Search

Mohammad Abul Kashem,1– 4 Xin-Yong Yuan,4 Lin Li,2 Joshua Kimani,5 Francis Plummer1 ,†Ma Luo1,2,4 1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada; 2JC Wilt Infectious Diseases Research Centre, Winnipeg, Mb, Canada; 3Department of Microbiology and Veterinary Public Health, Chittagong Veterinary and Animal Sciences University, Chittagong, Bangladesh; 4National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; 5Institute for Tropical and Infectious Diseases, University of Nairobi, Nairobi, Kenya†In memoriam (passed away on February 4, 2020).Correspondence: Ma LuoDepartment of Medical Microbiology, University of Manitoba, Winnipeg, MB, CanadaTel +204-789-5072Fax +204-789-2018Email maluo@canada.ca; Ma.Luo@umanitoba.caPurpose: TILRR (Toll-like interleukin-1 receptor regulator), a variant of FREM1 (Fras-related extracellular matrix 1), is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and inflammatory responses. It enhanced the expression of multiple genes in the NF-κB signaling pathway and promoted the production of multiple pro-inflammatory cytokines/chemokines. TILRR is an extracellular matrix protein and expressed in cells and tissues, and has never been considered to exist in the blood. The study aimed to identify circulating TILRR protein in human plasma as a biomarker of systemic inflammation.Methods and Results: We developed a multiplex bead array method (Bio-Plex) using 4 monoclonal antibodies targeting different protein domains of FREM1/TILRR to investigate whether TILRR can be detected in blood plasma. The results of the multiplex bead array method were validated by Western blot analysis of affinity-purified TILRR from patient plasma samples. We subsequently analyzed 640 plasma samples from women enrolled in the Pumwani Sex Worker cohort (PSWC) (Nairobi, Kenya). Our study showed that TILRR exists in all patient plasma samples, but its quantities vary greatly among the patients, ranging from 2.38 ng/mL to 5196.79 ng/mL. The plasma TILRR below 2.38 ng/mL can only be detected by affinity purification and Western blot analysis.Conclusion: Our in-house developed multiplex bead array method can successfully quantify TILRR protein in plasma samples. Because TILRR is an important modulator of many inflammation-responsive genes, it may be an inflammation biomarker in blood and play a role in modulating systemic inflammation.Keywords: TILRR, soluble biomarker, human plasma, mouse anti-FREM1 mAbs, multiplex bead array method, Western blot analysis

Clear cell renal cell carcinoma (ccRCC) is the most common and the most aggressive histopathological subtype of kidney cancer, with patients exhibiting high mortality rates for metastatic tumors. The Sonic Hedgehog (SHH) pathway serves a crucial role in embryonic development. The abnormal activity of SHH signaling is observed in a broad range of malignancies. However, its role in ccRCC is still undetermined. The aim of the present study was to assess the expression of the SHH pathway genes in ccRCC. Neoplastic and morphologically unchanged kidney tissues were obtained during radical nephrectomy from 37 patients with ccRCC. The SHH, PTCH1, SMO and GLI1 mRNA levels were assessed using the reverse transcription-quantitative PCR. Western blot analysis was used to assess the full-length and C-terminal SHH protein level. The mRNA levels of SHH, SMO and GLI1 were approximately 2-, 2,5- and 7-fold higher in ccRCC tissue compared with control kidney tissue, respectively. Correlational analysis between the mRNA levels of SHH pathway genes and patients' clinicopathological factors revealed decreased and increased mRNA levels of PTCH1 and SMO respectively, in tumor samples derived from older patients (age >62). Furthermore, the level of C-terminal SHH protein in ccRCC samples was significantly lower in a group of males compared with females. No correlation was exhibited between molecular data and patient survival. Western blot analysis indicated a ~3-fold higher level of SHH full-length protein, and a 4-fold lower level of the C-terminal SHH protein domain, in ccRCC tumor tissues compared with normal kidney samples. The current study indicated an involvement of the SHH pathway in ccRCC development.

