Effect of Homologous Peptides of Differentiation Factors HLDF and PEDF on Preimplantation Development of Mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351–356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84–89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2–4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

Biological role of a neurotrophic factor fragment from pigment epithelium: structure-functional homology with a differentiation factor for the HL-60 cell line

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in its C-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41-46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm of Xenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with the C-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.     

The biological function of a fragment of the neurotrophic factor from pigment epithelium: Structural and functional homology with the differentiation factor of the HL-60 cell line

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in itsC-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41–46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm ofXenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with theC-terminal fragment split off tby the cleavage of the Leu³⁸⁰-Thr³⁸¹ bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.     

Different Mechanisms of Protective and Differentiative Activities of Homological Peptides TGENHR and TQVEHR

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.     

Biologically active fragment of the differentiation factor from HL-60 cell line. Identification and properties

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1 beta, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.     

A biologically active fragment of the differentiation factor of the HL-60 line cells: Identification and properties

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1β, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.     

Proteolytic Hydrolysis of the Antitumor Peptide HLDF-6-AA in Blood Plasma

Russian Journal of Bioorganic Chemistry - HLDF-6 hexapeptide, which corresponds to the 41TGENHR46 fragment of human leukemia differentiation factor (HLDF), shows a wide range of neuroprotective,...     

Neuroprotective effect of the hexapeptide HLDF-6 on neurons of rat hippocampus on the model of Alzheimer's disease in vivo and in vitro

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer's disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by administration of the peptide corresponding to the 25-35 sequence of beta-amyloid peptide (25-35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the orientation-investigation activity of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of neurons of the hippocampus and cerebellum of rats under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer's disease and in the medium of the culture of hippocampus neurons in the presence of the Abeta(25-35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.     

HLDF-6 peptide affects behavioral reactions and organism functions dependent on androgen hormones in normal and castrated male mice

The hexapeptide Thr-Gly-Glu-Asn-His-Arg (HLDF-6), which was first identified as an active fragment of the human leukemia differentiation factor (HLDF) molecule, displays differentiation-inducing, neuroprotective and anti-drug abuse activities. Most of its in vivo effects were revealed only on male animals. We have studied HLDF-6 effects on a variety of organism functions and behavioral reactions, which are known to be dependent on androgen steroid hormones, both on castrated and normal (sham-operated) animals. Male NMRI mice were castrated or sham-operated at the age of 55 days (after puberty). After that, HLDF-6 peptide was injected daily during 3 weeks, followed by behavioral, morphological and biochemical testing. HLDF-6 increased testosterone level (1.5- to 2-fold) both in sham-operated and castrated animals. Sexual activity and pain sensitivity, which are strongly reduced in castrates, were completely or partially recovered by HLDF-6. At the same time, the peptide caused some effects similar to castration in sham-operated animals: aggression and locomotor activity were decreased; oral grooming was prolonged. Morphological studies of accessory sex glands showed that HLDF-6 partially normalizes the morphology and functional activity of seminal vesicles in castrates, but it does not prevent castration-induced apoptosis of prostate epithelial cells. Based on these observations, we can assume that HLDF-6 peptide displays at least two effects on androgen hormones metabolism in males: it stimulates testosterone biosynthesis by both testes and adrenals and simultaneously inhibits its conversion to dihydrotestosterone (DHT), most probably by diminution of 5alpha-reductase isoform 1 mRNA expression.     

Neuroprotective effect of the hexapeptide HLDF-6 on rat hippocampal neurons on the in vivo and in vitro models of alzheimer’s disease

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer’s disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by injection of beta-amyloid peptide (25–35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the exploratory behavior of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of the rat hippocampal and cerebellar neurons under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer’s disease and in the medium of the culture of hippocampal neurons in the presence of the Aβ(25–35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.     

Oral—aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral–aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral–aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oralaboral axis during sea urchin development.     

Chromosomal control of early embryonic development in mice I. Experiments on embryos with autosomal monosomy

The effects of autosomal monosomy on early embryonic development were studied in mice with Robertsonian and reciprocal (non-Robertsonian) translocations. It was found that monosomy for autosomes 1, 2, 3, 5, 6, 16, 17 and 19 had substantially different effects on preimplantation development and survival of mouse embryos. Monosomy for autosomes 1, 3, 6, 16 and 19 does not affect cleavage, compaction and blastulation and in some cases is compatible with implantation. Most of these embryos, however, die as early blastocysts (Ms 3, 6 or 19) and some of them are eliminated at early postimplantation stages (Ms 1 or 16). The embryos with monosomy for autosomes 2, 5 or 17 can be identified during cleavage owing to the reduced blastomere number and pathological changes in the nuclei. Most of these monosomies do not survive beyond the morula stage. The results indicate that differential genetic activity of autosomes in mice becomes already evident in very early embryonic development. A hypothetical mechanism for homologous autosome activation at the onset of embryonic development in mice is suggested.     

The Administration of Semax and HLDF-6 Peptides to Rats Regulates Protein Synthesis Rhythm in Hepatocytes and Corrects Senescent Disturbances

Russian Journal of Developmental Biology - To clarify the organizing effect of Semax and HLDF-6 peptides on the kinetics of protein synthesis in hepatocytes, in addition to an in vitro study...     

HLDF-6-Amide Reduces the Cytotoxicity of Doxorubicin and Activates the Proliferation of Tetrahymena pyriformis

Russian Journal of Bioorganic Chemistry - How a number of biological effects of the peptide neuroprotector HLDF-6-amide work are still unknown and require further study. It was found that...     

The protease inhibitor, MG132, blocks maturation of the amyloid precursor protein Swedish mutant preventing cleavage by beta-Secretase

Amyloid (Abeta) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by beta- and then by gamma-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Abeta peptide secretion by interfering with gamma-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the beta-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented beta-secretase cleavage. This block in beta-secretase cleavage was not observed with clasto-lactacystin beta-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of beta-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of beta-secretase cleavage was immediate and did not affect alpha-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of alpha-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the beta-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in beta-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Abeta peptide secretion.     

The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link.

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.     

不同激活方法对牛体细胞核移植胚胎体外发育的影响（简报）

Effects of different activation methods on the cleavage and in vitro development of bovine somatic cloned embryos constructed by intracytoplasmic nuclear injection were compared. The results show that the cleavage and in vitro development rate were not different significantly for constructed embryos cultured in 6-DMAP comparing with those in 6-DMAP + cytochalasin B (CCB) after activation with Ionomycin. Culture duration (3 to 4 h) in 6-DMAP or 6-DMAP + CCB had no significant effects on the cleavage and in vitro development ability of reconstructed embryos. CCB addition in the activation medium was benefit to the development of constructed embryos, although the effect wasn't significant. Within 1 to 4 h, the longer interval duration of nuclear injection and reconstructed embryo activation was, the higher cleavage and the blastocyst development rate of reconstructed embryos were.