Cloning of Humanα-defensin-1(HNP-1) Gene and Construction of Its Eukaryotic Expression Vector

1 IntroductionDefensins are small cationic antimicrobial peptides that function in the host‘s innate immune system. The human defensin family includes three small peptides from the azurophil granules of polymorphonuclear cells named human neutrophil peptide (HNP)-1, HNP-2, HNP-3, which consist 5%-7% of the protein of human neutrophil. HNP-4 is approximately one hundred times less abundant. They demonstrate antibacterial, antifungal and antiviral properties in vitro. HNPs are important component of nonoxidat...     

Cloning of Human α-defensin-1 (HNP-1) Gene and Construction of Its Eukaryotic Expression Vector

1 Introduction Defensins are small cationic antimicrobial peptides that function in the host's innate immune system. The human defensin family includes three small peptides from the azurophil granules of polymorphonuclear cells named human neutrophil peptide (HNP)-1, HNP-2, HNP-3,which consist 5%-7% of the protein of human neutrophil. HNP-4 is approximately one hundred times less abundant. They demonstrate antibacterial, antifungal and antiviral properties in vitro. HNPs are important component of nonoxidative mechanism in macrophages, and can direct inactivate the enveloped viruses. Because only special cells express defensins. And it is hard to extract them naturally and the production is few. So researcher expect to obtain defensins highly through heterogenous expression by gene engineering technology. In order to express HNP-1, we cloned the cDNA of HNP-1 from human polymorphonuclear cells in peripheral blood, and constructed its eukaryotic expression vector, which provided a base for the further study on its mechanism of antimicrobial effect.     

Anti-CD137 monoclonal antibody promotes the direct anti-tumor effect mediated by peripheral blood-derived human dendritic cells in vitro

CD137,a costimulatory factor of TNFR family,is expressed on activated T cells and freshly isolated mousedendritic cells (DCs).To date,there are only limited data dealing with the expression and the effect of CD137 onhuman DCs.We report in this work that CD137 can coexpress with CD137L on immature peripheralblood-derived human DCs (9.77%).The mature DCs stimulated by LPS showed a much higher level of CD137expression (36.06%).Ligation of CD137 on the surface of DCs with anti-CD137 monoclonal antibody (mAb)could enhance the direct anti-tumor effect mediated by human DCs in vitro.The agonistic anti-CD137 mAb wasable to elevate by about 20% of the DC-mediated inhibition of tumor growth in five tumor cell lines.Theseresults indicate that the appliance of anti-CD137 mAb might be used as a new strategy for tumorimmunotherapy.Cellular & Molecular Immunology.2004;1(1):71-76.     

Construction of a Mammary-specific Expression Vector of Human α- defensin- 1 ( HNP- 1) Gene

1 Introduction Defensins, also called human neutrophil peptides(HNP), are small cationic peptides with broad antimicrobial activity[1]. Human defensins are highly abundant in the cytoplasmic granules of polymorphonuclear neutrophils. Alpha-defensin-1 is an important mediator in either innate immunity or anti-infection. It can be developed to be an ideal new type antibiotic and may provide a better solution for the present situation of extensive antibiotics-resistence. It is difficult to achieve amount of antimicrobial peptides from nature sources. Transgenic mammary gland bioreactors offer a safe and cost effective source to produce important proteins. The purpose of this study was to construct a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter and human α-defensin-1 (HNP-1) gene.     

DAB2IP 基因的研究进展

Human DAB2 interaction protein (DAB2IP) is a novel member of Ras GTPase-activating protein family. It interacts directly with disabled-2 protein (DAB2/DOC2) which suppresses growth of cancers derived from different tissues, including mammary, prostate and ovarian cancers. DAB2IP was identified as an immediate downstream effector mediated by DAB2/DOC2. DAB2IP and DAB2/DOC2 form a unique protein complex that has a negative regulatory effect on the Ras-mediated signal pathway. It is demonstrated that DAB2IP is a tumor suppressor gene inactivated by methylation in several cancers. This article reviews the structure and biological functions of DAB2IP gene as well as its potential roles in carcinogenesis and evolution.     

