Long noncoding RNA UCA1 promotes proliferation and metastasis of thyroid cancer cells by sponging miR-497-3p.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Long noncoding RNA UCA1 promotes proliferation and metastasis of thyroid cancer cells by sponging miR-497-3p, by H. Gao, J.-Y. Yang, L.-X. Tong, H. Jin, C.-Z. Liu, published in Eur Rev Med Pharmacol Sci 2020; 24 (2): 728-734-DOI: 10.26355/eurrev_202001_20052-PMID: 32016975" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20052.

Long noncoding RNA UCA1 promotes proliferation and metastasis of thyroid cancer cells by sponging miR-497-3p.

OBJECTIVE: Recently, long noncoding RNAs (lncRNAs) have attracted much attention for their roles in tumor progression. The aim of this study was to investigate the exact role of lncRNA UCA1 in the development of thyroid cancer (TC) and to explore the possible underlying mechanism. PATIENTS AND METHODS: UCA1 expression in both TC cells and tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Colony formation assay, cell proliferation, and transwell assay were conducted in vitro. Subsequent luciferase reporter gene assay was applied to investigate the underlying mechanism. Furthermore, the function of UCA1 in vivo was monitored as well. RESULTS: UCA1 expression level in TC samples was significantly higher than that of corresponding normal tissues. After UCA1 was knocked down in vitro and in vivo, the proliferation, migration, and invasion of TC cells were significantly inhibited. Moreover, the expression of miR-497-3p was repressed after the knockdown of UCA1. Furthermore, miR-497-3p was directly targeted by UCA1. CONCLUSIONS: Knockdown of UCA1 could inhibit TC cell proliferation and metastasis via sponging miR-497-3p. Our findings might offer a new therapeutic intervention for TC patients.

[Expression of nucleolar protein 14 and CD31 in pancreatic cancer mouse model and its correlation with tumor progression].

Objective: To investigate expression of nucleolar protein 14(NOP14) and CD31 in pancreatic cancer mouse model and its correlation with tumor progression. Methods: Clinicopathological data of 5 patients with pathologically confirmed pancreatic ductal adenocarcinoma(PDAC) and hepatic metastasis between January 2013 and December 2015 was collected in Department of General Surgery, Peking Union Medical College Hospital. Immunohistochemistry staining was employed to detect the expression of NOP14 in matched primary PDAC and relevant metastasis.Pancreatic cancer cells with NOP14 stably knocked down were established by transfecting lentivirus with NOP14 targeted silencing RNA.The inhibition efficacy was detected by quantitative real time PCR and western blot.Microvascular density(MVD) in pancreatic cancer transplantation mouse model was determined by CD31 immunohistochemistry staining analysis and correlated with NOP14 expression and tumor progression. Results: NOP14 had a significant higher expression in liver metastasis than primary pancreatic adenocarcinoma (2.09±0.45 vs. 1.31±0.27, P=0.028). NOP14 was knocked down 86 percent on mRNA level determined by qPCR and 78 percents on protein level detected by western blot. MVD was significantly decreased in NOP14-inhibited tumor from both pancreatic cancer cells subcutaneously and orthotopically grafted tumor mouse model with the value of 61.40±13.85 vs. 85.53±14.59 (P=0.041) and 38.33±10.91 vs. 59.33±15.37(P =0.037), respectively. Besides, MVD was positively associated with tumor volume(r=0.842, P<0.01) and metastasis (r=0.726, P=0.008). Conclusion: NOP14 presents higher expression in hepatic metastasis of pancreatic adenocarcinoma and might promote tumor progression by increasing microvascular density.

Effect of tobacco smoke on hydrogen sulfide-induced rat thoracic aorta relaxation.

