Cathepsin Research Articles

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection.

Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.

A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells

Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory.

Corrigendum to “Structure of a Kunitz-type potato cathepsin D inhibitor” [J. Struct. Biol. 192(3) (2015) 554–560

Corrigendum to “Structure of a Kunitz-type potato cathepsin D inhibitor” [J. Struct. Biol. 192(3) (2015) 554–560

The application of a novel, cell permeable activity-based probe for the detection of cysteine cathepsins

Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.

Targets in systemic molecular targeted therapies for bone metastases and their pharmacologic agents

Tumor cells can secrete various cytokines which can enhance the activity of osteoclast in the bone microenvironment, and osteoclast can promote the release of many growth factors buried in bone matrix which would promote the growth and invasion of tumor cells. Thus, a ' vicious cycle' of bone destruction is developed in the bone metastatic microenvironment. Bone metastatic microenvironment facilitate this ' vicious cycle', while it also provides potential targets for the treatment of bone metastases. Osteoprotegerin, receptor activator of nuclear factor-κB and its ligand system are the typicality of molecular targets. Bone metastasis can promote the secretion of RANKL and the expression of OPG. The disbalance of RANKL/OPG is an important inducing factor for bone destruction. Many studies have shown that transforming growth factor-β which is produced by osteoclast plays an important role in mediating ' vicious cycle'. Src family tyrosine kinase, endothelin A receptor, matrix metalloproteinase, and cathepsin K are the potential targets of bone metastasis. Pharmacologic agents such as denosumab, can inhibit the ' vicious cycle' of bone metastasis. In addition to suppress bone destruction by Pharmacologic agents, they also can produce direct antitumor effect. They can delay the occurrence of skeletal related events, prolong the overall survival, and play an important role in patient's quality of life at last. Patients with bone metastasis have already benefited from systemic molecular targeted therapies, and further researches would be of great importance in improving patient therapeutically selections and enhancing the effect.

2'-Hydroxychalcones and their Cyclized Derivatives as Cathepsin L Inhibitors

2'-Hydroxychalcones and their Cyclized Derivatives as Cathepsin L Inhibitors

A novel missense mutation in cathepsin K (CTSK) gene in a consanguineous Pakistani family with pycnodysostosis.

Deficiency of cathepsin K (CTSK), a lysosomal cysteine protease, has been shown earlier as a cause of an autosomal recessive osteosclerotic skeletal dysplasia pycnodysostosis. The objective of the present study was to identify the potential sequence variants in CTSK gene in a large consanguineous Pakistani family with pycnodysostosis. Genotyping of 4 affected and 6 unaffected members of the family was performed using polymorphic microsatellite markers linked to CTSK gene on chromosome 1q21. To screen for pathogenic mutation, exons and splice junctions of CTSK gene were polymerase chain reaction amplified from genomic DNA and sequenced directly in an automated DNA sequencer. Microsatellite analysis showed linkage of the family to CTSK gene on chromosome 1q21. Sequence analysis revealed a novel missense mutation c.728G>A (p.G243E) in exon 6 of the CTSK gene. A novel missense mutation was identified in CTSK gene in a Pakistani family with 5 individuals affected with autosomal recessive pycnodysostosis.

Nephroprotective effect of arjunolic acid on cytosolic lipids and mitochondrial enzymes against cyclosporine induced renal complications in rats

