In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

Mouse models of type 1 and type 2 diabetes derived from the same closed colony: genetic susceptibility shared between two types of diabetes

Except for rare subtypes of diabetes, both type 1 and type 2 diabetes are multifactorial diseases in which genetic factors consisting of multiple susceptibility genes and environmental factors contribute to the disease development. Due to complex interaction among multiple susceptibility genes and between genetic and environmental factors, genetic analysis of multifactorial diseases is difficult in humans. Inbred animal models, in which the genetic background is homogeneous and environmental factors can be controlled, are therefore valuable in genetic dissection of multifactorial diseases. We are fortunate to have excellent animal models for both type 1 and type 2 diabetes--the nonobese diabetic (NOD) mouse and the Nagoya-Shibata-Yasuda (NSY) mouse, respectively. Congenic mapping of susceptibility genes for type 1 diabetes in the NOD mouse has revealed that susceptibility initially mapped as a single locus often consists of multiple components on the same chromosome, indicating the importance of congenic mapping in defining genes responsible for polygenic diseases. The NSY mouse is an inbred animal model of type 2 diabetes established from Jcl:ICR, from which the NOD mouse was also derived. We have recently mapped three major loci contributing to type 2 diabetes in the NSY mouse. Interestingly, support intervals where type 2 diabetes susceptibility genes were mapped in the NSY mouse overlapped the regions where type 1 diabetes susceptibility genes have been mapped in the NOD mouse. Although additional evidence is needed, it may be possible that some of the genes predisposing to diabetes are derived from a common ancestor contained in the original closed colony, contributing to type 1 diabetes in the NOD mouse and type 2 diabetes in the NSY mouse. Such genes, if they exist, will provide valuable information on etiological pathways common to both forms of diabetes, for the establishment of effective methods for prediction, prevention, and intervention in both type 1 and type 2 diabetes.     

Needleless in vivo gene transfer into muscles by jet injection in combination with electroporation

Previously, we have established an in vivo electroporation method for gene transfer into muscle by injection of DNA with a needle followed by electric pulse delivery using needle-type electrodes and proved that this method is effective for the systemic delivery of cytokines. To perform the needleless gene delivery, we combined jet injection of DNA with electroporation using plate-type electrodes. For delivery of beta-galactosidase- and enhanced green fluorescent protein (EGFP)-expressing plasmids into muscles, there was no significant difference between the previous needle-mediated method and the newly developed jet-injection method. When pCAGGS-IL-5 was introduced into tibialis anterior, quadricipital and back sural muscles by this new method, the serum IL-5 levels reached 3.4 +/- 0.9, 5.7 +/- 1.7 and 8.4 +/- 2.7 ng/ml at day 5, respectively. Although the peak values of IL-5 achieved by the jet-injection method in these muscles were lower than that of the highest value achieved by needle-mediated gene delivery into anterior tibial muscle, this new method could deliver plasmid into relatively large muscles with better efficiency than the needle-mediated method. Thus the jet-injection method provides a useful means of gene delivery into large muscles, which is essential for future use in human gene therapy.     

Six new constituents from the roots of Erythrina variegata

Three new isoflavonoids, eryvarins M-O (1-3), two new 2-arylbenzofurans, eryvarins P and Q (4 and 5), and a new 3-aryl-2,3-dihydrobenzofuran, eryvarin R (6), together with three known compounds, were isolated from the roots of Erythrina variegata. The structures of these compounds were established by spectroscopic analysis. Eryvarin R (6) is an unusual 3-aryl-2,3-dihydrobenzofuran derivative with a formyl (CHO) group. Eryvarin Q (5) showed potent antibacterial activity against methicillin-resistant Staphylococcus aureus.     

Antimicrobial activity of hydrophobic xanthones from Cudrania cochinchinensis against Bacillus subtilis and methicillin-resistant Staphylococcus aureus

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against B. subtilis (MIC = 1.56 microg/ml). Four xanthones, gerontoxanthone I, toxyloxanthone C, cudraxanthone S, and 1,3,7-trihydroxy-2-prenylxanthone, showed weak antibacterial activity against the bacterium (MICs = 3.13-6.25 microg/ml). These compounds also exhibited similar MIC values against methicillin-sensitive S. aureus, MRSAs, and Micrococcus luteus.     

The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.Communicated by W. Goebel     

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

An estrogen receptor (ER)alpha deoxyribonucleic acid-binding domain knock-in mutation provides evidence for nonclassical ER pathway signaling in vivo

We created a nonclassical estrogen receptor (ER) knock-in mouse model by introducing a mutation that selectively eliminates classical ER signaling through estrogen response elements, while preserving the nonclassical ER pathway. Heterozygous nonclassical ER knock-in (NERKI) females are infertile. Their ovaries contain no corpora lutea, reflecting a defect in ovulation, and the stromal cells contain lipid droplets, suggesting altered steroidogenesis. The uteri are enlarged with evidence of cystic endometrial hyperplasia, and the mammary glands are hypoplastic. These phenotypic features indicate differential ER effects on growth and development in various estrogen-responsive tissues. These findings suggest that nonclassical ER signaling pathways play an important physiological role in the development and function of the reproductive system.     

Synergistic effect of deoxyanthocyanins from symbiotic fern Azolla spp. on hrmA gene induction in the cyanobacterium Nostoc punctiforme

The hrmA gene of the N2-fixing cyanobacterium Nostoc punctiforme functions in repressing the formation of transitory motile filaments, termed hormogonia, by plant-associated vegetative filaments. Here, we report that anthocyanins can contribute to induction of hrmA expression. Aqueous extract from fronds of the fern Azolla pinnata, a host of symbiotic Nostoc spp., was found to be a potent inducer of hrmA-luxAB in N. punctiforme strain UCD 328. The hrmA-luxAB inducing activities of A. pinnata, as well as Azolla filiculoides, were positively correlated with levels of frond deoxyanthocyanins. Analyses of the deoxyanthocyanins in frond extracts revealed, in order of predominance, an acetylated glycoside derivative of luteolinidin (m/z 475) and of apigeninidin (m/z 459) and minor amounts of a second luteolinidin derivative. At up to 150 microM, a purified preparation of deoxyanthocyanins only weakly induced hrmA-luxAB on its own, but mixtures with hrmA-luxAB inducers (A. filiculoides extract or the flavonoid naringin) synergistically doubled to tripled their inducing activities. These results suggest that appropriately localized deoxyanthocyanins could function in plant-mediated mechanisms for repressing Nostoc spp. hormogonium formation.