Aucubin, an iridoid glucoside isolated from the leaves of Aucuba japonica and other herbs is a pharmacologically active natural compound that possesses anti-inflammatory, antioxidative, and other beneficial properties. This study aimed to evaluate the protective effect of aucubin against cisplatin (CP)-induced acute kidney injury in mice and the mechanism of its action. Aucubin was administrated to mice orally or intraperitoneally (i.p.) (1.5 and 5 mg/kg) for two consecutive days, two days after i.p. injection of cisplatin (11 mg/kg). Treatment with aucubin by both routes of administration ameliorated histopathological changes and reduced elevated serum markers of kidney injury (blood urea nitrogen and creatinine). CP administration increased renal expression of heme oxygenase-1 (HO-1) and 4- hydroxynonenal (4-HNE), as well as tumour necrosis factor-alpha (TNF-α), determined by western blot analysis, which was dose-dependently ameliorated by aucubin showing attenuation of CP-induced oxidative stress in kidneys. Moreover, aucubin reduced increased renal expression of cleaved caspase-3 and -9 and decreased poly (ADP-ribose) polymerase (PARP) cleavage, indicating the attenuation of CP-induced apoptosis in kidneys. Mechanistically, aucubin suppressed the activation of several signalling pathways involved in inflammation and apoptosis, including the immunohistochemically determined nuclear localisation of nuclear factor-kappa B (NF-κB) and by western blot analysis of signal transducer and activator of transcription 3 (STAT3), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and forkhead box O3a (FOXO3a) showing that aucubin suppresses CP-induced renal inflammation and suppresses CP-induced activation of ERK1/2, Akt/ mTOR, and FOXO3a signalling. The parenteral application was marginally but statistically more effective in reducing CP-induced kidney injury than oral administration. These findings suggest that aucubin acts as a protective agent against CP-induced nephrotoxicity, which should be further investigated.

Background This study is intended to clarify the influence of exogenous bovine lactoferrin (bLf) treatment on glycemic homeostasis, gene expression, and production of lactoferrin (Lf) in rats with experimentally induced diabetes. Methodology Fifty adult male rats were used; 15 rats were used as the normal control rats (group A) and were injected an intraperitoneal (I/P) with 1 ml of isotonic saline daily for 3 months. Thirty-five rats were administered I/P injection of 60 mg of streptozotocin per kilogram body weight to induce diabetes. Thirty streptozotocin-treated rats were selected and used as diabetic rats, and they were subdivided into 2 equal groups (15 rats each): the untreated diabetic group (group B) and treated diabetic group (group C). The rats in the diabetic group were administered an I/P injection of 1 ml of isotonic saline daily for 3 months, while rats in the diabetic treatment group were administered an I/P injection of 300 mg of bLf/kg body weight/day/3 months. At the end of the experiment, blood and renal tissue samples were collected from all rats. The levels of insulin, glucose, and lactoferrin in plasma were measured. Homeostatic model assessment for insulin resistance (HOMA-IR) was performed. The numbers of Lf-mRNA copies in renal tissue were assessed using quantitative RT-PCR. To measure Lf production in renal tissue, Western blot analysis was applied. Results The obtained data demonstrated that the treatment of diabetic rats with bLf maintained glycemic homeostasis at normal levels but increased the mRNA expression of renal Lf. Conclusion In diabetic rats, bovine lactoferrin treatment offers the potential for protection against incidences of insulin resistance (IR) by stimulating the suppressed expression and production of the LTF gene.

Background: Capsaicin (CPS) is a highly selective agonist of the transient receptor potential vanilloid type 1 (TRPV1) with a nanomolar affinity. High doses or prolonged exposure to CPS induces TRPV1 defunctionalization and, although this effect is currently used for the treatment of thermal hyperalgesia in chronic pain conditions, it is responsible of detrimental effects, such as denervation of sensory fibers. The aim of the present study was to formulate CPS loaded lipid nanocarriers (CPS-LN) in order to optimize CPS release, thus preventing TRPV1 internalization and degradation. Methods: CPS-LNs were formulated and characterized by in vitro studies. The activation of TRPV1 receptors after CPS-LN administration was evaluated by measuring spontaneous pain that was induced by local injection into the plantar surface of the mouse hind-paw. Moreover, the expression of TRPV1 in the skin was evaluated by western blot analysis in CPS-LN injected mice and then compared to a standard CPS solution (CPS-STD). Results: CPS inclusion in LN induced a lower pain response when compared to CPS-STD, further, it prevented TRPV1 down-regulation in the skin, while CPS-STD induced a significant reduction of TRPV1 expression. Conclusions: Drug encapsulation in lipid nanoparticles produced an optimization of CPS release, thus reducing mice pain behavior and avoiding the effects that are caused by TRPV1 defunctionalization related to a prolonged activation of this receptor.

