Microarray analysis of the developing rat mandible

To analyze the molecular events that occur in the developing mandible, we examined the expression of 8803 genes from samples taken at different time points during rat postnatal mandible development. Total RNA was extracted from the mandibles of 1-day-old, 1-week-old, and 2-week-old rats. Complementary RNA (cRNA) was synthesized from cDNA and biotinylated. Fragmented cRNA was hybridized to RGU34A GeneChip arrays. Among the 8803 genes tested, 4344 were detectable. We identified 148 genes with significantly increased expression, and 19 genes with significantly decreased expression. A comprehensive analysis appears to be an effective method of studying the complex process of development.     

Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.Key words: blastocyst, embryo implantation, apoptosis, cell adhesion, signal transduction     

Comparison of analytical methods for profiling <Emphasis Type="Italic">N-</Emphasis> and <Emphasis Type="Italic">O-</Emphasis>linked glycans from cultured cell lines

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.     

Possible role of coexpression of CD9 with membrane-anchored heparin-binding EGF-like growth factor and amphiregulin in cultured human keratinocyte growth

CD9 is a protein with 4 transmembrane domains, and functions as a cell surface antigen. We have previously reported that CD9 functions as an up-regulator of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) activity, which is a potent mitogen as well as a soluble HB-EGF. Anti-CD9 antibodies can neutralize the juxtacrine activity of proHB-EGF when both CD9 and proHB-EGF are coexpressed. We demonstrated here: (1) the CD9 gene was transcribed and translated in the cultured human keratinocytes; (2) anti-CD9 antibody inhibited the approximately 50% growth of human keratinocytes in culture; (3) CD9 was coprecipitated with proHB-EGF and membrane-anchored amphiregulin (proAR), and (4) the transient coexpression of CD9 with proHB-EGF or proAR in mouse L cells up-regulated their juxtacrine growth factor activities. These results suggest that CD9 would make a heterodimer and/or trimer complex with proHB-EGF and proAR, and might cooperate with proHB-EGF and proAR for human keratinocyte growth in a juxtacrine manner. J. Cell. Physiol. 171:291–298, 1997. © 1997 Wiley-Liss, Inc.     

Increased plasma plant sterol levels in heterozygotes with sitosterolemia and xanthomatosis

Plasma sterol levels in a family of sitosterolemia and xanthomatosis were determined by a high performance liquid chromatography. Three affected siblings manifested marked xanthomatosis including subcutaneous soft tissues and generalized atherosclerosis. Two other siblings as well as children of the patients did not show such clinical symptoms and signs. Plasma levels of cholesterol, sitosterol, campesterol, and cholestanol in three affected subjects were 190 +/- 18.5, 25.9 +/- 11.6, 16.1 +/- 7.8, 1.84 +/- 0.92 mg/dl (mean +/- SD), respectively. Four daughters of the affected subjects, who should be considered as obligatory heterozygotes, showed moderately increased levels of these sterols (195 +/- 41.7, 1.33 +/- 0.44, 1.56 +/- 0.69, 0.80 +/- 0.28 mg/dl), which were significantly higher than those of normal subjects. Treatment with cholestyramine had little effect on the increased plasma plant sterol levels, but markedly decreased plasma cholestanol concentrations in two affected siblings. This report presents the clinical features of the patients with sitosterolemia and xanthomatosis and also demonstrates that heterozygotes with this disorder have increased plasma levels of plant sterols as well as cholestanol, and suggests that this rare disease might be inherited as an autosomal co-dominant trait in certain cases. The data also indicate that cholestyramine administration was not effective in this family for treatment of sitosterolemia.     

Major Enteropathogenic Bacteria Isolated from Diarrheal Patients in Bolivia: A Hospital-Based Study

A total of 1,234 fecal samples from diarrhea cases were examined for etiological bacterial agents at medical facilities in La Paz and Sucre, Bolivia. Eighty strains of Shigella spp., 39 strains of Salmonella spp., 29 strains of Vibrio cholerae, and 222 strains of enteropathogenic Escherichia coli (139 EPEC, 55 ETEC, 29 EIEC, and 1 EHEC) were isolated. With regard to the serovars of Shigella, S. flexneri 2a, 3a, and 1b were predominant. In the case of Salmonella, S. enteritidis was the most common, followed by S. typhi, S. poona, and S. paratyphi B. Out of 29 cholera strains, 25 belonged to biovar El Tor, serovar Ogawa while the remaining 4 were serovar Inaba. Among 55 strains of ETEC serotypes, 5 showed ST producers but none showed LT producers. Likewise, among 55 strains of enterohemorrhagic serotypes, only one strain (O157:H7) produced verocytotoxin (VT 2). The results of drug sensitivity tests revealed the predominance of Shigella, EPEC, and ETEC strains resistant to aminobenzil-penicillin (ABPC) and trimethoprim. Since diarrheal patients in Bolivia are treated mainly with ABPC or sulfamethoxazole/trimethoprim (SXT) and rarely with gentamicin, kanamycin, or other drugs, it is possible that ABPC- and SXT-resistant strains will increase and persist in the near future.     

MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences

The Molecular Evolutionary Genetics Analysis (MEGA) software is a desktop application designed for comparative analysis of homologous gene sequences either from multigene families or from different species with a special emphasis on inferring evolutionary relationships and patterns of DNA and protein evolution. In addition to the tools for statistical analysis of data, MEGA provides many convenient facilities for the assembly of sequence data sets from files or web-based repositories, and it includes tools for visual presentation of the results obtained in the form of interactive phylogenetic trees and evolutionary distance matrices. Here we discuss the motivation, design principles and priorities that have shaped the development of MEGA. We also discuss how MEGA might evolve in the future to assist researchers in their growing need to analyze large data set using new computational methods.     

Different Modulation of Protein Kinase C Isozymes in Dibutyryl Cyclic AMP-Differentiated PC12h Cells

Abstract: PC12h cells can be differentiated into sympathetic neuron-like cells by various agents, including nerve growth factor, basic fibroblast growth factor, cyclic AMP analogues, and protein kinase C (PKC) activators. To study the involvement of PKC in the process of PC12h cell differentiation by cyclic AMP treatment, PKC isozymes (α, βI, βII, and γ) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(α) and PKC(βII), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(α) levels declined in the cytosolic fraction of the cells, whereas PKC(βII) levels increased. Increased PKC(βII) levels were also detected in the particulate fraction, whereas particulate PKC(α) levels did not change. The total PKC activity decreased in the cytosolic fraction following cyclic AMP stimulation of PC12h cells, whereas it stayed constant in the particulate fraction. Fractionation on a hydroxyapatite column showed a decreased level of PKC(α) activity and a transient increase followed by a decreased level of PKC(βII) activity. This discrepancy between increased PKC(βII) immunoreactivity and reduced PKC(βII) activity suggested the presence of nonactivatable PKC(βII) in cyclic AMP-treated PC12h extract. These findings indicate that PKC(α) and PKC(βII) are differentially regulated during the differentiation of PC12h cells. In addition, the differentiation of PC12h cells triggered by cyclic AMP seems to involve characteristic alterations of PKC isozymes.     

Nervous system development of the sea cucumber Stichopus japonicus

The nervous system development of the sea cucumber Stichopus japonicus was investigated to explore the development of the bilateral larval nervous system into the pentaradial adult form typical of echinoderms. The first nerve cells were detected in the apical region of epidermis in the late gastrula. In the auricularia larvae, nerve tracts were seen along the ciliary band. There was a pair of bilateral apical ganglia consisted of serotonergic nerve cells lined along the ciliary bands. During the transition to the doliolaria larvae, the nerve tracts rearranged together with the ciliary bands, but they were not segmented and remained continuous. The doliolaria larvae possessed nerves along the ciliary rings but strongly retained the features of auricularia larvae nerve pattern. The adult nervous system began to develop inside the doliolaria larvae before the larval nervous system disappears. None of the larval nervous system was observed to be incorporated into the adult nervous system with immunohistochemistry. Since S. japonicus are known to possess an ancestral mode of development for echinoderms, these results suggest that the larval nervous system and the adult nervous system were probably formed independently in the last common ancestor of echinoderms.     

A perennial ryegrass CBF gene cluster is located in a region predicted by conserved synteny between Poaceae species

CBF/DREB1 proteins are the most important regulators of the cold temperature signaling pathway in many plants. CBF genes are candidates for low-temperature tolerance QTL in wheat and barley. Ten novel putative CBF cDNAs of perennial ryegrass (Lolium perenne L.) have been isolated from cold-treated leaf tissue. Their primary structures contain some conserved motifs, characteristic of the gene class. Phylogenetic analysis revealed that LpCBF genes were attributable to the HvCBF3-, and HvCBF4-subgroups following the previously proposed classification of barley CBF genes. RT-PCR analysis revealed that the expression of LpCBF genes was rapidly induced in response to low temperature and that the expression pattern under the low-temperature conditions for a long period was different between the various LpCBF genes. Five of the ten LpCBF genes were assigned to the genetic linkage map using the p150/112 reference mapping population. LpCBFIb, LpCBFII, LpCBFIIIb and LpCBFIIIc were mapped on LG5 forming a cluster within 2.2 cM, while LpCBFVb was located on LG1. Based on comparative genetic studies, conserved synteny for CBF gene family was observed between the Triticeae cereals and perennial ryegrass. Information on the perennial ryegrass CBF genes at both the molecular and genetic level obtained in this study would be useful for the further study on the role of CBF genes and low-temperature tolerance in grasses.