Variability of chromosomal DNA contents in maize (Zea mays L.) inbred and hybrid lines

The flow karyotypes of different maize (Zea mays L.) inbred and hybrid lines were analyzed. The accumulation and isolation of large quantities of high-quality metaphase chromosomes from root tips was achieved from many kinds of maize lines. The chromosome suspensions were prepared by a simple slicing method from synchronized maize root tips and analyzed by flow cytometry. Variations of experimental flow karyotypes were detected among inbred and hybrid lines in terms of the positions and/or the numbers of chromosome peaks. The 2C DNA amount among eight inbred lines ranged from 5.09 to 5.52 pg. The selection of appropriate maize lines is critical for sorting specific single chromosome types. At least five different chromosome types can be discriminated and sorted from five maize lines. The variability of DNA content in maize chromosome 1 was 9.1%, ranging from 0.685 to 0.747 pg. Differences were detected in the DNA content of homologous chromosome 1 of hybrid lines.     

Chromosome 4q;10q translocations; Comparison with different ethnic populations and FSHD patients

Background  

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by the weakness of facial, shoulder-girdle and upper arm muscles. Most patients with FSHD have fewer numbers of tandem repeated 3.3-kb KpnI units on chromosome 4q35. Chromosome 10q26 contains highly homologous KpnI repeats, and inter-chromosomal translocation has been reported.     

Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA

Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts. The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution. We developed a new method of transforming yeast using chromosome-sized DNA. The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA. Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA. The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.     

Up-regulation of the peroxisomal beta-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid beta-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system.     

Link protein as an enhancer of cumulus cell-oocyte complex expansion

To investigate the specific components involved in regulating cumulus cell-oocyte complex (COC) expansion in an in vitro mouse experiment, freshly-isolated COC were cultured in the presence of various combinations of FSH (1.0 microg/ml), proteins of the inter-alpha-inhibitor (I alpha I) family (a light chain, also known as bikunin, heavy chains [HC1 + HC2] and I alpha I [0.01-2.0 microg/ml]) and link protein (LP) (0.016-10 microg/ml) for 24 h and were observed for expansion of their cumulus cells (percent of COC with + 3 and + 4 expansion and average projected area). The COC were videotaped in real time at the initiation of culture and after 24 h of culture. FSH alone did not stimulate cumulus expansion under serum-free conditions; however, treatment with I alpha I (0.1-2.0 microg/ml) or heavy chains (10 microg/ml), but not bikunin (10 micro g/ml), in the presence of FSH significantly increased COC expansion, with maximal promotion occurring at 1.0 microg/ ml of I alpha I. Addition of LP (2.0 micro g/ml) to the medium containing I alpha I (1.0 microg/ml) and FSH resulted in significantly higher expansion levels than were observed in response to I alpha I alone, although LP alone (10 microg/ml) had no or very little effect by itself. Anti-I alpha I or anti-LP polyclonal antibody, which inhibits binding of I alpha I and LP, respectively, to hyaluronic acid (HA), markedly reduced expansion of the surrounding cumulus cell extracellular matrices. Therefore, in vitro, LP might serve, in part, to enhance the COC expansion possibly by stabilizing HA-I alpha I (or heavy chains) complex on the surrounding cumulus cell matrices.     

Regulation of hyaluronan synthases in mouse uterine cervix

We examined the expression pattern of hyaluronan synthase (HAS) mRNAs in the uterine cervix of pregnant mice. The expression levels of HAS-1 and -2 mRNAs peaked at delivery, whereas that of HAS-3 mRNA peaked on the 15th day of pregnancy. The regulation of HAS mRNA expression was examined in pregnant mouse uterine cervical fibroblasts. The expression of HAS-1, -2, and -3 mRNAs was significantly augmented by interleukin-1beta (IL-1beta). Progesterone significantly interfered with expression of HAS-1 and -2 mRNAs, but significantly increased the expression of HAS-3 mRNA. Low-molecular-weight hyaluronan significantly enhanced only the expression of HAS-1 mRNA. These results indicate that HAS in the uterine cervix is regulated in a complex manner by IL-1beta, progesterone, and low-molecular-weight hyaluronan, of which changes in the cervical tissue and serum closely participate in uterine cervical ripening and/or inflammation.     

Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.     

Genetic down-regulation of phosphoinositide 3-kinase by bikunin correlates with suppression of invasion and metastasis in human ovarian cancer HRA cells

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.     

Phenotypic stability of mature dendritic cells tuned by TLR or CD40 to control the efficiency of cytotoxic T cell priming

It is generally accepted that after stimulation immature DCs turn into mature DCs, which present exogenous antigens together with their MHC class I molecules and then activate the antigen-specific CTLs. Although both TLR and CD40 stimulation appeared to provide the same effects on DC maturation, CD40-dependent CTL activation is much more potent than CTL activation through LPS stimulation. Despite their different outcomes, the factors that lead mature DCs to different functions remain largely undefined. In this study, we defined the transient maturation and subsequent deactivation of DCs by TLR stimuli, including those by LPS and CpG-ODN. In contrast, CD40 stimulation induced stable mature DCs that elicited sufficient CTL proliferation. The deactivated DCs, which we defined as "expired DCs," were phenotypically similar to immature DCs, except for their phenotype stability, MHC class I expression level and IL-10 production. Moreover, the functions of expired DCs were comparable to those of immature DCs in terms of CTL induction and tolerogenicity. These results may provide an explanation for the role of CD40 stimulation in antigen-specific CTL induction.