Background/Aims: Approximately 10%-20% of patients with acute cardiovascular disease who have received coronary intervention suffer restenosis and high inflammation. The stent compound paclitaxel+hirudin was prepared for the treatment of post-intervention restenosis. This study aimed to explore the anti-inflammatory and anti-restenosis mechanisms of paclitaxel+hirudin with regard to the TLR4/MyD88/NF-κB pathway. Methods: Human coronary artery smooth muscle cells (HCASMCs) at 4-6 generations after in vitro culture were used as a model. Lipopolysaccharide (LPS) was used as an inducer to maximally activate the TLR4/MyD88/NF-κB inflammation pathway. After MyD88 knockdown and selective blocking of MyD88 degradation with epoxomicin, the effects of paclitaxel+hirudin stenting on key sites of the TLR4/MyD88/NF-κB pathway were detected using ELISA, Q-PCR, and western blot analysis. Results: LPS at 1 μg/mL for 48 h was the optimal modeling condition for inflammatory activation of HCASMCs. Paclitaxel+hirudin inhibited the levels of key proteins and the gene expression, except for that of the MyD88 gene, of the TLR4-MyD88 pathway. The trend of the effect of paclitaxel+hirudin on the pathway proteins was similar to that of MyD88 knockdown. After epoxomicin intervention, the inhibitory effects of paclitaxel+hirudin on the key genes and proteins of the TLR4-MyD88 pathway were significantly weakened, which even reached pre-intervention levels. Paclitaxel+hirudin affected the MyD88 protein in a dosage-dependent manner. Conclusion: The paclitaxel+hirudin compound promotes MyD88 degradation in the TLR4/MyD88/NF-κB pathway to reduce the activity of TLR4 and NF-κB p65 and to weaken the LPS-initiated inflammatory reactions of IL-1β, IL-6, and TNF-α.

The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the Western blot signal against the mouse Bcnt/Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.

Transcranial alternating current stimulation (tACS) is a neuromodulation procedure that is currently studied for the purpose of improving cognitive function in various diseases. A few studies have shown positive effects of tACS in Alzheimer’s disease (AD). However, the mechanism underlying tACS has not been established. The purpose of this study was to investigate the mechanism of tACS in five familial AD mutation (5xFAD) mouse models. We prepared twenty 4-month-old mice and divided them into four groups: wild-type mice without stimulation (WT-NT group), wild-type mice with tACS (WT-T group), 5xFAD mice without stimulation (AD-NT group), and 5xFAD mice with tACS (AD-T group). The protocol implemented was as follows: gamma frequency 200 μA over the bilateral frontal lobe for 20 min over 2 weeks. The following tests were conducted: excitatory postsynaptic potential (EPSP) recording, Western blot analysis (cyclic AMP response element-binding (CREB) proteins, phosphorylated CREB proteins, brain-derived neurotrophic factor, and parvalbumin) to examine the synaptic plasticity. The EPSP was remarkably increased in the AD-T group compared with in the AD-NT group. In the Western blot analysis, the differences among the groups were not significant. Hence, tACS can affect the long-lasting enhancement of synaptic transmission in mice models of AD.

Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell–matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies.

The aim of the present study was to investigate the possible mechanism of Janus kinase (JAK)-signal transduction and activator of transcription (STAT)/PSMβ3 signaling in the occurrence of asthenozoospermia. We examined seminal fluid samples from 30 cases of asthenozoospermia and 30 healthy controls. Sperm was collected using the Percoll density gradient centrifugation method. The expression of JAK, STAT and PSMβ3 mRNA was assessed by reverse-transcription quantitative PCR and the protein levels of p-JAK, p-STAT and PSMβ3 were measured by western blot analysis. The PSMβ3 mRNA and protein expression levels were also measured after application of a JAK inhibitor, AG-490, to the control group, with a FITC-labeled monoclonal rabbit anti-human PSMβ3 primary antibody. The cells were observed under a laser confocal microscope. The mRNA levels of JAK, STAT and PSMβ3 in asthenozoospermia were decreased significantly (P