Recent advances in targeting the autotaxin-lysophosphatidate-lipid phosphate phosphatase axis in vivo

Extracellular lysophosphatidate(LPA) is a potent bioactive lipid that signals through six G-protein-coupled receptors.This signaling is required for embryogenesis,tissue repair and remodeling processes.LPA is produced from circulating lysophosphatidylcholine by autotaxin(ATX),and is degraded outside cells by a family of three enzymes called the lipid phosphate phosphatases(LPPs).In many pathological conditions,particularly in cancers,LPA concentrations are increased due to high ATX expression and low LPP activity.In cancers,LPA signaling drives tumor growth,angiogenesis,metastasis,resistance to chemotherapy and decreased efficacy of radiotherapy.Hence,targeting the ATX-LPA-LPP axis is an attractive strategy for introducing novel adjuvant therapeutic options.In this review,we will summarize current progress in targeting the ATX-LPA-LPP axis with inhibitors of autotaxin activity,LPA receptor antagonists,LPA monoclonal antibodies,and increasing low LPP expression.Some of these agents are already in clinical trials and have applications beyond cancer,including chronic inflammatory diseases.     

Lumican基因在大肠癌中的表达及其预后的关系

Objective To investigate the expression of Lumican in human colorectal cancer tissues and normal colorectal tissues, and its relationship with biological behavior and survival rate. Methods By using immunohistochemistry, we studied the Lumican expression patterns in 205 cases of colorectal cancers with rarious clinico-pathologic characteristics, and 50 normal colorectal tissues specimens. The relationship of Lumican expression with the biological behavior and survival of patients was evaluated. Results The expression rate of Lumican in normal colorectal tissues was 12.0%(6/50), and their expression was weaker expression. In sharp contrast, Lumican expression rate in colorectal cancer lesions was 75.61%(155/205). Lumican protein expression was closely related to the location of tumor, the depth of tumor invasion and histological types(x2 =16. 201, P = 0. 013;x2 = 9. 506, P = 0. 029), but not related to the number of metastatic lymph nodes and stage (x2 = 15. 457, P = 0. 079;x2 = 1. 887,P = 0. 596). The medium survival time in patients who had a tumor with negative, weak, postive and strong Lumican expression was 64.9、56.6、41.5 and 36.4 months, respectively (x2 =35. 066,P =0.000). Conclusion Normal and malignant colorectal tissues have unique Lumican expression patterns.Lumican might be served as an independent prognostic marker.     

利钠肽结构、受体的生理和病理作用

Five natriuretic peptides (NP) have been identified: A- type, B- type, C- type, D-type and V - type. AU of them have tinged- structures. The actions of the natriuretic peptides are modulated through their cognate receptors, which are named as NPR- A, NPR- B and NPR- C. The receptors are in-volved in the cGMP- dependent signaling cascades, which take part in many different physiological regulation,such as diureses, vasedilatation, depressing blood pressure, improving immunity, promoting development of bone, regulating nerve system and so on. At present, AhT and BNP have become a marker in diagnostic some cardiovascular diseases.     

The testis-specifically expressed gene Trim69 is not essential for fertility in mice

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis.The tripartite motif(TRIM)family proteins,most of which have E3 ubiquitin ligase activity,are highly conserved in mammals.They are involved in important cellular processes such as embryonic development,immunity,and fertility.Our previous studies indicated that Trim69,a testis-specific expressed TRIM family gene,potentially participates in the spermatogenesis by mediating testicular cells apoptosis.In this study,we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology.Here,we reported that the male Trim69 knockout mice had normal fertility.The adult knockout mice have shown that the appearance of testes,testis/body weight ratios,testicular histomorphology,and the number and quality of sperm were consistent with wild-type mice.These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility,and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis.This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.     

Characterization of Immunological Cross-Reactivity between Enterotoxigenic Escherichia coli Heat-Stable Toxin and Human Guanylin and Uroguanylin

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.     

