentially derived variety. An EDV is a variety being clearly distinct from, but conforming in the expression of the essential characteristics of, an ’initial variety’ (IV) from which it is found to have been predominantly derived) relationships, subject to establishing standardised protocols and statistical techniques. Some suggestions for such a protocol, including a statistical test for distinctness, are given.     

Rest length and compliance of non-immobilised and immobilised rabbit soleus muscle and tendon

The first aim of this study was to measure the contributions of muscle and tendon to the total compliance of resting muscle-tendon units. A second aim was to determine whether the decrease in muscle-tendon unit rest length produced by prolonged immobilisation in a shortened position is mediated primarily by adaptations of the muscle or tendon. One ankle joint from each of five rabbits was immobilised in a plantarflexed position for 14 days. The passive length-tension properties of soleus muscle fascicles and tendons from both hindlimbs were measured using a video-based tensile-testing system. In non-immobilised muscles, muscle fascicle strains exceeded tendon strains by up to four times. However, because the rest length of tendon was much greater than that of muscle fascicles, changes in tendon length accounted for nearly half of the total change in muscle-tendon unit length. The rest length of immobilised muscle-tendon units was less than that of non-immobilised muscle-tendon units from contralateral limbs. Most of this difference was attributable to a change in the rest length of the tendon; there was little change in the rest length of muscle fascicles. It is concluded that the tendon is responsible for a large part of the compliance of rabbit soleus muscle-tendon units at physiological resting tensions, and that adaptation of tendon rest length is the primary mechanism by which the rabbit soleus shortens in response to immobilisation at short lengths. Accepted: 7 May 1997     

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%–64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.     

Practical one-step glucuronidation via biotransformation

We herein report a practical one-step glucuronidation method by biotransformation using Streptomyces sp. SANK 60895. This novel direct method of biotransformation has been shown to be more practical and scalable for glucuronidation than previously reported chemical and enzymatic procedures given its simplicity, high β-selectivity, cost-effectiveness, and reproducibility. We applied the present method to the synthesis of acyl glucuronide and hydroxy-β-glucuronide of mycophenolic acid and compound 4, respectively. This method was also shown to be applicable to the N-glucuronidation of various compounds.