Compounds were isolated from Ecklonia stolonifera Okamura, a marine brown alga widely consumed as food. Among the isolated compounds, 974-A was demonstrated for the first time to be a potent competitive inhibitor of mushroom tyrosinase activity towards l-tyrosine and l-DOPA (IC50 values = 1.57 &plusmn, 0.08 and 3.56 &plusmn, 0.22 &micro, M, respectively). Molecular docking simulations clarified that the hydroxyl residues of the isolated compounds formed hydrogen bonds with residues at the catalytic and allosteric sites of tyrosinase, while other residues participated in hydrophobic interactions. Moreover, 974-A, phlorofucofuroeckol-A and eckol reduced the cellular melanin content and tyrosinase activity, and downregulated the expression of melanogenesis enzymes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 in B16F10 melanoma cells. These compounds also effectively scavenged radicals at the cellular level. Thus, our results revealed that compounds isolated from E. stolonifera are potent tyrosinase inhibitors with potential applications in the cosmetic industry for treatment of hyperpigmentation and for the anti-browning effect in the agricultural field.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell cancer, characterized by the highest mortality rate among other RCC subtypes due to the occurrence of metastasis and drug resistance following surgery. The Von Hippel-Lindau tumor suppressor (VHL)-hypoxia-inducible factor 1 subunit α (HIF1A)/hypoxia-inducible factor 2α (HIF2A)-vascular endothelial growth factor A (VEGFA) protein axis is involved in the development and progression of ccRCC, whereas sunitinib, a tyrosine kinase inhibitor, blocks the binding of VEGFA to its receptor. The aim of the present study was to examine the possible association of the gene expression of VHL, HIF1A, HIF2A, VEGFA and tumor protein P53 (P53) in cancer tissue with the outcome of ccRCC patients who were treated with sunitinib as first-line therapy following nephrec-tomy. A total of 36 ccRCC patients were enrolled, 11 of whom were administered sunitinib post-operatively. Tumor and control samples were collected, and mRNA and protein levels were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. High mRNA and protein expression levels of HIF2A and VEGFA were found to be associated with shorter overall survival (OS) and progression-free survival (PFS) rates, as well as with unfavorable risk factors of cancer recurrence and mortality. Resistance to sunitinib was also observed; the OS and PFS rates were shorter (median OS and PFS: 12 and 6 months, respectively, vs. undetermined). Sunitinib resistance was associated with high HIF2A and VEGFA protein levels (b=0.57 and b=0.69 for OS and PFS, respectively; P

Previous studies have demonstrated that the expression of CARD10 is closely associated with the occurrence of tumors, and its role is mainly to promote tumor progression by activating the transcription factor NF-κB. However, the signaling pathway in renal cancer remains unclear. The objective of the present study was to investigate the ability of caspase recruitment domain 10 (CARD10) to regulate the NF-κB signaling pathway and promote the progression of renal cell carcinoma (RCC). Expression of CARD10 in ACHN, 786-O and HK-2 cells was evaluated via western blot analysis, as was the epidermal growth factor (EGF)-induced activation of NF-κB signaling pathway-related proteins in cells. The expression of CARD10 was inhibited by CARD10 short hairpin RNA transfection. Cell cycle analysis and MTT assays were used to evaluate cell proliferation. Cell apoptosis was analyzed via flow cytometry. The invasion of renal cell lines was detected via Transwell cell migration and invasion assays in vitro. The results showed that CARD10 expression was significantly higher in RCC cells than in normal renal tubular epithelial cells. CARD10 silencing inhibited the proliferation, invasion and migration of RCC cells. EGF stimulation upregulated the activation of the NF-κB pathway in RCC cells. Inhibition of CARD10 expression inhibited NF-κB activation in RCC cells. Taken together, these data suggested that CARD10 promotes the progression of renal cell carcinoma by regulating the NF-κB signaling pathway. Thus, this indicated that CARD10 may be a novel therapeutic target in RCC.

