Extraction method for preparing pyridylamino sugar derivatives and application to porcine gastric mucus glycoprotein analysis

For the sensitive detection of free sugars and oligosaccharides, the use of their pyridylamino derivatives has now found general acceptance. To remove excess 2-aminopyridine from this derivative in a reaction mixture, gel filtration and ion-exchange chromatography were conducted. It was found in the present study that contaminated 2-aminopyridine could be selectively removed from the reaction mixture by adjusting the pH with saturated sodium bicarbonate at above 8.5 followed by extraction with benzene. By using this method, fewer purification steps and less time are required, with minimum loss of pyridylamino sugar derivatives.     

Application of L-DNA to the study of the specific DNA recognition mechanism of bleomycin.

The hexadeoxynucleotide analog, L-d(CGCGCG) composed of L-deoxyribose was synthesized and clearly shown to have the same conformation and dynamic properties with natural D-d(CGCGCG) except for chirality with CD spectra. This unnatural hexanucleotide was not cleaved by bleomycin, an antitumor DNA cleaving drug, but was able to bind to the DNA binding domain of bleomycin to a similar extent with the natural one. These results strongly suggest the importance of the other moiety than the DNA binding domain for the specific DNA recognition of bleomycin. Thus, L-oligonucleotides are useful for the study of DNA-drug interactions.     

Effects of Notch glycosylation on health and diseases

Notch signaling is an evolutionarily conserved signaling pathway and is essential for cell-fate specification in metazoans. Dysregulation of Notch signaling results in various human diseases, including cardiovascular defects and cancer. In 2000, Fringe, a known regulator of Notch signaling, was discovered as a Notch-modifying glycosyltransferase. Since then, glycosylation—a post-translational modification involving literal sugars—on the Notch extracellular domain has been noted as a critical mechanism for the regulation of Notch signaling. Additionally, the presence of diverse O-glycans decorating Notch receptors has been revealed in the extracellular domain epidermal growth factor-like (EGF) repeats. Here, we concisely summarize the recent studies in the human diseases associated with aberrant Notch glycosylation.     

Thermal stability of oligodeoxynucleotide duplexes containing l-deoxynucleotide at termini

The effects of substituting l-deoxynucleotide for d-deoxynucleotide at duplex termini were evaluated and the terminal substitutions were found to show much less effects on duplex destabilization and to show a similar tendency in base pairing selectivity, compared with internal chiral substitutions.     

TGF-beta mediated Msx2 expression controls occipital somites-derived caudal region of skull development

Craniofacial development involves cranial neural crest (CNC) and mesoderm-derived cells. TGF-beta signaling plays a critical role in instructing CNC cells to form the craniofacial skeleton. However, it is not known how TGF-beta signaling regulates the fate of mesoderm-derived cells during craniofacial development. In this study, we show that occipital somites contribute to the caudal region of mammalian skull development. Conditional inactivation of Tgfbr2 in mesoderm-derived cells results in defects of the supraoccipital bone with meningoencephalocele and discontinuity of the neural arch of the C1 vertebra. At the cellular level, loss of TGF-beta signaling causes decreased chondrocyte proliferation and premature differentiation of cartilage to bone. Expression of Msx2, a critical factor in the formation of the dorsoventral axis, is diminished in the Tgfbr2 mutant. Significantly, overexpression of Msx2 in Myf5-Cre;Tgfbr2flox/flox mice partially rescues supraoccipital bone development. These results suggest that the TGF-beta/Msx2 signaling cascade is critical for development of the caudal region of the skull.     

Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Detection of underlying characteristics of nuclear chromatin patterns of thyroid tumor cells using texture and factor analyses

BACKGROUND: Aspiration biopsy cytology of thyroid tumors has been used more frequently in recent times to differentiate between malignant and benign lesions. Chromatin patterns of the tumor cell nuclei are one of most important factors for cytologic diagnosis. The interpretation of nuclear chromatin patterns is subjective and more difficult than that of nuclear size or shape. In the present report, we investigated how to detect underlying chromatin characteristics of benign and malignant thyroid tumor cells by means of texture and factor analyses. METHODS: We employed a computer-aided system in which light microscopy was combined with an image processor and monochrome camera. Using this system, 100 randomly selected cells in a Papanicolaou stained, aspiration biopsy cytologic smear in each case of 39 benign and malignant thyroid tumor cases were digitized. We applied two-dimensional and higher texture analyses with the use of co-occurrence and run-length matrices to analyze the chromatin patterns. Factor analysis was used to determine whether a large number of independent variables actually measured one or more underlying common variables. RESULTS: According to parameters with high factor-loading values, the morphologic chromatin characters were classified into three categories according to heterogeneity, contrast, and homogeneity of chromatin patterns. On the basis of analyses with these morphologic categories, nuclei of papillary carcinoma showed higher contrast of chromatin patterns than did those of the benign group. Moreover, there was a variety of contrasting chromatin patterns among cells in each papillary carcinoma case in comparison with the benign group. In contrast, follicular carcinomas showed a significant difference in the standard deviation of factor 3, which indicated more monotonous chromatin patterns among cells in each follicular carcinoma case than in each benign case. CONCLUSION: We believe that this technique, using texture and factor analyses, is useful in the detection of underlying characteristics of nuclear chromatin patterns in aspiration biopsy cytology.     

Inhibition mechanisms of hematophagous invertebrate compounds acting on the host blood coagulation and platelet aggregation pathways

To facilitate blood feeding, hematophagous invertebrates have evolved a sophisticated array of physiological compounds that counteract homeostatic systems and inflammatory reactions of the vertebrate host. For this reason, hematophagous invertebrates possess a variety of anticoagulation components that are inhibitors of coagulant factors or antagonists of the platelet receptor. The examination of kinetic data and the crystal structure analysis have exposed the inhibition mechanisms for many of these anticoagulant reagents. Here, we attempt to classify the antihemostatic molecules and to focus on the kinetic approaches that have been instrumental in defining these mechanisms.     

Attenuation of expression of gamma-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic beta cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 microg/10(6) cells/h in control cells (CC) to 1.48 microg/10(6) cells/h. This was associated with increased intracellular Ca(2+) concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca(2+) concentration.