Tilted peptides: a motif for membrane destabilization (Hypothesis)

Cell life depends on the dynamics of molecular processes: molecule folding, organelle building and transformations involving membrane fusion, protein activation and degradation. To carry out these processes, the hydrophilic/hydrophobic interfaces of amphipathic systems such as membranes and native proteins must be disrupted. In the past decade, protein fragments acting in the disruption of interfaces have been evidenced: they are named the tilted or oblique peptides. Due to a peculiar distribution of hydrophobicity, they can disrupt hydrophobicity interfaces. Tilted peptides should be present in many proteins involved in various stages of cell life. This hypothesis overviews their discovery, describes how they are detected and discusses how they could be involved in dynamic biological processes.     

Coitinuous cell lines from embryonic tissues of ticks (Acari: Ixodidae)

Summary Six new cell lines were established in continous culture from embryonic tissues of ixodid ticks. Four were fromDermacentor variabilis and two fromD. parumapertus. The cells are mostly fibroblastic and diploid. Mosquito-borne viruses (Chikungunya, O'nyong, yellow fever, and St. Louis encephalitis) as well as tick-borne ones (Langat, Powassan, Colorado tick fever, Kemerovo, and Sawgrass) replicated in certain of these cell lines, but a nonvector-borne flavivirus, Modoc, did not. An undescribed virus fromD. occidentalis ticks, which could not be isolated in Vero cells or newborn mice, was readily isolated in theD. variabilis cell line.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.     

Differential response of satellite cells and embryonic myoblasts to a tumor promoter

Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) reversibly suppressed myotube formation and expression of acetylcholine receptors in cultures of Day 15 mouse embryo presumptive myoblasts, but was totally ineffective in cultures of adult mouse satellite cells. A subpopulation of TPA-resistant myogenic cells became apparent in cultures prepared from older embryos or newborn mice. Thus, limb presumptive myoblasts are a heterogeneous population, and part of the distinct TPA-resistant subpopulation may represent satellite cell precursors.     

Use of glycine as a non-enzymic procedure for separation of mouse embryonic tissues and dissociation of cells

A non-enzymic procedure for separation and cell dissociation of mouse embryonic tissues is described, utilizing solutions of either 1 or 0.5 M glycine with EDTA. Incubation of blastocysts in glycine resulted in decompaction of cells without complete dissociation. Post-implantation egg cylinders were readily separated into component tissue layers, with subsequent dissociation into single cells. Cell viability was high after such treatment, and all cell types adhered rapidly and spread out on plastic culture dishes. Parietal endoderm, visceral yolk sac and amnion tissues from 10th and 14th day embryos were incubated in glycine, with resulting cell dissociation and/or tissue separation. Changes in cell adhesion properties appear to take place during development of these tissues. PSA1 embryoid bodies readily separated into the outer ‘endoderm’ layer and inner embryonal carcinoma cell core after a brief incubation in glycine. However, this procedure was unsuccessful for dissociation of cells from a plastic or gelatin substrate in vitro, suggesting that cell-substrate adhesion in vitro may differ from that in vivo.     

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) ⁺ RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.Abbreviations HRGP(s) hydroxyproline-richglycoprotein(s) - DAP days after pollination The present work was supported by grants from Plan Nacional de Investigation Cientifica y Técnica (grant BI088-0242) and European Communities (grant BAP-374). L.R.-A. is the recipient of a fellowship from the Plan Nacional de Formación de Personal Investigador.     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs)

Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). We envision that during steady state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) possess a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). This supports that VSELs are related to epiblast-derived migrating PGC-like cells and, despite their pluripotent stem cell character, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors.     

The chicken alpha 1 (XI) collagen gene is widely expressed in embryonic tissues.

Complementary DNA and genomic DNA clones corresponding to the chicken alpha 1 (XI) collagen gene were isolated and characterized. These recombinant DNA clones covered 2667 base pairs of the mRNA and encode 624 amino acids of the triple helical region plus the entire carboxyl-terminal propeptide. Northern blot analysis showed a major band of approximately 6.5 kilobases and a minor band of approximately 7.5 kilobases. A combination of Northern blot and in situ hybridization analyses showed that, in addition to its presence in cartilage, this mRNA also is present in a wide variety of chicken noncartilaginous embryonic tissues including brain, heart, skeletal muscle, calvaria, and skin, but was not detected in liver. Type II collagen mRNA has also been detected at low levels in these same tissues. Also, similar to the mRNA for the alpha 1 chain for type II collagen, the alpha 1 (XI) collagen mRNA is detected in limb mesenchyme prior to condensation and differentiation of the core mesenchyme into cartilage.     

Differential distribution of poly(A)-containing RNA in the embryonic cells of Oncopeltus fasciatus: Analysis by in situ hybridization with a [3H]poly(U) probe

The regional distribution of poly(A)⁺ RNA was examined in the embryonic cells of the milkweed bug, Oncopeltus fasciatus, by in situ hybridization of histological sections with a [³H]poly(U) probe. As shown by a number of control experiments, this probe interacts specifically with poly(A) sequences preserved in the sections. Using this method, it was shown that labeling of periplasmic and vitellophage nuclei increases markedly early during syncytial blastoderm formation. At this time, label also increases in the vitellophage cytoplasm but not in the cytoplasm surrounding the blastodermal nuclei. Labeling continues to increase in the blastodermal nuclei during cellularization and germ band differentiation without a concomitant accumulation in the blastodermal cell cytoplasm. At the time of germ band invagination, the region of the most intense subcellular labeing shifts from the nucleus to the cytoplasm of the invaginated cells. This shift is not evident in the blastodermal cells which remain at the surface of the egg to become the serosa. In the serosa and the vitellophage energids, labeling then decreases as histogenesis proceeds. Significant labeling of the nuclei and cytoplasm of the invaginated germ band cells continues through germ layer formation. It is concluded that poly(A)⁺ RNA, probably synthesized de novo following oviposition, is subject to differential intracellular distribution in three types of Oncopeltus embryonic cells which may reflect cell-specific patterns of mRNA or poly(A) metabolism.     

Insulin-like 4 (INSL4) gene expression in human embryonic and trophoblastic tissues

Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues. Mol. Reprod. Dev. 51:123–129, 1998. © 1998 Wiley-Liss, Inc.