Cyclosporine (CsA) has considerably modified the graft survival in solid organ and bone marrow transplantations. Renal toxicity is the major adverse effect of chronic CsA administration. Deterioration of renal function and renal histopathology are the basic elements of the diagnosis.The present study aimed at investigating the protective effects of Arjunolic acid (10mg/kg body weight) against Cyclosporine (25mg/kg body weight) on the activities of TCA cycle enzymes - SDH (succinate dehydrogenase), MDH (malate dehydrogenase) Acotinase, Citrate synthase and examined the activities of respiratory chain complexes I, II, III, and IV. Mitochondrial enzymes of NADH DH, ICDH and ?KG DH activities in cytosol, Lysosomal enzymes of ?-Cathepsin, ?-Glucuronidase, ?-Galactosidase, ?-D-N-Acetyl Glucosaminidase in cytosol, lipids of Cholesterol, Triglycerides (TG), Free fatty acids (FFA), and phospholipids were estimated. Urea, Uric acid, and Creatinine were also estimated in 24hrs urinary sample. Results showed that CsA caused a marked decrease in the level of SDH, MDH, Acotinase, Citrate synthase, Respiratory chain complexes of I, II, III, IV, NADH DH, ICDH, ?KG DH, in the kidney mitochondrial tissue homogenate and also urea, uric acid and creatinine in urine sample. The lysosomal enzymes of ?-Cathepsin, ?-Glucuronidase, ?-Galactosidase, ?-D-N-Acetyl Glucosaminidase in cytosol, lipids of Cholesterol, Triglycerides (TG), Free fatty acids (FFA), and phospholipids were found to be increased in the cytosol of kidney tissue homogenates. But AA successfully prevented the alterations of these effects in the experimental animals. Our study demonstrated that AA could protect the mitochondrial kidney tissues against CsA induced oxidative stress probably by increasing antioxidative defense activities.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages.

BackgroundParticulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.MethodsPrimary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss −/− mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.ResultsWe demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss −/− mice.ConclusionsThis study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0129-5) contains supplementary material, which is available to authorized users.

Renal cell carcinoma with t(6;11)(p21.2;q13)/MALAT1-TFEB fusion: a clinical and pathological analysis

To study the clinicopathologic features, immunophenotype, differential diagnosis and prognosis of renal cell carcinoma (RCC) associated with t(6;11)(p21.2;q13)/MALAT1-TFEB gene fusion. A total of 9 cases of such rare tumor were selected for clinicopathologic, immunohistochemical and molecular analysis, with review of literature. The age of the patients ranged from 21 to 42 years (mean=31.3 years). The patients included four men and five women. Histologically, 4 of the 9 cases studied showed classic morphologic features of TFEB RCC, with hyaline material, pigments and psammoma bodies frequently identified. The remaining 5 cases demonstrated uncommon morphology, mimicking perivascular epithelioid cell neoplasm, clear cell RCC, chromophobe RCC or papillary RCC. Immunohistochemical study showed that TFEB and vimentin were positive in all cases. Most of the tumors studied also expressed Ksp-cadherin, E-cadherin, CD117, HMB45, Melan A and Cathepsin K. CKpan showed immunostaining in only 1 case. The staining for TFE3, CD10 and CK7 were all negative. TFEB gene rearrangement was detected in all the 9 cases studied using fluorescence in-situ hybridization. MALAT1-TFEB fusion gene was identified in 2 cases by polymerase chain reaction and direct sequencing. TFEB RCC seemed to be an indolent tumor. During a mean follow-up of 31 months, none developed tumor recurrence, progression, or metastasis. TFEB fusion-associated RCC is a rare neoplasm, tends to occur in young age group and carries an indolent behavior. Diagnosis relies on clinicopathologic findings and immunohistochemical analysis. TFEB break-apart FISH assay is a reliable tool in confirming the diagnosis.

Papillon-Lefèvre syndrome: a case report and mutation analysis of the cathepsin C gene

Objective To analyze mutations in the cathepsin C (CTSC) gene in a patient with Papillon-Lefevre syndrome (PLS). Methods Clinical data were collected from a patient with PLS. Two milliliters of venous blood samples were obtained from the patient, his parents and 100 unrelated healthy controls separately. DNA was extracted from these blood samples, and PCR was performed to amplify all the 7 exons of the CTSC gene followed by direct DNA sequencing. Results Two heterozygous mutations were observed in the CTSC gene of the patient. One was a novel mutation c.824C > T at position 824 in the exon 6, which resulted in a substitution of ACC (threonine) by ATC (isoleucine) at codon 275 (p.T275I). The other one was the mutation c.1040A > G at position 1040 in the exon 7, causing the substitution of TAT (tyrosine) by TGT (cysteine) at codon 347 (p.Y347C). His father and mother carried the heterozygous mutation c.824C > T and c.1040A > G respectively. Neither of the two mutations was observed in the 100 healthy controls. Conclusions CTSC mutations are responsible for the clinical phenotype of PLS. Identification of the c.824C > T mutation extends the spectrum of mutations in the CTSC gene and provides a basis for genetic diagnosis of PLS. Key words: Mutation; Cathepsin C gene; Papillon-Lefevre syndrome