In this work, four phenolic compounds, N1,N5-di-[(E)-p-coumaroyl]-spermidine (1), p-coumaric acid (2), ferulic acid (3), and rutin (4) were isolated from n-butanol fraction of acetone extract of defatted adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) seed meal. Compound 1 was identified from adlay for the first time. The structures of these compounds were characterised by ESI-MS, 1H NMR, 13C NMR and UV–vis techniques. These compounds were evaluated for their anti-tumour activities against HepG-2, MCF-7 and CaCo-2 cancer cell lines by MTT assay. Of all purified compounds, compound 1 possessed the highest anti-tumour activity than other compounds with IC50 values between 46.34 ± 3.99 and 92.69 ± 5.63 µg/ml. Furthermore, activation of the caspase cascade in HepG-2 cells for intrinsic pathways caused by compound 1 were observed using western blot analysis. The result also showed that the expression of caspase-3, caspase-9, cytochrome c and Bax protein increased and the protein level of Bcl-2 decreased.

The aim of the present study was to investigate the PAQR3 gene expression and its methylation level in colorectal cancer tissues, as well as the association with colorectal cancer clinical data. In total, 54 cases of colorectal cancer tissue samples and normal adjacent tissue samples were collected between June, 2013 and July, 2014. RT-PCR and western blot analysis were used to detect the mRNA and protein levels of PAQR3 in colorectal samples, respectively. MSP was used to detect the methylation level of PAQR3 gene in colorectal samples, which was compared with colorectal data. The results showed that a decreased expression level of PAQR3 mRNA in colorectal cancer tissues and the expression reduction rate was 57.4% (31/54). Similarly, the expression level of PAQR3 protein was reduced in cancer tissues, and the reduction rate was 46.3% (25/54), while the protein expression reduction rate in cancer adjacent tissue was 5.6% (3/54), and the difference was statistically significant (P

Cross-reactive immunological material (CRIM) status is an important prognostic factor in patients with infantile Pompe disease (IPD) being treated with enzyme replacement therapy. Western blot analysis of cultured skin fibroblast lysates has been the gold standard for determining CRIM status. Here, we evaluated CRIM status using peripheral blood mononuclear cell (PBMC) protein. For 6 of 33 patients (18%) CRIM status determination using PBMC was either indeterminate or discordant with GAA genotype or fibroblast CRIM analysis results. While the use of PBMCs for CRIM determination has the advantage of a faster turnaround time, further evaluation is needed to ensure the accuracy of CRIM results.

In the present study, the in vitro cytotoxicity of daidzein was evaluated in human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. BEL-7402 cells were sensitive to daidzein treatment, with an IC50 value of 59.7±8.1 µM. Daidzein showed no cytotoxic activity toward A549, HeLa, HepG-2 and MG-63 cells. Daidzein increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Morphological and comet assays showed that daidzein effectively induced apoptosis in BEL-7402 cells. Additionally, daidzein caused cell cycle arrest at the G2/M phase in the BEL-7402 cell line. Daidzein downregulated the expression of Bcl-2, Bcl-x and Baid proteins and upregulated the levels of Bim protein in the BEL-7402 cells. The results demonstrated that daidzein induced BEL-7402 cell apoptosis through an ROS-mediated mitochondrial dysfunction pathway.

Epithelial cell transforming 2 (Ect2) protein is a member of the human diffuse B-cell lymphoma family of guanine nucleotide exchange factors, which activate the Ras homolog gene family of small GTPases; however, the clinical implications of Ect2 in colorectal carcinoma (CRC) are unclear. The present study aimed to determine the relationship between Ect2 expression and prognosis in patients with CRC. Western blot analysis and immunohistochemistry assays were used to determine the expression of Ect2 in CRC and paired non-cancerous tissues from 66 patients. The correlation between Ect2 expression and clinicopathological parameters was assessed using χ2 tests. Patient survival was determined using the Kaplan-Meier method and log-rank test. Cox regression was used for multivariate analysis of prognostic factors. Results demonstrated that Ect2 protein was highly expressed in human CRC samples [29/45 (64.45%)] and significantly correlated with a poor prognosis (P