Levels of hydrogen sulfide (H2S), a gaseous signaling molecule, are reduced in the serum of individuals who smoke. We hypothesized that tobacco smoke influenced smooth muscle relaxation by decreasing H2S levels and this effect could also influence expression of cystathionine γ-lyase (CSE) and sulfonylurea receptor-2 (SUR-2). The aim of this study was to explore the effect of tobacco smoke on H2S-mediated rat thoracic aorta relaxation and its possible mechanism. Thirty-two Sprague-Dawley rats were divided into four groups: control (C) group, short-term smoker (SS) group, mid-term smoker (MS) group, and long-term smoker (LS) group. H2S concentrations in serum, action of H2S on rat aortic vascular relaxation, and expression of CSE and SUR-2 in thoracic aortic smooth muscle were measured. Although there was no significant difference in H2S between the C and the SS groups, concentration of H2S was significantly reduced in both the LS and MS groups compared to control (P<0.01). Furthermore, H2S was significantly lower in the LS than in the MS group (P<0.05). Rat aortic vascular relaxation was lower in all three treatment groups compared to the control, with the most significant decrease observed in the LS group (P<0.05 compared to the MS group). Expression of CSE and SUR-2 was reduced in the LS and MS groups compared to control (P<0.05), with the lowest levels observed in the LS group (P<0.05). Therefore, tobacco smoke reduced expression of CSE and SUR-2 in rat thoracic aorta, which may inhibit H2S production and vascular dilation.

Alleviating the ischemia-reperfusion injury of donor liver by transfection of exogenous hTERT genes.

BACKGROUND: Ischemia-reperfusion (I/R) injury had been linked to primary graft dysfunction in transplantation. To find effective methods to alleviate donor liver injury from I/R, we transferred exogenous human telomerase reverse transcriptase (hTERT) genes into donor rats before liver transplantation. METHODS: SD rats (age, 16 months) were divided into 3 groups: group A were donors pretreated with exogenous hTERT gene; group B were donors pretreated with adenovirus vector only; and group C were donors pretreated with physiologic saline. Alanine aminotransferase (ALT), apoptotic index, telomerase activity, and histological evaluation were calculated after liver transplantation. RESULTS: The levels of ALT and apoptotic index of group A were significantly lower than those of group B or group C (P < .05), at the same time, a mild histological injury and increased telomerase activity were also observed in group A. CONCLUSIONS: Exogenous hTERT gene provides protection against I/R injury, which depends on exogenous hTERT gene-mediated inhibition of apoptosis.

Increased expression of type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) and its relationship with androgen receptor in prostate carcinoma.

Type 2 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) is a multi-functional enzyme that possesses 3alpha-, 17beta- and 20alpha-HSD, as well as prostaglandin (PG) F synthase activities and catalyzes androgen, estrogen, progestin and PG metabolism. Type 2 3alpha-HSD was cloned from human prostate, is a member of the aldo-keto reductase (AKR) superfamily and was named AKR1C3. In androgen target tissues such as the prostate, AKR1C3 catalyzes the conversion of Delta(4)-androstene-3,17-dione to testosterone, 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), and 3alpha-diol to androsterone. Thus AKR1C3 may regulate the balance of androgens and hence trans-activation of the androgen receptor in these tissues. Tissue distribution studies indicate that AKR1C3 transcripts are highly expressed in human prostate. To measure AKR1C3 protein expression and its distribution in the prostate, we raised a monoclonal antibody specifically recognizing AKR1C3. This antibody allowed us to distinguish AKR1C3 from other AKR1C family members in human tissues. Immunoblot analysis showed that this monoclonal antibody binds to one species of protein in primary cultures of prostate epithelial cells and in LNCaP prostate cancer cells. Immunohistochemistry with this antibody on human prostate detected strong nuclear immunoreactivity in normal stromal and smooth muscle cells, perineurial cells, urothelial (transitional) cells, and endothelial cells. Normal prostate epithelial cells were only faintly immunoreactive or negative. Positive immunoreactivity was demonstrated in primary prostatic adenocarcinoma in 9 of 11 cases. Variable increases in immunoreactivity for AKR1C3 was also demonstrated in non-neoplastic changes in the prostate including chronic inflammation, atrophy and urothelial (transitional) cell metaplasia. We conclude that elevated expression of AKR1C3 is highly associated with prostate carcinoma. Although the biological significance of elevated AKR1C3 in prostatic carcinoma is uncertain, AKR1C3 may be responsible for the trophic effects of androgens and/or PGs on prostatic epithelial cells.