Abstract 16994: RNA Editing Controls Gene Expression in Atherosclerotic Heart Disease by Enabling HuR-mediated Posttranscriptional Regulation

Background: Adenosine to inosine (A-to-I) RNA editing is catalysed by ADARs (adenosine deaminases acting on RNA) and is an important posttranscriptional regulator of RNA metabolism. Although RNA editing is essential for life, its role in cardiovascular disease is unknown. Methods and Results: Next generation RNA-sequencing (RNA-seq) of human endothelial cells revealed that ADAR1 edits almost 25% of endothelial transcriptome, regulates gene expression (135 transcripts were at least 2-fold regulated after ADAR1 knockdown) and controls endothelial cell function. Among the highest edited transcripts is cathepsin S (CTSS), an extracellular matrix degradation enzyme with an established role in cardiovascular and inflammatory diseases. The number of edited nucleotides in the 3’UTR of CTSS mRNA was increased under hypoxic conditions and was strongly associated with increased CTSS expression (r=0.971, P=0.029). Most importantly, absence of RNA editing resulted in a 3-fold decrease of CTSS mRNA expression under hypoxic conditions. RNA-seq analysis of 8 patients with coronary heart disease (CHD) and 4 healthy subjects revealed that A-to-I RNA editing of CTSS 3’UTR is significantly increased in patients with CHD compared to control healthy subjects (P<0.001). Most importantly, a strong association was observed between RNA editing in the CTSS 3’UTR and mRNA expression in our cohort (r>0.7, P<0.001). Mechanistically, RNA-immunoprecipitation, mRNA stability, luciferase reporter assays, mutagenesis experiments and NMR spectroscopy revealed that silencing of ADAR1 completely inhibited RNA editing of the 3’UTR of CTSS mRNA, which consequently affected CTSS RNA secondary structure, HuR binding to the CTSS 3’UTR and finally CTSS mRNA stability and protein expression (P<0.001 for all). Taken into consideration that 82% of edited genes in 3’UTR are enriched for HuR binding sites, RNA editing may critically regulate HuR-mediated posttranscriptional regulation and thus gene expression. Conclusion: This study suggests a novel primate-specific mechanism that may account for the increased cathepsin S expression in ischemic/inflammatory diseases and it may serve as a prototypical example for the evaluation of RNA-based mechanisms in health and disease.

Association Between Polymorphism in the Human Cathepsin L (CTSL1) Promoter with Hypertension in the Uygur, Kazak and Han Populations in China.

To systemically investigate the association between the polymorphism (rs3118869) in cathepsin Lenzyme gene with hypertension in three ethnic groups (Han, Kazak and Uygur) in China. Case-control study. Department of Cardiology, The First Affiliated Hospital, Shihezi Medical College, Shihezi University and Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from January 2013 to May 2014. This case-control study included 1224 patients (422 Uygur, 425 Kazak and 377 Han individuals) with hypertension and 967 healthy unrelated individuals (339 Uygur, 337 Kazak and 291 Han individuals) as controls. The participants came from three ethnic groups (Han, Kazak and Uygur) which were recruited from Xinjiang Province of China. The polymorphism (rs3118869) of the human cathepsin Lgene was genotyped using the TaqMan 5' nuclease assay. Binary logistic regression was built to determine the association of polymorphism with hypertension. The genotype distribution of polymorphism was not significantly different in three ethnic groups. The rs3118869 polymorphism was significantly associated with Essential Hypertension (EH) in co-dominant model (A/C vs. C/C) in total people (OR = 0.697, 95% CI = 0.520 -0.932, p = 0.015), the same result was obtained in recessive model (C/C + A/C vs. A/A) in total people (OR = 0.689, 95% CI = 0.522 -0.910, p = 0.009). Similar finding of rs3118869 in recessive model (C/C + A/C vs. A/A) was also observed after adjusting the variable to the covariates age (OR = 0.629, 95% CI = 0.464 0853, p = 0.003). The study results indicate the A-allele of rs3118869 is a protective factor in hypertension.