Silencing of tumor suppressor genes by hypermethylation in gene promoter regions is a crucial mechanism in carcinogenesis. Gene methylation may be reversible and evaluated easily, thus providing a promising substrate for the development of biomarkers for the detection and prevention of cancer, including colorectal cancer (CRC). In the present study, the protein expression and methylation status of smooth muscle protein 22α (SM22α) was investigated in 78 cases of CRC and adjacent normal tissue. The aim of the study was to investigate the function of SM22α in the pathogenesis of CRC and to identify a candidate biomarker for the early detection of CRC. The methylation status of promoter of SM22α gene was detected using methylation-specific polymerase chain reaction. The protein expression of SM22α was evaluated using western blot analysis. The results demonstrated a significant decrease of SM22α protein expression in 50 (68.5%) cases of CRC compared with that in adjacent normal tissues (P

This study was designed to investigate the biological effects of fish collagen peptide (FCP) on human osteoblasts. Human osteoblasts were treated with 0.1% FCP, which was the optimal concentration confirmed by the increase in alkaline phosphatase activity. After one, three, five and seven days of culture, the number of FCP-treated cells increased significantly compared with untreated cells. In a real-time PCR analysis, the expression of osteocalcin, osteopontin, BMP-2 and integrin β3 mRNAs in FCP-treated cells showed increases compared with untreated cells after three days of culture. After seven days of culture, the expression levels of osteopontin and integrin β3 were still higher in the FCP-treated cells than in untreated cells. The production of osteocalcin, osteopontin and integrin β3 proteins in FCP-treated cells also showed increases after seven days of culture. Furthermore, FCP accelerated matrix mineralization in the cultures. The present study indicates the potential utility of FCP as a biomaterial., Dental Materials Journal, 32(2), pp.233-240; 2013

The outbreak of severe acute respiratory syndrome (SARS) in 2002 affected thousands of people and an efficient diagnostic system is needed for accurate detection of SARS coronavirus (SARS CoV) to prevent or limit future outbreaks. Of the several SARS CoV structural proteins, the nucleocapsid protein has been shown to be a good diagnostic marker. In this study, an ssDNA aptamer that specifically binds to SARS CoV nucleocapsid protein was isolated from a DNA library containing 45-nuceotide random sequences in the middle of an 88mer single-stranded DNA. After twelve cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, 15 ssDNA aptamers were identified. Enzyme-linked immunosorbent assay (ELISA) analysis was then used to identify the aptamer with the highest binding affinity to the SARS CoV nucleocapsid protein. Using this approach, an ssDNA aptamer that binds to the nucleocapsid protein with a Kd of 4.93 ± 0.30 nM was identified. Western blot analysis further demonstrated that this ssDNA aptamer could be used to efficiently detect the SARS CoV nucleocapsid protein when compared with a nucleocapsid antibody. Therefore, we believe that the selected ssDNA aptamer may be a good alternative detection probe for the rapid and sensitive detection of SARS.

Asbestos-induced mesothelioma is a challenging social problem in many countries, and oxidative stress via iron is closely associated with its carcinogenesis. Mesothelioma is thought to originate from the mesothelial cells that cover the somatic cavity such as pleural, pericardial and peritoneal cavities. They are single layered and so flat that it is extremely difficult to obtain pure mesothelial cells as control samples from experimental animals. Here we describe a novel method to collect mesothelial cells from animals by the use of simple equipments. Surface of the most organs including lung, spleen and liver are covered with a single layer of mesothelial cells. Scraping the surface of those organs with razor blades after snap-freeze in liquid nitrogen satisfactorily confers almost pure population of mesothelial cells. This simple method would be helpful for obtaining mesothelial control samples from animals to elucidate the molecular mechanisms of a variety of mesothelial pathology.

To study the function of the 49 kDa excretory-secretory (ES) protein gene (P49) of Trichinella, the genes was amplified by RT-PCR from RNA of Trichinella spiralis and Trichinella nativa and several Chinese Trichinella isolates of domestic animals, and sequenced after being cloned. The amplified products of these parasites produced bands of about 950 bp. The 97.2 % to 100 % nucleotides identity and 94.3 % to 100 % identity of deduced amino acids among P49 gene of these Trichinella strains showed the close relationship of these parasites. The P49 gene of T. nativa was cloned into the BamHI site of the prokaryotic expression vector pET-30a, and the recombinant vector was expressed. The expressed product was 40.8 kDa in size. In Western blot analysis, the expressed product was reactive to sera of mice infected with T. nativa, T. spiralis and their Chinese geographical strains.

