Evidence of programmed cell death in post-phloem transport cells of the maternal pedicel tissue in developing caryopsis of maize

We present cellular- and ultracellular-level studies here to show developmental programmed cell death (PCD) of placento-chalazal (P-C) cell layers in maternal pedicel tissue in developing caryopses of normal seed (Mn1) and in the invertase-deficient miniature (mn1) seed mutant in maize (Zea mays). PCD was evidenced by loss of nuclei and all subcellular membranous organizations in many P-C layers. The terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) stain that is diagnostic of apoptotic-like PCD identified spatially and temporally two distinctive subdomains, which coincided with nucellar and integumental P-C layers based on their developmental origins. The early phase of PCD in the nucellar P-C was TUNEL negative and was specific to only the fertilized caryopses, indicating that the signaling for PCD in these maternal cells originated in the zygotic tissues. In fact, the initiation of PCD coincided with endosperm cellularization and was rapidly and coordinately completed prior to the beginning of the major storage phase in endosperm. Cell shape in these cell layers was also influenced by the genotype of filial endosperm. The later phase of PCD was restricted to the integumental P-C layers underneath the nucellar cells and was TUNEL positive in both genotypes. The two subdomains of the P-C layers were also distinguishable by unique cell wall-associated phenolic compounds. Based on collective evidence, we infer that the nucellar PCD may have osmolytic etiology and may lead to activation of the post-phloem transport function of the P-C layer, whereas the integumental PCD was senescent related, in particular, protecting the maturing seed against microbes that may be transported from the maternal tissue.     

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.     

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.     

Neural differentiation of mouse embryonic stem cells grown in monolayer

To drive neural differentiation of mouse embryonic stem (ES) cells, various culture protocols have been previously developed that all require the formation of embryoid bodies, usually combined with a treatment by all-trans retinoic acid (aRA). Here, we investigated whether or not neural differentiation can also occur in a simplified monolayer culture. Mouse ES cells were plated in serum-containing DMEM media with and without aRA and grown under these conditions for 2 days. Then, the cells were transferred to fresh serum-containing DMEM media and/or to serum-free DMEM/F12 media supplemented with a mixture of insulin, transferrin, selenium, and fibronectin (ITSF) for further culture. The changes in cell morphology and in the expression of selected molecular markers were monitored. Generally, in contrast to all the others, the protocol consisting of a 2-day culture in serum-containing DMEM followed by continuous exposure to the ITSF supplement in DMEM/F12 drove a vast majority of ES cells to generate phenotypic signs of neural lineage. Altogether, neural differentiation can be achieved in vitro without the step involving the formation of embryoid bodies as well as the treatment by aRA.     

Pax6 heterozygous eyes show defects in chamber angle differentiation that are associated with a wide spectrum of other anterior eye segment abnormalities

The development of the chamber angle was studied in the eyes of heterozygous Pax6(lacZ/+) mutant mice (Nature 387 (1997) 406). Mutations in PAX6 cause aniridia, a condition that is frequently associated with glaucoma, a blinding disease that may be associated with chamber angle defects. Mesenchymal cells were seen in the chamber angle at P1-P5. In wild-type mice, these cells differentiated into typical trabecular meshwork (TM) cells next to Schlemm's canal. In Pax6(lacZ/+) mice, TM cells remained undifferentiated and Schlemm's canal was absent. From P1 to P4, staining for beta-galactosidase and immunoreactivity for Pax6 were observed in chamber angle mesenchyme, but were absent later. Cultured murine TM cells expressed Pax6. The defects in chamber angle and TM differentiation were associated with a wide spectrum of other anterior eye defects, which included various degrees of iris hypoplasia and corneal haze, isolated iridocorneal adhesions and atypical coloboma, and a vascularized cornea in all adult animals. A third of the animals showed Peters' anomaly including corneal opacity and iridocorneal adhesions. The separation of the lens from the cornea was incomplete, and epithelial layers of lens and cornea were continuous. Pax6 activity is directly required for differentiation of the chamber angle. Variations in phenotype of Pax6(lacZ/+) mice appear not to involve direct dominant-negative or dose-dependent effects.     

