Genetic synthetic lethality screen at the single gene level in cultured human cells

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.     

Leafless autumnal-flowering geophytes in the Mediterranean region — phytogeographical,ecological and evolutionary aspects

Numerous Mediterranean geophytes are hysteranthous, i.e. they flower in a leafless stage during autumn. On the basis of their life cycles and other features two types can be recognized: In the Crocus type flowers with subterranean ovary develop at the start of the new reproductive cycle from an annual storage organ; seed dispersal and germination are delayed to the spring and the next autumn. In the Urginea type, flowers with supraterranean ovary originate from a perennial storage organ at the end of the life cycle; seed dispersal and germination follow flowering immediately. Similar rhythms exist in other seasonal climates. Field observations and some experiments demonstrate flower stimulation by temperature decrease or increase in different geophytes, and relationships between water supply and synanthous versus hysteranthous leaf development. The origin, ecological importance, and horticultural implication of the hysteranthous syndrome is discussed.     

THE CYTOTOXIC PRINCIPLE OF THE PHYTOFLAGELLATE PRYMNESIUM PARVUM

The cytotoxic events leading to lysis induced in Ehrlich ascites tumor (E.A.) cells by Prymnesium parvum cell extracts were followed microscopically and measured quantitatively as changes in E.A. cell volume, uptake of trypan blue, and release of macromolecular constituents from the cells. Cell swelling was the most immediate response to P. parvum cytotoxin, while cell death and lysis were later events distinguished by a decline in cell volume, uptake of dye, and appearance of cellular macromolecules free in the incubation medium. The pH and temperature were shown to affect the outcome of the lytic sequence. At either low pH or temperature, cells swelled but did not lyse until the pH or temperature was raised. On the other hand, cells swollen at the higher pH or temperature could be protected from lysis by lowering either the pH or the temperature.     

A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Galβ1–4GlcNAc) and LacdiNAc (GalNAcβ1–4GlcNAc) Motifs Found on Vertebrate and Insect Cells

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1–4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1–4GlcNAcβ(CH₂)₈CONH(CH₂)₂NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1–4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1–6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1–6 bond in LacNAcβ1–6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.     

Ontological differences in first compared to third trimester human fetal placental chorionic stem cells

Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.     

Partially hydrated pollen: taxonomic distribution, ecological and evolutionary significance

 The problem of the water content of pollen is reconsidered, especially the distinction between “partially hydrated pollen” (PH pollen), pollen with a water content greater than 30%, and “partially dehydrated pollen” (PD pollen), which has a water content of less than 30%. Both types have been found even in systematically contiguous groups or the same genus. Partially hydrated pollen, encountered in at least 40 families of angiosperms, has the advantage of germinating quickly, normally in a few minutes to less than an hour. Dispersal of highly hydrated pollen also occurs in orchids but for a different reason, i.e. to enable packaging of massulae. The disadvantage of pollen dispersed with a high water content is that water is readily lost and the pollen may desiccate and die unless it has biochemical or anatomical devices to retain water or phenological strategies, such as flowering when temperatures are not too high and when relative humidity is high. Most pollen of Gymnosperms and Angiosperms studied has, however, been found partially dehydrated. Received March 8, 2002; accepted April 8, 2002 Published online: November 14, 2002 Addresses of the authors: G.G. Franchi (e-mail: franchi@unisi.it), Department of Pharmacology, Università di Siena, Via delle Scotte 6, I-53100 Siena, Italy; M. Nepi (e-mail: nepim@unisi.it) and E. Pacini (e-mail: pacini@unisi.it), Department of Environmental Sciences, Università di Siena, Via P.A. Mattioli 4, I-53100 Siena, Italy; A. Dafni (e-mail: adafni@research.haifa.ac.il), Laboratory of Pollination Ecology, Institute of Evolution, University of Haifa, 31905 Israel.     

Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth-supporting tissues, alveolar bone, periodontal-ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM⁺), induced in vivo regeneration of all tooth-supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM⁺ induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio-temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM⁺.