Differential expression of the TFF-peptides xP1 and xP4 in the gastrointestinal tract of Xenopus laevis

TFF-peptides (formerly P-domain peptides, trefoil factors) represent major secretory products of the mammalian gastrointestinal tract. A molecular cloning approach revealed the existence of two TFF-peptides, xP1 and xP4, also in the stomach of Xenopus laevis. Here, the localization of these two peptides by Western blot analysis as well as immunohistochemistry is presented. xP1 is found predominantly in the surface mucous cells of the stomach, whereas xP4 is mainly localized to a specific population of goblet cells in the esophagus, to mucous neck cells of the stomach, and to closely resembling cells in antral glands. xP4 in the esophagus and in the stomach differ by their N-glycosylation patterns. Compared to mammalian TFF-peptides, xP1 obviously represents the frog homologue of human TFF1 (formerly pS2) and xP4 seems to be the amphibian equivalent of human TFF2 (formerly hSP).     

The P2X(7) receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes

The purinergic P2X(7) receptor is an ATP-receptor channel predominantly expressed in immune cells. P2X(7) has been cloned from human, rat and mouse. Here we report cloning of the Xenopus laevis P2X(7) receptor (xP2X(7)). xP2X(7) is only about 50% identical to the mammalian homologues, shows a broad tissue expression pattern, and has the electrophysiological characteristics typical of a P2X(7) receptor: low agonist affinity (EC(50) about 2.6 mM) and a non-desensitizing current. Moreover, expression of xP2X(7) in Xenopus oocytes is sufficient to induce the formation of a large pore, which is permeable to large cations such as NMDG(+). Identification of a non-mammalian P2X(7) receptor may help to identify functionally important parts of the protein.     

Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.     

Molecular medicine of TFF-peptides: from gut to brain

TFF-peptides (i.e. TFF1, TFF2, TFF3; formerly P-domain peptides, trefoil factors) have been established as secretory products typical of the gastrointestinal tract. Their synthesis has recently been recognized in a number of mucin-producing epithelial cells, for example, of the respiratory tract, the salivary glands, the uterus and of the conjunctiva. They have a pivotal role in maintaining the surface integrity of these delicate epithelia as constituents of mucus gels as well as by their anti-apoptotic properties and their motogenic activity modulating cell migratory processes. The latter is important for rapid healing in particular of gastrointestinal and respiratory epithelia by a process termed "restitution". On the other hand, one of these peptides--namely TFF3--has been detected as a new neuropeptide of the human hypothalamo-pituitary axis where it is synthesized in oxytocinergic neurons of the paraventricular and supraoptic nuclei. From there it is transported to the posterior pituitary where it is released into the blood stream. Synthesis of TFF-peptides also occurs pathologically as result to chronic inflammatory diseases, for example of the gastrointestinal tract. Aberrant synthesis of TFF-peptides is observed in many tumors.     

The mouse one P-domain (pS2) and two P-domain (mSP) genes exhibit distinct patterns of expression

We have previously shown that the human pS2 gene, which codes for a secreted peptide of 60 amino acids, is expressed in a number of human carcinomas, including carcinomas of the breast, the pancreas, and the large bowel. Strong pS2 gene expression was also observed in the normal gastric mucosa and in the regenerative tissues surrounding ulcerous lesions of the gastrointestinal tract. A number of pS2 similar peptides, designated as P-domain peptides, have been described, notably the porcine (PSP), murine (mSP), and human (hSP) spasmolytic polypeptides, which correspond to duplicated pS2 proteins. We have now cloned a mouse homolog of the human pS2 cDNA to dispose of an animal model to study the pS2 protein function, which remains unknown at the present time. We show that the mouse putative pS2 protein sequence and the physiological pattern of expression of the mouse pS2 gene are well conserved. The mouse pS2 gene is highly expressed in the stomach mucosa cells, whereas no pS2 gene expression could be detected in the mouse mammary gland, even during postnatal development processes dependent on growth factors or hormones. Using in situ hybridization, we show that although coexpressed in the fundus, the antrum and the antrum-pyloric regions of the stomach, the mouse pS2 and mSP genes exhibit distinct and complementary cellular patterns of expression.     

A signal peptide secretion screen in<Emphasis Type="Italic"> Fucus distichus</Emphasis> embryos reveals expression of glucanase,EGF domain-containing,and LRR receptor kinase-like polypeptides during asymmetric cell growth

Zygotes of the brown alga Fucus distichus (L.) Powell develop polarity prior to the first embryonic cell division and retain a pattern of asymmetric growth during early embryogenesis. In order to identify F. distichus polypeptides secreted during asymmetric cell growth, we used a functional assay in Saccharomyces cerevisiae to screen a cDNA library generated from asymmetrically growing Fucus embryos for sequences encoding polypeptides that function as signal peptides for secretion. We isolated and sequenced 222 plasmids containing Fucus cDNAs encoding signal peptide activity. The cDNA inserts from these plasmids were translated in silico into 244 potential polypeptide sequences, 169 of which are predicted to contain signal peptides. BlastP analysis of the Fucus sequences revealed similarity between many Fucus proteins and cell surface proteins that function in development in other eukaryotes, including epidermal growth factor (EGF)-like repeat-containing proteins, plant leucine-rich repeat (LRR)-receptor kinases, and algal -1, 3-exoglucanase. However, most of the isolated Fucus polypeptides lack similarity to known proteins. The isolation of cDNAs encoding secreted Fucus proteins provides an important step toward characterizing cell surface proteins important for asymmetric organization and growth in fucoid embryos.Abbreviations ECM extracellular matrix - EGF epidermal growth factor - ER endoplasmic reticulum - LRR leucine-rich repeat - SST signal sequence trap - WAK wall-associated kinase     

Pineal antigonadotrophic substances: polypeptides and indoles.

