Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF)

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in a rabbit. Two immunoassays were compared: a radioimmunoassay (RIA) using 125I aFGF and an enzyme immunoassay (EIA) using aFGF coupled to the tetrameric form of acetylcholinesterase (aFGF-AchE) as tracer. With EIA, the detection limit was 1.5 ng/ml, versus 2.2 ng/ml with RIA, while the dose at 50% was 5.9 ng/ml for EIA and 9.6 ng/ml for RIA. Using a modified EIA procedure where aFGF-AchE was added 2 h after the other reagents, the dose at 50% binding was 1.5 ng/ml. Examples of the performance of both immunoassays are presented for various brain extracts of different species including human. The aFGF content obtained by these methods correlates (CR = 0.987) with the values obtained by biological assay.     

Androgen-dependent messenger RNA(s) related to secretory proteins in the mouse epididymis

Total RNA from mouse epididymides was translated in a cell-free system derived from rabbit reticulocyte lysate. The androgen dependence of a highly represented mRNA(s) was detected. This mRNA(s) encoded for a band of Mr 26,000 visualized by denaturing gel electrophoresis (SDS-PAGE). No other mouse sexual tissues (testis, vas deferens and seminal vesicle), liver, kidney or striated muscle presented such a band. Furthermore, this mRNA(s) was restricted to the caput epididymidis. Two-dimensional gel electrophoresis (2D-PAGE) showed that this band of Mr 26,000 was composed of 6 basic polypeptides. This translated protein may correspond to a newly synthesized secretory protein of Mr 24,000. The concentration of translatable mRNA(s) encoding for the band of Mr 26,000 dropped to 31% of the normal level at 3 days after castration and to 7%, 20 days later. Administration of testosterone to 30-day-castrated mice partly reversed these changes: 32% of the control value was reached after 10 days of treatment. Accumulation of this mRNA(s) was also just detectable (7% of the mature level) at 10 days of age. The high levels attained by 20 days of age (60% of the control value) suggest that the greatest increase of accumulation for this mRNA(s) occurs during the prepubertal period.     

In vitro properties of various growth factors and in vivo effects

This article summarizes some of the data that have been accumulated on several growth factors. Biochemical and biological properties of the Epidermal, Fibroblast, Astrocytes and Tumor growth factors (EGF, FGF, AGF, TGF) and those of growth factors derived from Platelets (PDGF), Brain (BDGF, ECGF), Eye (EDGF) and Cartilage (CDGF) are reviewed, as well as the in vitro mechanism of action of EGF and PDGF. The in vivo effects of these growth factors, particularly the experiments achieved to understand the physiological or physiopathological significance are described. The potential interest of these molecules in pharmacology and their use as wound healing agents is discussed.     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.     

Tumor cells can escape DNA-damaging cisplatin through DNA endoreduplication and reversible polyploidy

Cancer chemotherapy can induce tumor regression followed, in many cases, by relapse in the long-term. Thus this study was performed to assess the determinants of such phenomenon using an in vivo cancer model and in vitro approaches. When animals bearing an established tumor are treated by cisplatin, the tumor initially undergoes a dramatic shrinkage and is characterized by giant tumor cells that do not proliferate but maintain DNA synthesis. After several weeks of latency, the tumor resumes its progression and consists of small proliferating cells. Similarly, when tumor cells are exposed in vitro to pharmacological concentrations of cisplatin, mitotic activity stops initially but cells maintain DNA duplication. This DNA endoreduplication generates giant polyploid cells that then initiate abortive mitoses and can die through mitotic catastrophe. However, many polyploid cells survive for weeks as non-proliferating mono- or multi-nucleated giant cells which acquire a senescence phenotype. Prolonged observation of these cells sheds light on the delayed emergence of a limited number of extensive colonies which originate from polyploid cells, as demonstrated by cell sorting analysis. Theses colonies are made of small diploid cells which differ from parental cells by stereotyped chromosomal aberrations and an increased resistance to cytotoxic drugs. These data suggest that a multistep pathway, including DNA endoreduplication, polyploidy, then depolyploidization and generation of clonogenic escape cells can account for tumor relapse after initial efficient chemotherapy.     

Live analysis of endodermal layer formation identifies random walk as a novel gastrulation movement

During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.     

Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel,potent and selective GABAA α5 inverse agonist series

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA_A α5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.     

Analysis of two human pre-ribosomal factors,bystin and hTsr1, highlights differences in evolution of ribosome biogenesis between yeast and mammals

Recent studies reveal that maturation of the 40S ribosomal subunit precursors in mammals includes an additional step during processing of the internal transcribed spacer 1 (ITS1), when compared with yeast Saccharomyces cerevisiae, even though the protein content of the pre-40S particle appears to be the same. Here, we examine by depletion with siRNA treatment the function of human orthologs of two essential yeast pre-ribosomal factors, hEnp1/bystin and hTsr1. Like their yeast orthologs, bystin is required for efficient cleavage of the ITS1 and further processing of this domain within the pre-40S particles, whereas hTsr1 is necessary for the final maturation steps. However, bystin depletion leads to accumulation of an unusual 18S rRNA precursor, revealing a new step in ITS1 processing that potentially involves an exonuclease. In addition, pre-40S particles lacking hTsr1 are partially retained in the nucleus, whereas depletion of Tsr1p in yeast results in strong cytoplasmic accumulation of pre-40S particles. These data indicate that ITS1 processing in human cells may be more complex than currently envisioned and that coordination between maturation and nuclear export of pre-40S particles has evolved differently in yeast and mammalian cells.