Estrogen-deficient osteoporosis enhances the recruitment and activity of osteoclasts by breast cancer cells.

To reduce the burden of bone metastases, the pathophysiology of the metastatic niche should be elucidated and targeted. The aim of the present study was to assess the effect of tumor cells on osteoclast (OC) recruitment and activity in the presence of altered bone remodelling. Peripheral blood mononuclear cells (PBMC) were isolated from healthy and ovariectomized (OVX) rats and co-cultured with MRMT-1 rat breast carcinoma cells or with their conditioned medium for 1 and 2 weeks. Alamar Blue viability test, synthesis of cathepsin K, transforming growth factor-beta 1 (TGF-β1), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-7, MMP-9, FITC-conjugate phalloidin staining and tartrate-resistant acid phosphatase (TRAP) staining were evaluated. The results indicate that breast carcinoma cells induced different responses in PBMC derived from rats affected by estrogen deficiency osteoporosis (OP) in comparison with healthy ones, with a significant increase in proliferation rate, OC differentiation, synthesis of TNF-α, MMP-7 and MMP-9. The data support the "proof of concept" that OP due to estrogen deficiency might offer a receptive site for cancer cells to form bone metastases.

Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210.

BackgroundAutophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed.MethodsBMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining.ResultsThe experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages.ConclusionsAutophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0974-3) contains supplementary material, which is available to authorized users.

Picomolar aggregated, but not soluble, Aβ inhibits cathepsin b and l activities

Picomolar aggregated, but not soluble, Aβ inhibits cathepsin b and l activities

Autophagy-Regulated ROS from Xanthine Oxidase Acts as an Early Effector for Triggering Late Mitochondria-Dependent Apoptosis in Cathepsin S-Targeted Tumor Cells.

Cathepsin S (CTSS), which is highly expressed in various malignant tumor cells, has been proposed to promote tumor progression, migration, and invasion. CTSS inhibition not only blocks tumor cell invasion and endothelial tube formation but also induces cellular cytotoxicity. In our previous studies, we have observed that CTSS inhibition induces autophagy, which is responsible for up-regulating xanthine oxidase for early ROS generation and consequent cell death. However, whether the autophagy-regulated early ROS triggers apoptosis remains unclear. We conducted a long-term follow-up study to investigate the relationship between early autophagy and late mitochondria-dependent apoptosis. We demonstrated that early ROS generation is critical for mitochondria damage and the activation of intrinsic apoptotic pathway. Attenuating the early ROS level diminished later mitochondrial damage and downstream apoptotic signaling. Collectively, mitochondria-dependent apoptosis is regulated by autophagy-regulated early ROS, which serves as an early effector that triggers mitochondrial signaling for late apoptosis. The data emphasize the essential role of autophagy-regulated early ROS in triggering late apoptotic signaling.

Determinants of chronic obstructive pulmonary disease in patients with intermediate levels of alpha-antitrypsin.