Transient atypical monocytosis mimic acute myelomonocytic leukemia in post-chemotherapy patients receiving G-CSF: report of two cases.

Granulocyte colony-stimulating factor (G-CSF) is now widely used in patients with malignant disorders receiving intensive chemotherapy to increase leukocyte count and to upregulate phagocyte function during neutropenia. Monocytosis associated with G-CSF has been reported in anecdotal literature. We report two cases of pseudoleukemia secondary to G-CSF administration. Both patients initially presented with myelodysplastic syndrome with chromosome 7 abnormalities that evolved into acute myeloid leukemia. Case one had deletion 7q while case two initially had monosomy 7 and subsequently developed a balanced translocation between the short (p) arm of chromosome 1 and long (q) arm of chromosome 15. Following the induction chemotherapy and G-CSF administration, both of these patients developed pseudoleukemia. Patient 1 had white blood cell (WBC) count of 26 x 10(9)/l with 72% monocytes, while patient two had WBC of 14.1 x 10(9)/l with 30% monocytes. In both patients the monocytosis resolved after the discontinuation of G-CSF therapy. In summary, patients treated with G-CSF should be followed closely. In those cases with pseudoleukemia discontinuation of the drug with no supplemental chemotherapy is probably enough to control the atypical monocytosis.

Expression of lymphomagenic oncogenes in T-cell lymphomas of HPV 16 transgenic mice.

We have previously established that a dimer repeat of the complete HPV 16 genome is sufficient to cause multiple organ malignancies, either carcinomas or T-cell lymphomas, in transgenic mice. Here, we report the expression of oncogenes supporting the notion that these tumors arose via multiple oncogenic pathways. In these mice, the transgenic HPV 16 genome cosegregated with the tumor phenotype. E6/E7 expression was observed in both carcinomas and T-cell lymphomas, while E2 expression was observed only in T-cell lymphomas. Some of the T-cell lymphomas revealed E2 expression alone, implying that oncogenic pathways of HPV other than the one involving E6/E7 existed in these transgenic mice. To establish that this is the case, expression of genes downstream from E6/E7 and oncogenes involved in T-cell lymphoma formation were analyzed. p53 mutations were observed in two of five tumors that lacked E6 expression. High levels of c-myc gene expression were observed in five of six tumors with E7 expression, suggesting that a pathway involving E7, inactivation of Rb, and activation of c-myc is important in tumorigenesis of HPV 16 in these transgenic animals. High levels of expression of the c-Pim gene were also noted in two of three c-myc-expressing T-cell lymphomas, suggesting cooperation between these two proto-oncogenes. Activation of Hox-11, Tal2/SCL-2, and Rbtn1/Ttg1 expression, which are highly associated with human T-cell acute lymphoblastic leukemia (T-ALL), was observed in three of three T-cell lymphomas with E2 expression but not E6/E7 expression, showing that pathways to tumor formation not involving E6/E7 exist in these transgenic animals. At least two oncogenic pathways to tumors in HPV 16 transgenic mice exist, one involving E6/E7 and c-myc and the other involving E2 and lymphomagenic oncogenes.

Characterization of the promoter region and genomic organization of GLI, a member of the Sonic hedgehog-Patched signaling pathway.

GLI is the prototype for the Gli-Kruppel gene family characterized by a consensus C2-H2 zinc finger domain and is believed to function as a transcription activator in the vertebrate Sonic hedgehog-Patched signal transduction pathway. Understanding GLI gene regulation may be of importance to understanding causes of human birth defects and cancer. To begin to understand the regulation of this developmentally important gene we have cloned the human GLI gene and functionally characterized its 5' flanking region. The GLI gene is composed of 12 exons and 11 introns and in the zinc finger coding region shares a highly conserved splicing pattern with several other Gli family members in both vertebrates and C. elegans. A major transcription initiation site was identified upstream of the GLI translation start site along with three minor transcription initiation sites. The region surrounding the transcription initiation sites lacks TATA and CCAAT consensus sequences, has a high GC content, includes a CpG island, and contains several GC boxes. A 487bp segment surrounding the transcription initiation sites increased expression of a luciferase reporter gene 15-fold in Tera-1 cells and was defined as the core promoter region of human GLI. In transgenic mice this region directed beta-galactosidase expression to the central nervous system on embryonic days 10.5-12.5 and to sites of endochondral ossification on embryonic days 12.5 and 13.5 in a pattern comparable to the endogenous expression pattern of mouse gli within these tissues. The previously identified gastrointestinal expression of gli was not driven by this region and may require elements outside of the core promoter. Sequence analysis of the 5' flanking region of the mouse gli gene and the full-length mouse gli cDNA demonstrated high homology with human GLI, suggesting conservation of GLI regulation and function.