Duodenal bicarbonate secretion in rats: stimulation by intra-arterial and luminal guanylin and uroguanylin

Aim: Uroguanylin and guanylin are endogenous ligands for guanylate cyclase C, an upstream regulator of the cystic fibrosis transmembrane resistance (CFTR) anion channel, and both peptides increase intestinal anion export in vitro. We have compared the effects of close intra‐arterial and luminal administration of uroguanylin and guanylin on duodenal bicarbonate secretion in vivo and studied the interactions with melatonin and cholinergic stimulation. Methods: Lewis × Dark Agouti rats were anaesthetized and a segment of the proximal duodenum with intact blood supply was cannulated in situ. Mucosal bicarbonate secretion (pH stat) was continuously recorded and peptides were infused intra‐arterially or added to the luminal perfusate. Results: Intra‐arterial (50–1000 pmol kg^?1 h^?1) as well as luminal administration (50–500 nmol L^?1) of guanylin or uroguanylin caused dose‐dependent increases in the duodenal secretion. Luminal administration induced more rapidly appearing rises in secretion and the two peptides induced secretory responses of similar shape and magnitude. The melatonin MT₂‐selective antagonist luzindole (600 nmol kg^?1) significantly depressed the response to intra‐arterial guanylins but did not affect secretion induced by luminal guanylins. Similarly, the muscarinic antagonist atropine (0.75 μmol kg^?1 followed by 0.15 μmol kg^?1 h^?1) abolished the response to intra‐arterial uroguanylin but caused only slight suppression of the response to luminal uroguanylin. Conclusions: Intra‐arterial as well as luminal uroguanylin and guanylin are potent stimuli of duodenal mucosal bicarbonate secretion in vivo. The response to luminal guanylins reflects an action at apical receptors. Stimulation by parenteral guanylins, in contrast, is under cholinergic influence and interacts with melatonin produced by mucosal enteroendocrine cells.     

Sudden infant death syndrome: is it a transepithelial transport disorder?

The first limiting factor of dietary zinc deficiency has been described as a loss of the protective role of zinc against auto-oxidation of membrane sulfhydryl (SH) compounds. It has now been established that the prohormones (nutriuretic peptides) of the intestinal guanylin family are activated extracellularly by conversion of cysteines in the peptide to disulfide bridges. The induction of uroguanylin mRNA is elevated in intestinal zinc deficiency and nutriuretic peptides regulate epithelial transport of salt and water. Nitric oxide (NO) is also a modulator of salt and water transport. The constitutive forms of nitric oxide synthase (cNOS) in neurons and endothelial cells are calcium-dependent. The inducible form of nitric oxide synthase (iNOS) is activated by bacterial entero-toxins and damaged mucosa with NO penetrating the cell and acting directly on guanylate cyclase. The activated receptor-guanylate cyclases initiating the intracellular cycle 3'-5' guanasine monophosphate (cyclic GMP) cascade in target cells results in a flux of chloride and water into the intestinal lumen. Most of the actions of NO are mediated by activation of cyclic GMP. High-altitude pulmonary edema (HAPE) is associated with a defect in transepithelial water transport. It is suggested that dietary zinc, by modulating thiol oxidation to disulfides in guanylin prohormones to active hormones, is associated with salt and water secretion such that the overworked heart in hypoxemia increases the production and release of natriuretic peptides to activate guanylate cyclase receptors in target tissue in sudden infant death syndrome.     

Guanylyl cyclase C is a specific marker for differentiating primary and metastatic ovarian mucinous neoplasms

Aims:  The aim was to assess the value of GCC in distinguishing primary ovarian mucinous neoplasms from metastatic mucinous adenocarcinomas with ovarian involvement. Guanylyl cyclase C (GCC) is a brush border membrane receptor for the endogenous peptides guanylin and uroguanylin, and the homologous diarrhoeagenic bacterial heat-stable enterotoxins that is selectively expressed by epithelial cells from the duodenum to the rectum, but not by normal epithelia of the stomach or oesophagus, or normal extramucosal cells in humans.
Methods and results:  Fifty ovarian tumours: 27 primary ovarian mucinous neoplasms (seven cystadenomas, 10 borderline tumours and 10 cystadenocarcinomas) and 23 metastatic mucinous adenocarcinomas with ovarian involvement [13 colorectal adenocarcinomas, four gastric adenocarcinomas, six appendiceal mucinous tumours (four adenocarcinomas, one with neuroendocrine features, and two appendiceal mucinous cystadenomas)] were studied. For primary ovarian mucinous neoplasms, 25 of 27 were negative for GCC. Twelve of 13 cases of colorectal adenocarcinoma (except for one neuroendocrine adenocarcinoma) were positive for GCC. Three of four appendiceal mucinous adenocarcinomas were positive for GCC in both the primary and metastatic tumours (except for one neuroendocrine adenocarcinoma). Two of two appendiceal mucinous cystadenomas were positive for GCC. Of four cases of gastric adenocarcinoma with ovarian involvement, only one (primary tumour) exhibited focal GCC staining.
Conclusions:  GCC is a useful marker for differentiating between primary and secondary ovarian mucinous neoplasms.     

Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer

IGF2 is the first gene discovered to be imprinted and expressed exclusively from the paternal allele in both human and mouse. IGF2 is also the first imprinted gene displaying loss of imprinting (LOI) or aberrant imprinting in human cancers. Evidently, LOI or reactivation of the maternal allele of IGF2 is associated with an increase of IGF2 expression that may subsequently play an important role in the onset of human cancers. The most important discovery was the association of LOI of IGF2 with the risk of developing human colorectal cancer. LOI occurs not only in colon cancer tissues, but also in matched normal tissues and peripheral blood cells. A pilot study indicated a significant relationship between LOI of IGF2 and family history as well as personal history of colorectal cancer, suggesting that LOI of IGF2 might be a valuable biomolecular marker of predicting an individual's risk for colon cancer. A recent epigenetic progenitor model suggested that human cancers might have a common basis that involves an epigenetic disruption of progenitor cells mediated by "tumor progenitor genes" and proposed that non-neoplastic but epigenetically disrupted progenitor cells might be an important target for cancer risk assessment and prevention.     

Alu element insertion in the MLH1 exon 6 coding sequence as a mutation predisposing to Lynch syndrome

Lynch syndrome (LS) is the most frequent cause of hereditary colorectal cancer. A subset of patients with a history of LS shows no causal germline pathogenic alteration and are identified as having Lynch‐like syndrome (LLS). Alu retrotransposons are the most abundant mobile DNA sequences in the human genome and have been associated with numerous human cancers by either disrupting coding regions or altering epigenetic modifications or splicing signals. We report a family first classified as having LLS by Sanger sequencing analysis. Next‐generation sequencing (NGS) analysis identified an AluY5a insertion in MLH1 exon 6 that led to exon skipping. This splicing alteration inducing a pathogenic frameshift was found in patients who developed colorectal adenocarcinomas. Retroelement insertion might thus be an important but underestimated mechanism of cancer genetics that could be systematically tested in patients with a phenotype suggesting LS to accurately assess family risk and surveillance approaches.     

Mutational analysis of the CASP6 gene in colorectal and gastric carcinomas

Failure of apoptosis is one of the hallmarks of cancer. As an execution-phase caspase, caspase-6 plays a crucial role during apoptosis. To explore the possibility that the genetic alterations of CASP, which encodes caspase-6, might be involved in the development of human cancers, we analyzed the entire coding region and all splice sites of the human CASP6 gene for the detection of somatic mutations in 100 colorectal carcinomas and 50 gastric carcinomas. Overall, we detected three somatic mutations of the CASP6 gene, including two missense mutations and one splice-site mutation. The mutations were observed in two of the 100 colorectal carcinomas (2.0%) and one of the 50 gastric carcinomas (2.0%). Of note, one colorectal carcinoma with the CASP6 mutation harbored CASP3 and CASP8 gene mutations as well. We also analyzed caspase-6 expression by immunohistochemistry, and found that caspase-6 was expressed in 60% of the gastric cancers and 90% of the colorectal cancers. This is the first report on CASP6 gene mutations in human cancers, and these data indicate that the CASP6 gene is occasionally mutated in gastric and colorectal carcinomas. Also, the data suggest the possibility that deficiency of caspase-6 expression might contribute to the pathogenesis of gastric cancers.     

Microsatellite instability in early onset and familial colorectal cancer.