Two categories of compounds, polypeptides and indoles, are discussed in terms of their potential importance as pineal antigonadotrophic substances. One of the polypeptides has been structurally identified as arginine vasotocin. Arginine vasotocin and other peptides of unknown structural identity have been found to be strongly antigonadotrophic in experimental animals. Besides melatonin, the gonad-inhibiting ability of several other pineal indoles including serotonin, 5-hydroxytryptophol, 5-methoxytryptophol and N-acetylserotonin was considered. In addition to its gonad suppressing ability, in three species of rodents melatonin has been shown to negate the antigonadotrophic effects of the pineal gland, i.e., it acts as a counter antigonadotrophic factor. A theory is presented in which the indoles may interact with the polypeptides in the control and/or the synthesis of the latter compounds. A mechanism for the secretion of pineal polypeptides is also considered. It is concluded that it is premature to classify any pineal constituent, be it a polypeptide or an indole, as $\text{the}$ antigonadotrophic factor of the mammalian pineal gland.     

B cell growth modulating and differentiating activity of recombinant human 26-kd protein (BSF-2, HuIFN-beta 2, HPGF).

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.     

Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors

In addition to acting as a physical barrier against microorganisms, the skin produces antimicrobial peptides and proteins. After wounding, growth factors are produced to stimulate the regeneration of tissue. The growth factor response ceases after regeneration of the tissue, when the physical barrier protecting against microbial infections is re-established. We found that the growth factors important in wound healing, insulin-like growth factor I and TGF-alpha, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human beta-defensin 3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. Both an individual and a synergistic effect of these growth factors were observed. These findings offer an explanation for the expression of these peptides/polypeptides in the skin disease psoriasis and in wound healing and define a host defense role for growth factors in wound healing.     

Protein and Peptide Factors from Bacillus sp. 739 Inhibit the Growth of Phytopathogenic Fungi

Thermolabile peptides inhibiting the growth of Helminthosporium sativum, a facultative phytopathogen, have been isolated from the low-molecular-weight fraction of extracellular metabolites of the strain Bacillus sp. 739. Paper chromatography of the fraction, followed by bioautography, revealed the presence of three components exhibiting antifungal activity. These components were separated by gel chromatography on Toyopearl HW-40. SDS-Na-PAGE (the Laemmli procedure) demonstrated that only one component was a protein (MW, 14 kDa). The other two substances were polypeptides with molecular weights less than 6 kDa each. The protein factor inhibited the growth of H. sativum with a minimum effective concentration of 0.1 to 0.2 mg/ml.     

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the alpha(v)-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.     

Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.     

Targeting tumor micro-environment for design and development of novel anti-angiogenic agents arresting tumor growth

Angiogenesis: a process of generation of new blood vessels has been proved to be necessary for sustained tumor growth and cancer progression. Inhibiting angiogenesis pathway has long been remained a significant hope for the development of novel, effective and target orientated antitumor agents arresting the tumor proliferation and metastasis. The process of neoangiogenesis as a biological process is regulated by several pro- and anti-angiogenic factors, especially vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, hypoxia inducible factor 1 and transforming growth factor. Every endothelial cell destined for vessel formation is equipped with receptors for these angiogenic peptides. Moreover, numerous other angiogenic cytokines such as platelet derived growth factor (PGDF), placenta growth factor (PGF), nerve growth factor (NGF), stem-cell factor (SCF), and interleukins-2, 4, 6 etc. These molecular players performs critical role in regulating the angiogenic switch. Couple of decade's research in molecular aspects of tumor biology has unraveled numerous structural and functional mysteries of these angiogenic peptides. In present article, a detailed update on the functional and structural peculiarities of the various angiogenic peptides is described focusing on structural opportunities made available that has potential to be used to modulate function of these angiogenic peptides in developing therapeutic agents targeting neoplastic angiogenesis. The data may be useful in the mainstream of developing novel anticancer agents targeting tumor angiogenesis. We also discuss major therapeutic agents that are currently used in angiogenesis associated therapies as well as those are subject of active research or are in clinical trials.     

Expression of spasmolysin (FIM-A.1): an integumentary mucin from Xenopus laevis

In the past, a unique type of precursor for a secretory protein was discovered. It contains a central repetitive domain rich in threonine residues and terminal cysteine-rich domains. Due to striking homologies of these terminal domains with pancreatic spasmolytic polypeptide, originally the name "prepro-spasmolysin" was proposed. Here we show that the mature protein has a MW of about 130 kDa, consisting of about 70% carbohydrate and 30% protein. Similar O-linked glycoproteins have been found in mucins from human intestine. For this and numerous other reasons we decided to rename this glycoprotein "frog integumentary mucin A.1" (FIM-A.1). Furthermore, analysis of the protein with specific antibodies against the predicted C-terminal end indicates that FIM-A.1 is probably not processed at pairs of basic residues. In situ hybridization as well as immunofluorescence studies revealed that FIM-A.1 is expressed and stored exclusively in mature mucous glands of Xenopus laevis skin. Only cone cells at the proximal part of these glands do not synthesize FIM-A.1. In contrast, all other physiologically active peptides from X. laevis skin investigated so far are synthesized in granular glands. A hypothetical function of FIMs for defense against microbial infections is discussed.     

Symplekin,a constitutive protein of karyo- and cytoplasmic particles involved in mRNA biogenesis in Xenopus laevis oocytes

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo- and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV-VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the "Cajal bodies." However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3'-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions.