In vitro propagation of <Emphasis Type="Italic">Helleborus</Emphasis> species

A general in vitro cloning system was established for four Helleborus species: H. argutifolius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—based medium (Murashige and Skoog 1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The first results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in a solution of indole-3-butyric acid (IBA -3 mg l⁻¹) and 1-naphthaleneacetic acid (NAA-1 mg l⁻¹) at 5°C.     

Pleiotrophin Gene Therapy for Peripheral Ischemia: Evaluation of Full-Length and Truncated Gene Variants

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.     

Assessment of exploited fish species in the Lake Edward System,East Africa

The unknown status of inland fish stocks hinders their sustainable management. Therefore, increasing stock status information is important for sustainable inland fisheries. Fisheries reference points were estimated for five exploited fish species (11 stocks) in the Lake Edward system, East Africa, which is one of the most productive inland water systems. The aim was to ascertain the status of the fisheries and establish reference points for effective management. The reference points were based on four linked stock assessment approaches for data-limited fisheries. Estimates showed poor stock status with the stocks defined as either collapsed, recruitment impaired or overfished. However, higher catches could be obtained under sustainable management. Estimates of maximum sustainable yield (MSY) and supporting biomass (B_msy) are provided for 10 of the stocks as targets for rebuilding plans. The immediate target of management should be rebuilding biomass to B_msy. Applicable measures include shifting length at first capture to the length that maximizes catch without endangering size structure and biomass, and livelihood diversification out of fisheries.     

Competitive inhibition by Rauber's sickle of the primitive streak and/or (pre)neural plate inducing effects of sickle endoblast in avian blastoderms

When in unincubated chicken blastoderms the Rauber's sickle is (sub)totally mechanically removed by selective scraping, the further evolution of the blastoderm in culture is often profoundly disturbed, going from only expansion of the upper layer and preneural plate formation to the development of a slowly growing miniature embryo. Our results suggest that the developmental potencies of the embryo are related to the presence or absence of Rauber's sickle material left after its removal. This can be checked after culture by the presence or nonpresence of junctional endoblast (derived from Rauber's sickle) and the concomitant induction of blood islands in the immediate neighborhood. Our study thus indicates that without Rauber's sickle (in the cases of successful total selective removal), an avian blastoderm cannot develop normally, even in the presence of an intact caudal marginal zone. After placing a fragment of quail sickle endoblast on the anti-sickle region of unincubated chicken blastoderms from which the Rauber's sickle was (sub)totally removed, different developmental scenarios were seen, according to the degree of removal, both in the anti-sickle as in the sickle regions. 1) If Rauber's sickle activity is strongly reduced, then besides a centripetally directed miniature embryo, induced by the remnants of the autochthonous Rauber's sickle, an additional centripetally directed embryo or preneural plate (without accompanying blood islands) develops in the anti-sickle region under inductory influence of the apposed quail sickle endoblast. We make a distinction between a neural plate and a preneural plate. The latter consists of a thickening of the upper layer (with the same initial aspect as a neural plate) adjacent to endophyll or sickle endoblast in the absence of chordomesoblast and gastrulation phenomena. 2) If Rauber's sickle activity is totally absent, then the inducing power of the sickle endoblast fragment becomes maximal and, starting from the anti-sickle region, one single embryo (without blood islands) extending over the whole area centralis appears. 3) If much of the Rauber's sickle material has been left in the blastoderm, then the inducing activity of the sickle endoblast, placed on the anti-sickle region, will be totally suppressed (although the sickle endoblast remains intact) and neither a preneural plate nor a primitive streak was induced. After placing a fragment of quail sickle endoblast on the anti-sickle region of an unincubated chicken blastoderm from which the Rauber's sickle and surrounding tissues were completely excised, an embryo was always induced by the sickle endoblast in the adjacent upper layer of this anti-sickle region. In the absence of sickle endoblast, this never occurred. Thus, our experiments demonstrate that in the absence of the Rauber's sickle, a parent tissue (sickle endoblast) induces both gastrulation and neurulation phenomena, while in the full presence of Rauber's sickle these functions are totally suppressed. Moreover, Rauber's sickle not only organizes gastrulation and blood island formation by itself but also influences neurulation at a distance (in space and time) by part of its cell lineage (i.e., sickle endoblast). Our study suggests that the inhibitory effect of Rauber's sickle on its parent tissue (sickle endoblast) represents an early mechanism impairing polyembryony, so that only a single primary major organizer (Rauber's sickle) remains active in the young avian germinal disc.     

In the absence of Rauber's sickle material,no blood islands are formed in the avian blastoderm

Using the quail-chick chimera technique, we followed the fate of Rauber's sickle cells in older whole blastoderms (cultured for approximately 2 days): after removal of the autochthonous Rauber's sickle from an unincubated chicken blastoderm, a quail Rauber's sickle was grafted isotopically and isochronically in its place. In transverse sections through these chimeras, the grafted quail Rauber's sickle cells were seen to have transformed into a broad row or ridge of quail junctional endoblast cells extending at the inner border of the area containing blood islands. After unilateral removal of the junctional endoblast from an intermediate streak chicken blastoderm (Stage 3; Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed during further in vitro culture that at the operated side, in the area previously occupied by this junctional endoblast, blood islands no longer developed. If after such a unilateral removal of the chicken junctional endoblast quail junctional endoblast was apposed in its place, then blood islands reappeared in the operated area. The intimate contact between the apposed quail junctional endoblast and the recently formed blood islands, derived from peripherally migrating mesoderm, was very obvious on sections through such chimeras. We further demonstrate that Rauber's sickle vs. junctional endoblast is indispensable for the anlage of blood islands in avian blastoderms. Indeed, in the absence of Rauber's sickle material no blood islands develop (even when mesoderm is present after ingression of the upper layer via a primitive streak) in the isolated central region of the area centralis of unincubated chicken blastoderms after culture in vitro. Also, no junctional endoblast and no sickle canal appear in these explants. By contrast, if a Rauber's sickle fragment is placed on such an isolated central blastoderm region, then blood islands develop. These blood islands start to develop from peripherally migrating mesoderm in the neighborhood of the Rauber's sickle-derived junctional endoblast.     

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.     

The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis

Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wild-type lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.KEY WORDS: EPHA3, EPH receptor A3, GEMM, Adenocarcinoma, Lung morphogenesis     

Somatic embryogenesis from mature <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxburgh as basis for mass production of elite forestry bamboos

A reliable protocol for mass propagation via somatic embryogenesis in mature bamboos has been established using pseudospikelets of Bambusa balcooa. Fourty percent of the explants gave rise to multiple regenerants within 4 months. This conversion rate is sufficiently high to use the process in commercial mass production. Further, shoot apical meristems can also be used as primary explants without lost of efficiency. Regenerated plants were uniform and identical to the mother plant and to plants obtained by axillary branching with respect to growth characteristics and morphology. Furthermore, epigenetic changes could not be detected by Methylation Sensitive AFLP (MSAP). During the complete process no changes in ploidy level could be observed. The process allows for a cost reduction for this tropical bamboo for forestry of up to 57% compared to micropropagation via axillary branching. For the first time, a reliable process based on somatic embryogenesis has been developed that is well suited for commercial micropropagation of elite mature bamboos.