We examined elderly heterozygotes for alpha-antitrypsin deficiency (phenotype MZ) to determine which of several factors might be associated with the development of chronic obstructive pulmonary disease (COPD). Elderly heterozygotes who smoked had a very high incidence of COPD (70 per cent), whereas homozygotes (phenotype MM) who smoked had a significantly lower incidence of COPD (35 per cent). There was no difference in the incidence of COPD between subjects with MM and MZ phenotypes who did not smoke. To determine if leukocyte enzymes were related to the development of COPD in elderly heterozygotes, we measured the activities of monocytic acid cathepsin, neutrophil acid cathepsin, neutral cathepsin, and elastase. The activities of these enzymes were not associated with the development of COPD or with smoking history in the heterozygotes. In conclusion, cigarette smoking seemed to be a significant determinant of the development of COPD in heterozygotes for alpha-antitrypsin deficiency, but the leukocyte enzyme actitivty that we studied was unrelated.

Tomato root exudates induce transcriptional changes of Meloidogyne hispanica genes

Meloidogyne hispanica is a polyphagous root-knot nematode of emerging importance that has the ability to infect a broad range of plants, and tomato ( Solanum lycopersicum L.) crops can be severely damaged. This study investigated whether tomato root exudates regulate the expression of five candidate parasitism genes previously identified and sequenced in M. Hispanica . These were; calreticulin ( crt-1 ), cathepsin L cysteine protease ( cpl-1 ), β -1,4 endoglucanase-1 ( eng-1 ), fatty acid retinol binding protein ( far-1 ) and venom allergen-like protein ( vap-1 ). One thousand M. hispanica second-stage juveniles (J2) were exposed overnight to tomato root exudates, obtained from the root systems of 4-week-old plants during 4 h of agitation in sterilized distilled water, and the relative expression of the parasitism genes was determined by quantitative real-time PCR. The cpl-1 , crt-1 , far-1 and vap-1 genes were differentially up-regulated ( P <0.05) in the pre-parasitic J2 after exposure to tomato root exudates, while expression of eng-1 was largely unaffected ( P =0.05) by the treatment. This results suggests that tomato root exudates induce changes in the expression of candidate parasitism genes. Gene silencing by RNAi will elucidate the exact function of these candidate parasitism genes in nematode penetration and survival. Identification of the plant signal molecules in the tomato root exudates responsible for the up-regulation of these parasitism genes may lead to the development of novel approaches for the management of M. hispanica .

Osteoclast differentiation of Raw264.7 cells expressing enhanced green fluorescent protein in vitro

To observe the osteoclast differentiation of Raw264.7 strain stably expressing enhanced green fluorescent protein (EGFP). Raw264.7 cells were transfected with EGFP-Lifeact gene via retrovirus. The G3 cell clone was obtained by limited dilution technique which stably expressed EGFP under the fluorescence microscope. The morphology of G3 cells were observed. The effects of transfection on receptor activator of nuclear factor kappaB ligand (RANKL)--induced osteoclastogenesis and bone resorbing function of G3 cells were examined by tartrate resistant acid phosphatase (TRAP) staining, immunoblotting detection of cathepsin K and bone pit resorption assay. The real-time images of podosome dynamics were taken by laser confocal microscopy during osteoclast differentiation of G3 cells. The Raw264.7 cells were successfully transfected with EGFP-Lifeact gene. The G3-EGFP cloned strain which could stably express EGFP even after 20 passages was constructed. There was no significant difference in morphology between G3-EGFP and wild Raw264.7 cells. The fusion rates of the transfection group and of the wild control group were (35±5)% and (39±5)%, respectively, which were not significantly different (P>0.05). The semi-quantitative ratio of cathepsin K/β-actin in the wild control group and in the transfection group was 0.83±0.07 and 1.02±0.08 (P>0.05), respectively. Bone pit results showed that the total area of the bone resorption was respectively 272,252±36,193 and 262,408±23,243 (P>0.05) and the number of the bone pits was respectively 320±51 and 339±55 (P>0.05). The photos of laser confocal microscopy showed the constant cell-cell fusion during osteoclast differentiation of G3-EGFP cells. In addition, the dynamic self-organized podosome initially assembled podosome clusts, then dynamic rings, finally formed the characteristic podosome belt pattern in mature osteoclasts. Enhanced green fluorescent protein high effectively expressed in Raw264.7. Biological character does not change after transfection.