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is often considered to be the most common form of inherited colorectal cancer, although its precise incidence is unknown. The clinical diagnosis of HNPCC relies on a combination of family history and young age of onset of colorectal cancer, but as many familial aggregations of colorectal cancer do not fulfil the strict diagnostic criteria, HNPCC might be underdiagnosed. The majority of HNPCC families have germline mutations in mismatch repair (MMR) genes, such as MSH2 or MLH1, so that HNPCC cancers characteristically exhibit DNA replication errors (RERs) at microsatellite loci. Although an RER positive phenotype in tumours can also result from somatic mutations in an MMR gene, the prevalence of RER + tumours should provide a maximum estimate of the incidence of germline MMR gene mutations in patients with early onset and familial colorectal cancer. We investigated colorectal cancers for RERs from (1) a population based study of 33 patients with colorectal cancer aged 45 years or less, (2) 65 kindreds with familial colorectal cancer which only partially fulfilled the criteria for the diagnosis of HNPCC, and (3) 18 cancers from 12 HNPCC kindreds. Seven of 33 patients (21%) with colorectal cancer aged 45 years or less had an RER + cancer, with only two of these having a clear family history of HNPCC. A greater proportion of RER + tumours (5/7) occurred proximal to the splenic flexure than RER - tumours (4/26; chi2 = 6.14, p < 0.025). RERs were detected in all 18 cancers from HNPCC patients but in only six of 65 non-HNPCC familial colorectal cancer kindreds (9%; chi2 = 52.2, p < 0.0005). These findings suggest that most cancers in patients diagnosed at 45 years of age or less and familial aggregations of colorectal cancer which do not fulfil HNPCC diagnostic criteria do not have germline mutations in MSH2 and MLH1. Hence population screening for germline mutations in these genes is unlikely to be an efficient strategy for identifying people at high risk of developing colorectal cancer.     

Overexpression of the obesity hormone leptin in human colorectal cancer

BACKGROUND: Leptin is an adipocyte-derived neurohormone, high levels of which are found in obese individuals. Leptin controls energy expenditure, acting in the brain, and regulates different processes in peripheral organs. Recent studies have suggested that leptin may be involved in cancer development and progression. AIMS: To analyse leptin expression in human colorectal cancer as well as in colorectal mucosa and colorectal adenomas. METHODS: Leptin expression was assessed by immunohistochemistry in 166 colorectal cancers, 101 samples of colorectal mucosa and 41 adenomas. Leptin concentration in colorectal cancer was correlated with selected clinicopathological features. RESULTS: Immunoreactivity for leptin was observed in 51.2% (85/166) of primary colorectal cancers. In adenomas leptin expression was observed in 14.6% (6/41) of studied cases. In normal mucosa, leptin was present at low levels, except in tumour bordering areas where its concentration appeared to reflect levels in the adjacent cancer tissue. Leptin expression in colorectal cancer significantly correlated with tumour G2 grade (p = 0.002) as well as with histological type (adenocarcinoma) of tumours (p = 0.044). CONCLUSIONS: Results indicate that leptin is overexpressed in human colorectal cancer, which suggests that the hormone might contribute to colorectal cancer development and progression.     

Somatic mutations of caspase-2 gene in gastric and colorectal cancers

There is mounting evidence that evasion of apoptosis is a hallmark of cancer. Caspase-2, which plays roles in both extrinsic and intrinsic apoptosis pathways, is considered a candidate tumor suppressor. The aim of this study was to explore the possibility that genetic alterations of caspase-2 gene are present in human cancers. In this study, we analyzed the entire coding sequences of human caspase-2 gene for the detection of somatic point mutations in 90 gastric carcinomas and 100 colorectal carcinomas by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). Of the cancers analyzed, two gastric cancers (2/90; 2.2%) and two colorectal cancers (2/100; 2.0%) harbored somatic missense mutations of caspase-2. The mutations consisted of p.V46M (at prodomain), p.S157L (at prodomain), p.R357K (at p13 subunit), and p.R397L (at p13 subunit). We expressed these tumor-derived mutants in 293 T cells and found that three of the mutants decreased cell death activity of caspase-2. Our data indicate that somatic mutation of caspase-2 is rare in gastric and colorectal carcinomas. However, functional data of the caspase-2 mutations also suggest that caspase-2 gene mutation might affect the pathogenesis of some gastric and colorectal cancers by inactivating cell death function of caspase-2.