Evolution of Hawaiian Drosophilidae. II. Patterns and Rates of Chromosome Evolution in an Antopocerus Phylogeny

The phylogenetic relationships of seven species of the genus Antopocerus (Family Drosophilidae) have been determined by means of a study of the metaphase configurations and polytene chromosomes. Based on biogeographical, behavioral and cytogenetic information, A. longiseta from Molokai is tentatively identified as the primitive species of the genus. The metaphase karyotypes of all Antopocerus species are either five pairs of rod chromosomes and a pair of dots (5R1D), or six rods (6R). Heterochromatin additions converted the dots to rods. Chromosome breakpoints for inversions also are clustered at heterochromatic loci. The chromosome segments between heterochromatic loci may represent sets of functionally related loci, evolving as a unit. The rate of chromosomal inversion substitution is estimated in the origin of the taxon (probably a subgenus of Drosophila rather than a separate genus). It averages no greater than one substitution per 1,000 years, or one per 5,000 generations. The average genetic death rate per generation of one individual per hundred is required to achieve this substitution rate. The rate of inversion substitution during radiation of this taxon may be only 4.4 x 10^-3 times as fast as that present in forming the taxon. Alternatively, radiation may have required only 250,000 years if rates of substitution are the same as in the origination of the taxon. Average rates of substitution reflect genetic accidents, selection pressures and rates of adaptation to new niches, as well as the rate of encountering new niches. Rate of adaptation probably is much greater in this instance than rate of encountering new niches. Therefore, the average rate of evolution reflects more nearly biogeographic and ecological factors than genetic factors.     

Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC₅₀ 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.     

NMR studies on novel antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin

Artemisinin and its derivatives, which have been known as antimalarial drugs, have also demonstrated their cytotoxicity against tumor cells. It has been proposed that antitumor activity depends on the lipophilicity of functional group on artemisinin derivatives. Solution structures of two artemisinin derivatives as antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin, were determined by NMR spectroscopy to elucidate structure-activity relationship. According to biological assay, antitumor efficiencies are not dependent upon lipophilicity. Instead, these compounds demonstrated their distinctive structural features of boat/chair conformation and capability to interact with receptors, as they have different efficiencies on antitumor activity. Especially, carboxypropyl moiety or carbonyl moiety in artemisinin derivatives influences the conformation and stability of ring structure. Although the detailed mechanism of antitumor activity by artemisinin derivatives has not been addressed, we suggest that antitumor activity is not determined only with lipophilicity and that artemisinin derivatives have specific target proteins in each type of cancer.     

9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

Affinity biosensor for avidin using a double functionalized dendrimer monolayer on a gold electrode

We have developed an affinity biosensor system based on avidin-biotin interaction on a gold electrode. As the building block of an affinity-sensing monolayer, a fourth-generation (G4) poly(amidoamine) dendrimer having partial ferrocenyl-tethered surface groups was prepared and used. The unmodified surface amine groups from dendrimers were functionalized with biotinamidocaproate, and the biotinylated and electroactive dendritic monolayer was constructed on a gold electrode for the affinity-sensing surface interacting with avidin. An electrochemical signal from the affinity biosensor was generated by free glucose oxidase in electrolyte, depending on the degree of coverage of the sensing surface with avidin. The sensor signal decreased correlatively with increasing avidin concentration and approached a minimum level when the sensing surface was fully covered with avidin. The detection limit of avidin was about 4.5 pM, and the sensor signal was linear ranging from 1.5 pM to 10 nM under optimized conditions. From the kinetic analysis using the biotinylated glucose oxidase, an active enzyme coverage of 2.5 x 10(-12) mol/cm(2) on the avidin-pretreated surface was registered, which demonstrates the formation of a spatially ordered and compact protein layer on the derivatized electrode surface.     

Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production

L-arabinose isomerase (EC 5.3.1.4) mediates the isomerization of D-galactose into D-tagatose as well as the conversion of L-arabinose into L-ribulose. To investigate the properties of L-arabinose isomerase as a biocatalyst for the conversion of galactose to tagatose, the L-arabinose isomerase of Escherichia coli was characterized. The substrate specificity for L-arabinose was 166-fold higher than that for D-galactose. The optimal pH and temperature for the galactose isomerization reaction were 8.0 and 30 °C, respectively. The enzyme activity was stable for 1 h at temperatures below 35 °C and within a pH range of 8–10. The Michaelis constant, K _m, for galactose was 1480 mM, which is 25-fold higher than that for arabinose. The addition of Fe²⁺ and Mn²⁺ ions enhanced the conversion of galactose to tagatose, whereas the addition of Cu²⁺, Zn²⁺, Hg²⁺, and Fe³⁺ ions inhibited the reaction completely. In the presence of 1 mM Fe²⁺ ions, the K _m for galactose was found to be 300 mM.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.