GLI activates transcription through a herpes simplex viral protein 16-like activation domain.

Three proteins have been identified in mammals, GLI, GLI2, and GLI3, which share a highly conserved zinc finger domain with Drosophila Cubitus interruptus and are believed to function as transcription factors in the vertebrate Sonic hedgehog-Patched signaling pathway. To understand the role GLI plays in the Sonic hedgehog-Patched pathway and mechanisms of GLI-induced transcriptional regulation, we have characterized its transcriptional regulatory properties and contributions of specific domains to transcriptional regulation. We have demonstrated that GLI activates expression of reporter constructs in HeLa cells in a concentration-dependent manner through the GLI consensus binding motif and that a GAL4 binding domain-GLI fusion protein activates reporter expression through the GAL4 DNA binding site. GLI-induced transcriptional activation requires the carboxyl-terminal amino acids 1020-1091, which includes an 18-amino acid region highly similar to the alpha-helical herpes simplex viral protein 16 activation domain, including the consensus recognition element for the human TFIID TATA box-binding protein-associated factor TAFII31 and conservation of all three amino acid residues believed to contact directly chemically complementary residues in TAFII31. The presence of this region in the GLI activation domain provides a mechanism for GLI-induced transcriptional regulation.

Inhibition of RPE cell-mediated matrix adhesion and collagen gel contraction by crovidisin, a collagen-binding snake venom protein.

PURPOSE: Cell-mediated collagen gel contraction plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). Anti-adhesion therapy has been suggested as a promising strategy in the treatment of PVR. Crovidisin, a snake venom protein isolated from Crotalus viridis, has been shown to bind selectively to collagen and to inhibit collagen-induced platelet aggregation. In the present study, the effectiveness of crovidisin in inhibiting the attachment of retinal pigment epithelial (RPE) cells to collagen, and RPE cell-mediated collagen gel contraction, was evaluated. METHODS: Fluorescein isothiocyanate (FITC)-conjugated crovidisin was prepared and used to evaluate its binding affinity for collagen type I, fibronectin, vitronectin, and laminin. The inhibitory effect of crovidisin on RPE cell-mediated extracellular matrix attachment and collagen gel contraction was evaluated by cell adhesion and type I collagen gel contraction assays. The cytotoxic effect of crovidisin was examined with a cell proliferation assay, using the Alamar blue method. Flavoridin, an Arg-Gly-Asp-containing peptide from viper venom, was used for comparison. RESULTS: FITC-conjugated crovidisin bound selectively to collagen type I with high affinity. It did not bind to other matrix proteins, including fibronectin, vitronectin and laminin, nor to RPE cells. Crovidisin inhibited RPE cell attachment to type I collagen in a dose-dependent manner. This inhibitory effect was enhanced by the presence of flavoridin. Crovidisin also dose-dependently inhibited RPE cell-mediated type I collagen gel contraction. Crovidisin was non-toxic to RPE cells. CONCLUSIONS: Crovidisin, a snake venom-derived collagen-binding protein, possessing an inhibitory activity on RPE cell-collagen interaction and RPE cell-mediated collagen gel contraction, may be a useful tool for studying cell-collagen interaction, and a potential anti-adhesion therapeutic agent for ocular disorders in which cell-collagen interaction in involved, such as PVR.

Expression of human GLI in mice results in failure to thrive, early death, and patchy Hirschsprung-like gastrointestinal dilatation.