Female sex pheromone of Cameraria ohridella Desch. and Dim. (Lepidoptera: Gracillariidae): structure confirmation,synthesis and biological activity of (8E,10Z)-8,10-tetradecadienal and some analogues

Mass spectrometric investigations confirmed the structure of the female produced sex pheromone of the horse-chestnut leafminer Cameraria ohridella Desch. and Dim. to be (8E,IOZ)-8,10-tetradecadienal. Pure samples, prepared in a straightforward synthesis, were highly attractive in field tests and proved to be suitable for monitoring of flight activities and population dynamics. In mixtures with the synthetic pheromone, analogues like 9-tridecynal and 7-dodecynyl formate were shown to reduce trap catches. In electroantennographic experiments, pheromone analogues were less active than the pheromone. 9-Tridecynal was the most EAG active analogue tested, followed by 7-dodecyn-1-yl formate and 7-undecyn-1-yl formate.     

The influence of the biomechanical parameters of the hip on the outcome of treatment of hips subject to avascular necrosis of the femoral head

We were interested in whether or not the biomechanical status of the hip influences the course of avascular necrosis of the femoral head. To investigate this, we used a computer aided system based on a three dimensional mathematical model for determining the stress distribution in the hip joint from standard anteroposterior rentgenographs (X-ray images) of both hips and pelvis. Based on the results of our study, we suggest that the biomechanical parameters of the hip play an important role in the outcome of treatment of hips affected by avascular necrosis of the femoral head.     

The origin of tobacco's T genome is traced to a particular lineage within Nicotiana tomentosiformis (Solanaceae)

Nicotiana tabacum (tobacco) is a natural allotetraploid. The maternal genome donor is not controversial and is probably derived from an ancestor of N. sylvestris. The paternal, T-genome donor has been less clear, with N. tomentosiformis, N. otophora, or an introgression hybrid proposed. Here we provide evidence that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. We show that the repetitive sequences of geminiviral origin, GRD53 and GRD3, are present in the genomes of N. tabacum cultivars, a tobacco cell suspension culture TBY-2, and N. tomentosiformis ac. NIC 479/84. Surprisingly, they are not present in another three varieties of N. tomentosiformis. A detailed cytogenetic analysis also revealed that N. tomentosiformis ac. NIC 479/84 most closely resembles the N. tabacum T genome in the location of other tandem repetitive sequences. Thus, tobacco formed after divergence within N. tomentosiformis, and the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats. It is clear that future paternity studies in tobacco should use N. tomentosiformis ac. NIC 479/84 rather than any other accession.     

Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors

The activities'of the lysosomal cysteine proteinases cathepsin B and L are regulated by their endogenous inhibitors, stefins A and B, and cystatin C, and their imbalance may be associated with increased invasiveness and development of the malignant cell phenotype. The aim of this study was to investigate mRNA, protein and activity levels of the above proteins in relation to in vitro invasiveness and to the reported in vivo tumorigenicity of four human breast tumor cell lines: the spontaneously immortalized cell line MCF10A, its c-Ha-ras transfectant MCF10AT, and two tumorigenic derivative cell lines, MCF10AT-Ca1a and MCF10AT-Ca1d. Invasiveness did not correlate with tumorigenicity, since the MCF10AT cell was the most invasive and the remaining three were at about half of its level. Cathepsin B expression paralleled the in vitro invasiveness through matrigel at all levels of expression, but cathepsin L did not. Stefin levels were elevated several-fold in the tumorigenic cell lines, but not in MCF10AT. The hypothesis that cathepsin B plays an active role in the invasion of breast cancer cell lines was confirmed by the fact that synthetic cysteine proteinase inhibitors, particularly those selective for cathepsin B, significantly reduced the invasion of the MCF10AT cells.