Proteomic approach to apoptotic thymus maturation

Apoptosis is an essential process for selection of T lymphocytes specific for foreign antigen in the process of mammalian thymus maturation. Proteomics, a comprehensive study of proteins expressed in a cell, will facilitate the systematic analysis of protein molecules related to such a complicated biological system. Protein expression profiles including information about protein signatures, localization and their quantitative changes with extracellular stimulations are extremely useful to construct intracellular pathway models resulting in the apoptotic cell death.     

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4alpha causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4alpha in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4alpha protein, due, in part, to the limited availability of human HNF4alpha-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4alpha fusion proteins as the immunizing agent. The mouse anti-human HNF4alpha monoclonal antibody (K9218) generated against human HNF4alpha1/alpha2/alpha3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4alpha proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4alpha protein, but HEK293 showed no expression of HNF4alpha protein. Nuclear-specific localization of the HNF4alpha protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4alpha protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4alpha protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4alpha in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4alpha mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4alpha and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4alpha isoforms in humans and in several other mammalian species.     

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.     

Mother–young distance in Japanese Black cattle at pasture

Distance between dam and offspring (1–121 days old) in a herd of Japanese Black cattle (Bos taurus) grazing a tropical grass (Paspalum notatum) pasture (1.5 ha) was investigated during 7-h grazing periods over grazing seasons from May (spring) to October (autumn). The mother–young distance was not constant throughout the grazing period, repeatedly increasing and decreasing. Although significant periodicity was always detected in the mother–young distance, there was no consistent dominant cycle, indicating the complexity of the within-day pattern of mother–young distance. The mean mother–young distance over the grazing period increased as a calf aged, reaching a plateau at an age of about 33 days. The mean distance of a calf from its mother was usually shorter than that from a non-mother cow, with the difference between the mean distances decreasing sharply until a calf became about 35 days old. The results and literature show that mutual independence of mother and young rapidly develops in the first 30–50 days after parturition. Electronic Publication     

The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes

Drosophila glial cells missing (gcm) is a key gene that determines the fate of stem cells within the nervous system. Two mouse gcm homologs have been identified, but their function in the nervous system remains to be elucidated. To investigate their function, we constructed retroviral vectors harboring Drosophila gcm and two mouse Gcm genes. Expression of these genes appeared to influence fibroblast features. In particular, mouse Gcm1 induced the expression of astrocyte-specific Ca(2+)-binding protein, S100beta, in those cells. Introduction of the mouse Gcm1 gene in cultured cells from embryonic brains resulted in the induction of an astrocyte lineage. This effect was also observed by in utero injection of retrovirus harboring mouse Gcm1 into the embryonic brain. However, cultures from mouse Gcm1-deficient mouse brains did not exhibit significant reductions in the number of astrocytes. Furthermore, in situ hybridization analysis of mouse Gcm1 mRNA revealed distinct patterns of expression in comparison with other well-known glial markers. The mammalian homolog of Drosophila gcm, mouse Gcm1, exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells.     

Community dynamic models of two dioecious tree species

The effects of dioecy on community dynamics were examined by using transition matrix models for two dioecious tree species, one a superior competitor with a narrow dispersal range and the other an inferior competitor with a wide dispersal range. The models are based on tree-by-tree replacements in each identical microsite occupied by either male or female canopy trees of the superior competitor and canopy trees of the inferior competitor. Coexistence of the two species is possible not only because of a trade-off between competitive and dispersal abilities but also because of the existence of a competitor gap, which the superior competitor cannot occupy. The competitor gap is created under the male trees of the superior competitor. The inferior competitor occupies the competitor gap because of its wide dispersal range. The relative abundance of the two species depends on the dispersal ability and sex ratios of the superior competitor. The decreasing dispersal ability and the female abundance of the superior competitor increase the competitor gap, which allows the regeneration of the inferior competitor.An erratum to this article can be found at     

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry. Accepted: 1 December 1999