BACKGROUND: GLI is an oncodevelopmental gene in the vertebrate hedgehog/patched signaling pathway that is spatiotemporally regulated during development and is amplified in a subset of human cancers. GLI is the prototype for the Gli-Kruppel family of transcription factors, which includes the Drosophila segment polarity gene ci, the C. elegans sex-determining gene tra-1, and human and mouse GLI3, all of which contain a conserved domain of five C2-H2 zinc fingers. GLI3 mutations have been implicated in the mouse mutant extra toes, as well as in human Greig cephalopolydactaly syndrome and the autosomal dominant form of Pallister-Hall syndrome. As such, GLI and the vertebrate hedgehog/patched signaling pathway appear to play important roles in both normal development and neoplasia. MATERIALS AND METHODS: Since it is not known whether aberrant GLI expression is similarly linked to developmental disorders, we developed gain-of-function transgenic mice which express human GLI ectopically. RESULTS: Affected transgenic mice exhibit a phenotype of failure to thrive, early death, and Hirschsprung-like patches of gastrointestinal dilatation. The colons of affected mice have greatly attenuated smooth muscle layers and abnormal overlying epithelium. The density of myenteric plexuses is reduced in the colonic walls. The severity of the phenotype is related to the level of transgene expression. CONCLUSIONS: The transgenic mouse model supports a role for GLI in gastrointestinal development. As part of the vertebrate hedgehog/patched signaling pathway, GLI is essential to mesoderm and CNS ectoderm development and transgenic GLI expression affects neuronal, muscular, and epithelial cell differentiation in the gut. Expression of human GLI in mice results in impairment of enteric neuronal development and a Hirschsprung-like phenotype.

Measurement of glycoprotein IIb/IIIa blockade by flow cytometry with fluorescein isothiocyanate-conjugated crotavirin, a member of disintegrins.

The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Crotavirin, a potent platelet aggregation inhibitor purified from the venom of the snake Crotalus viridis.

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC50 (approximately 1.0 micrograms/ml, 0.11 microM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins, e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

The HPV 16 genome induces carcinomas and T-cell lymphomas in transgenic mice.

Human papillomavirus type 16 is highly associated with cervical carcinoma. Here we report families of transgenic mice produced by the microinjection of a dimer repeat sequence of the human papillomavirus type 16 genome. Thirty-two transgenic animals in four families developed multiple organ malignancies that appeared in middle age without other intervention. The tumor phenotype of poorly differentiated carcinomas or malignant lymphomas and the transgene cosegregate in these lineages. The tumors arise in the subcutaneous compartment, thoracic cavity, or abdomen; are widely metastatic; and grow rapidly in nude mice. No cervical lesions were identified in six females examined. No rearrangements in transgene E6/E7, E2, and E1 regions were found in tumor tissues, and the truncated E2 region, which was thought to play an important role in human cervical carcinogenesis, was not needed for tumorigenesis in these animals. The transgenic mice produce RNA from the E6/E7 open reading frames, which has been identified in both the carcinomas and the lymphomas, but RNA from the E2 open reading frame is present only in malignant T-cell lymphomas and not in carcinomas, hyperplastic lymphoid tissue, or normal lymphoid tissue.

Design and testing of novel cytotoxic polyamine analogues.

We designed three polyamine analogues, 1,14-diamino-N5-methyl-5,10- diazatetradecane (5me-4-4-4), 1,14-diamino-N5,N5-dimethyl-5,10-diazatetradecane (Q-Amm-4-4-4), and 1,14-bis-(ethylamino)-N5,N5-dimethyl-5,10-diazatetradecane (BE-Q-Amm-4-4-4), on the basis of computer modeling and physical-chemical studies of polyamine-DNA interactions. These analogues differ from natural polyamines and from one another in the charge distribution on their aliphatic backbone. We found that 10 microM 5me-4-4-4 did not inhibit growth and was not cytotoxic to the human brain tumor cell lines SF-767 and SF-126. The same concentrations of Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 inhibited cell growth and killed more than 90% of each cell type on day 7 of the experiment. BE-Q-Amm-4-4-4 was slightly more toxic than Q-Amm-4-4-4 in both cell lines. All three agents either decreased or completely depleted intracellular putrescine and spermidine. Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 each also lowered spermine. The fact that 5me-4-4-4 was nontoxic but that Q-Amm-4-4-4 was cytotoxic and inhibited growth suggests that the charge distribution along the surface of the aliphatic backbone of polyamines is important in determining growth inhibition and cytotoxicity.

Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry.

Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-IIIa complex on platelet membrane with a very high affinity (Kd, 10(-7)-10(-8) M). In this study, we analyzed GPIIb-IIIa complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-IIIa. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann's thrombasthenia were probed with FITC-disintegrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiproliferative properties of polyamine analogues: a structure-activity study.

A basis set of polyamine analogues was designed and synthesized. These compounds were used to initiate a systematic investigation of the role of chain length, terminal nitrogen alkyl group size, and symmetry of the methylene backbone in the antineoplastic properties of polyamine analogues. New synthetic methods predicated on our earlier polyamine fragment synthesis are described for accessing the tetraamines of interest. An unsymmetrically substituted diamine reagent, N-(tert-butoxycarbonyl)-N,N'-bis(mesitylenesulfonyl)-1,4-diaminobu tane, was developed for entry into unsymmetrical tetraamines. All of the tetraamines synthesized were first evaluated in a murine leukemia L1210 cell IC50 assay at 48 and 96 h. In an attempt to correlate this behavior with some aspect of polyamine metabolism, each compound was tested for its ability to compete with spermidine for the polyamine uptake apparatus, its impact on the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), and its effect on the polyamine-catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) and on polyamine pools. While there was no obvious correlation between the 48 and 96 h IC50's and the impact of the analogues on polyamine metabolism, there were other structure-activity relationships. Correlations were observed to exist between chain length and IC50's and between terminal alkyl substituents and impact on Ki, ODC, and AdoMetDC. Also, preliminary studies suggest a relationship may exist between the 48 and 96 h IC50 activities and the analogue's chronic toxicity in vivo. Finally, when the overall length of the polyamine backbone was held constant, the symmetry of the methylene chains of the polyamine fragments was shown to be unimportant to the compound's activity.

Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist.

By means of Sephadex G-75 and CM-Sephadex C-50 column chromatography and reverse-phase HPLC, a low molecular weight (Mr = 7500), cysteine-rich peptide, halysin, was purified from Agkistrodon halys (mamushi) snake venom. Halysin is a potent platelet aggregation inhibitor that concentration-dependently inhibited human platelet aggregation stimulated by ADP, thrombin and collagen (IC50 = 0.16 to 0.36 microM) without affecting platelet secretion. It was active in inhibiting platelet aggregation of platelet-rich plasma and whole blood. Halysin had no effect on thromboxane B2 formation of platelets or intracellular Ca2+ mobilization of Quin 2-AM loaded platelets stimulated by thrombin. It inhibited the fibrinogen-induced aggregation of elastase-treated platelets. Halysin concentration-dependently inhibited the 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50 = 0.16 microM). 125I-Halysin bound to resting platelets (Kd = 1.6 x 10(-7) M) and to ADP-stimulated platelets (Kd = 3.4 x 10(-8) M) in a saturable manner. EDTA, the Arg-Gly-Asp (RGD)-containing snake venom peptides trigamin and rhodostomin, Arg-Gly-Asp-Ser (RGDS), and Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val blocked both 125I-fibrinogen binding and 125I-halysin binding to ADP-stimulated platelets. The monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex blocked both 125I-fibrinogen and 125I-halysin binding, whereas 10E5 had no significant effect on halysin binding to ADP-stimulated platelets, indicating that 7E3 and halysin bind to an epitope which is different from that of 10E5. RGDS concentration-dependently inhibited 125I-halysin binding in a competitive manner. We determined the primary structure of halysin which is a single peptide chain of 71 amino acid residues. An RGD sequence appeared in the carboxy-terminal domain of halysin. Halysin showed about an 85% identical sequence with trigamin which is a specific antagonist of fibrinogen receptor associated with glycoprotein IIb-IIIa complex. In conclusion, halysin inhibited platelet aggregation by interfering with fibrinogen binding to the fibrinogen receptor of the activated platelets. The RGD sequence of halysin plays an important role in the expression of its biological activity.