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80 degrees C for at least 60 days. M30-Apoptosense plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.

Natural killer (NK) cell neoplasms are rare diseases. Frequent abnormalities of the tumor suppressor genes Rb, p53, p151NK4B, p161NK4A and p14ARF have been reported. However, no oncogenes associated with tumorigenesis of NK cell neoplasms have been reported so far. We analyzed the status of oncogenes including N‐ras, K‐ras, H‐ras, c‐myc, N‐myc and mdm2 by Southern blot, PCR‐SSCP, western blot analysis and immunohistochemical staining. We analyzed four cell lines derived from NK cell neoplasms and 31 clinical samples with five subclasses of NK cell neoplasms. We found no point mutations of the ras family genes. We detected no mutations in the c‐myc and N‐myc genes. No overexpression of c‐Myc protein was detected by western blot analysis. Although we found neither amplification nor rearrangement of the mdm2 gene, we found high expression of MDM2 protein in some cases by western blot analysis. Immunohistochemical staining confirmed the overexpression of MDM2 protein. We found 14 cases with overexpression of MDM2 protein out of 15 cases (93%) with four subclasses of NK cell neoplasms except chronic NK lymphocytosis. Our previous and these results suggested that the expression level of MDM2 protein is independent of the status of the p14ARF, p53, Rb genes. MDM2 protein might independently contribute to car‐cinogenesis of NK cell neoplasms. Although the number of the cases we analyzed was not large, alterations of ras and myc family genes may rarely contribute to tumorigenesis in NK cell neoplasms. In contrast, overexpression of MDM2 might be associated with tumorigenesis of NK cell neoplasms, especially aggressive subclasses.

We have evaluated whether cyclosporine A affects cell structure and cytoskeletal proteins of the thymus of Wistar rats. Immunohistochemical analysis showed that expression of the cytoskeletal proteins vimentin and desmin was much higher in epithelial cells, dendritic cells and lymphocytes in the thymus of treated rats than in untreated controls. Protein expression was observed as a positive condensation in a distinct area near the nucleus with a capping-like configuration. An ultrastructural study showed that the amount of cytoskeletal fibrillar structures was increased in the treated rats. The structures were assembled in a limited area of the cell with a nuclear capping-like configuration which was in agreement with the light microscopical observations. Immunoblotting analysis demonstrated that vimentin and desmin had a lower molecular weight in treated rats than in controls (57 and 53 kDa versus 55 and 51 kDa, respectively). The results clearly indicate that cyclosporine A affects the structure of the cytoskeleton suggesting that this could be the first step in its immunosuppressive effects by altering nucleus/cytoplasm signaling.

[Purpose] To report the clinical and immunological characterization of paraneoplastic retinopathy (PR) and to investigate the association between spectral-domain optical coherence tomography (SDOCT) findings and the targets of autoantibodies in PR. [Methods] We retrospectively enrolled eight patients (age range, 57–85 years; four men and four women) suspected of having PR. All patients underwent comprehensive ophthalmic examinations, including best-corrected visual acuity (BCVA) measurement, slitlamp examinations, kinetic visual field testing with Goldmann perimetry, electroretinography (ERG), fundus photography, fluorescein angiography, fundus autofluorescence (FAF), SDOCT, and serum sample tests (Western blot analysis and immunohistochemistry [IHC]). [Results] Three patients had a history of malignant tumors, and four patients were newly diagnosed as having neoplastic tumors (small cell lung carcinoma [SCLC], thymoma, pancreatic neuroendocrine neoplasm, and colon cancer). Another de novo malignancy (SCLC) was detected in a patient with a history of malignancy (bladder cancer and liposarcoma). The BCVA in these patients ranged from hand motion to 1.5. Goldmann perimetry revealed island, ring-shaped, concentric, or central scotoma. All patients showed nonrecordable or reduced amplitude results on ERG. Fluorescein leakage was detected in five patients. Hyperautofluorescence and/or hypoautofluorescence on FAF was detected in six patients. The serum sample tests identified anti-retinal antibodies in all patients. Patients whose serum contained anti-photoreceptor or anti–retinal pigment epithelium antibody on IHC showed damage of the outer retina on SDOCT. [Conclusions] In this case series, PR was associated with a variety of neoplasms and autoantibodies. Spectral-domain OCT can be used to characterize morphologic changes, and the changes were associated with the targets of autoantibodies.

