Construction of high-density bacterial colony arrays and patterns by the ink-jet method

We have developed a method for fabricating bacterial colony arrays and complex patterns using commercially available ink-jet printers. Bacterial colony arrays with a density of 100 colonies/cm(2) were obtained by directly ejecting Escherichia coli (E. coli) onto agar-coated substrates at a rapid arraying speed of 880 spots per second. Adjusting the concentration of bacterial suspensions allowed single colonies of viable bacteria to be obtained. In addition, complex patterns of viable bacteria as well as bacteria density gradients were constructed using desktop printers controlled by a simple software program.     

Semi-synthesis, topoisomerase I and kinases inhibitory properties, and antiproliferative activities of new rebeccamycin derivatives

In the course of structure-activity relationship studies, new rebeccamycin derivatives substituted in 3,9-positions on the indolocarbazole framework, and a 2',3'-anhydro derivative were prepared by semi-synthesis from rebeccamycin. The antiproliferative activities against nine tumor cell lines were determined and the effect on the cell cycle of murine leukemia L1210 cells was examined. Their DNA binding properties and inhibitory properties toward topoisomerase I and three kinases PKCzeta, CDK1/cyclin B, CDK5/p25 and a phosphatase cdc25A were evaluated. The 3,9-dihydroxy derivative is the most efficient compound of this series toward CDK1/cyclin B and CDK5/p25. It is also characterized as a DNA binding topoisomerase I poison. Its broad spectrum of molecular activities likely accounts for its cytotoxic potential. This compound which displays a tumor cell line-selectivity may represent a new lead for subsequent drug design in this series of glycosylated indolocarbazoles.     

Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep

Adeno-associated virus (AAV), unique among animal viruses in its ability to integrate into a specific chromosomal location, is a promising vector for human gene therapy. AAV Replication (Rep) protein is essential for viral replication and integration, and its amino terminal domain possesses site-specific DNA binding and endonuclease activities required for replication initiation and integration. This domain displays a novel endonuclease fold and demonstrates an unexpected structural relationship to other viral origin binding proteins such as the papillomavirus E1 protein and the SV40 T antigen. The active site, located at the bottom of a positively charged cleft, is formed by the spatial convergence of a divalent metal ion and two conserved sequence motifs that define the rolling circle replication superfamily.     

Alkylation of guanine in DNA by S23906-1, a novel potent antitumor compound derived from the plant alkaloid acronycine

The discovery of a new DNA-targeted antitumor agent is a challenging enterprise, and the elucidation of its mechanism of action is an essential first step in investigating the structural and biological consequences of DNA modification and to guide the rational design of analogues. Here, we have dissected the mode of action of the newly discovered antitumor agent S23906-1. Gel retardation experiments reveal that the diacetate compound S23906-1 and its monoacetate analogue S28687 form highly stable covalent adducts with DNA. The covalent adducts formed between S23906-1 and a 7-bp hairpin oligonucleotide duplex were identified by spectrometry. In contrast, the inactive compound S23907, lacking the two acetate groups of S23906-1, fails to yield covalent DNA adducts, indicating that the C1-C2 functionality is the DNA reactive moiety. DNase I footprinting and DNA alkylation experiments indicate that S23906-1 reacts primarily with guanine residues. A 30-mer oligonucleotide containing only G.C bp forms highly stable complexes with S23906-1 and S28687, whereas the equivalent A.T oligonucleotide is not a good substrate for these two drugs. The use of an oligonucleotide duplex containing inosines instead of guanosines identifies the guanine 2-amino group exposed in the minor groove of DNA as the potential reactive site. The reactivity of S23906-1 toward the guanine-N2 group was independently confirmed by fluorescence spectroscopy. Covalent DNA adducts were also identified in the genomic DNA of B16 melanoma cells exposed to S23906-1, and the specific accumulation of the drug in the nucleus of the cells was visualized by confocal microscopy. The elucidation of the mechanism of action of this highly potent anticancer agent opens a new field for future drug design.     

Comparison of static and dynamic medium environments for culturing of pre-implantation mouse embryos

The ability to manipulate mouse pre-implantation embryos in vitro has become a valuable tool in many scientific disciplines. However, fewer embryos maintain viability following in vitro manipulation compared with embryos in vivo. It has been suggested that use of dynamic medium environments may improve viability by simulating in utero environment. The objective of the study reported here was to compare a microdrop in vitro culture system with a microdevice in vitro culture system containing a static and two dynamic medium environments (0.1 and 0.5 microl/h) for culturing of mouse pre-implantation embryos. Results indicated that the static medium environment, in silicon-glass microdevices, was not significantly different from the microdrop control environment in proportion of embryos developing from the two-cell to the blastocyst stage. However, the static microdevice environment produced significantly (P < 0.05) more morulas than did that of the control group. Both of these treatment groups, under the presented conditions, consistently had significantly higher proportions of blastocysts (P < 0.05) and morulas (P < 0.05) and lower proportions of abnormal (P < 0.05) and eight-cell embryos (P < 0.05), compared with those of the high flow rate dynamic environment microdevice treatment groups. Studies exploring slower or pulsatile rates of medium delivery in a dynamic medium environment are indicated.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.     

RETRACTED: SENSORY SUGGESTIVENESS AND LABELING: DO SOY LABELS BIAS TASTE?1

Can labels suggestively influence sensory perceptions and taste? Using a “ Phantom Ingredient” taste test, we show that the presence or absence of a labeled ingredient (soy) and the presence or absence of a health claim negatively bias taste perceptions toward a food erroneously thought to contain soy. We found a label highlighting soy content made health claims believable but negatively influenced perceptions of taste for certain segments of consumers. Our results and discussion provide better direction for researchers who work with ingredient labeling as well as for those who work with soybean products.     

Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demonstrated (Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process. Here, it is shown that inhibition of the transport of endogenously synthesized metabolically radiolabeled free oligosaccharides out of the endoplasmic reticulum into the cytosol of permeabilized HepG2 cells occurs when assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharides (50 mM), differing from mannose at position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6 (mannoheptose), have little effect. N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect. It is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transport out of the endoplasmic reticulum is inhibited. These results demonstrate that the terminal nonreducing mannosyl residues of free polymannose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the free oligosaccharide with the transport machinery, and that this transport process requires the presence of calcium sequestered in the lumen of the endoplasmic reticulum.      

Development of Trypanosoma (M.) theileri in Tabanids

Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length.