MAGE genes encoding tumor antigens recognized by cytotoxic T lymphocytes are appropriate target molecules for specific immunotherapy of cancer. We have investigated whether the demethylating agent 5-aza-2'-deoxycytidine (DAC) induces MAGE-1, -2, -3, and -6 in normal and malignant lymphoid cells. DAC induced these MAGE genes in both PHA/interleukin-2 (IL-2)-activated T cells from healthy donors and MAGE-negative T and B cell leukemias in most cases. It also induced MAGE-1 in IL-2-dependent T cell clones and all MAGE genes tested in Epstein-Barr virus-transformed B cell lines. Expression of MAGE-1 protein in the cells was confirmed by western blot analysis with anti-MAGE-1 polyclonal antibody. Therefore, demethylation is a potent stimulus to induce MAGE genes in both normal and malignant lymphoid cells.

X-arrestin is a recently identified retina-specific gene of unknown function. Affinity-purified anti-peptide antibody to human X-arrestin was prepared, and used in Western blot analysis of human retinal proteins and for immunohistochemistry on human retinal sections. By Western blot analysis, the antibody specifically bound to an approximately 47 kDa protein, and by indirect immunofluorescence specifically labeled cone photoreceptors with greatest intensity in their outer segments. In single and double label experiments, the localization of X-arrestin immunoreactivity was compared with immunolabeling patterns obtained with antibodies to red/green cone opsin, rhodopsin, and S-antigen. The results showed that X-arrestin is expressed in red-, green- and blue-sensitive cones in the human retina.

Summary Cats exposed to feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) cannot be differentiated by serological analysis. Three synthetic peptides and anE. coli recombinant fusion protein generated from FIPV 79-1146 spike gene sequence were produced. Coronavirus positive cat sera reacted to peptide aa 950–990 but were non-reactive to aa137–151 and aa 150–180 peptides as demonstrated by ELISA. Amino acid sequence 97–222 expressed as a galk fusion protein inE. coli was tested against coronavirus positive cat sera by western blot analysis. Only sera from cats exposed to the FIPV type-II strains DF-2 or 79–1146 that were exhibiting signs of FIP recognized the fusion protein. Sera from FECV exposed cats did not recognize the 97–222 fusion protein in western blot analysis.

The capsular serotype has long been associated with the virulence of Streptococcus pneumoniae . Here we present an in-depth study of phenotypic and genetic differences between serotype 3 and serogroup 11 S. pneumoniae clinical isolates from both the general and indigenous populations of Australia. Both serotypes/groups included clonally unrelated strains with differences in well-known polymorphic virulence genes, such as nanA and pspA , as demonstrated by multilocus sequence typing and Western blot analysis. Nonetheless, the serotype 3 strains were consistently and significantly more virulent in mice than the serogroup 11 strains. Despite extensive genomic analysis, noncapsular genes common to one serotype/group but not the other were not identified. Nevertheless, following the conversion of a serotype 11A isolate to serotype 3 and subsequent analysis in an intranasal infection model, it was evident that both capsular and noncapsular factors determine the virulence phenotype in mice. However, it appears that these noncapsular factors vary from strain to strain.

The natural p300-specific histone acetyltransferase (HAT) inhibitor, curcumin (CUR), has been widely investigated for its potential therapeutic effect as an anticancer and anti-inflammatory agent. Notwithstanding this interesting pharmacological profile, CUR shows some drawbacks, such as poor absorption and a very fast metabolism and elimination, that limit its clinical use. Aim of the present study was to formulate CUR loaded nanostructured lipid carriers (NLC-CUR) in order to improve the bioavailability and stability of this compound after systemic administration with increased effects in the central nervous system (CNS). NLC-CUR were prepared and characterized on their physicochemical properties by PCS and DSC analyses. Thus, NLC-CUR were systemically injected and the effects in the CNS were compared with a CUR control formulation containing 0.05% DMSO (DMSO-CUR). Our results demonstrate that CUR is able to decrease histone acetylation in the CNS when included in NLCs. Western blot analysis shows that intraperitoneal injection of NLC-CUR (100mg/kg) in mice induces a marked hypoacetylation of histone 4 (H4) at lysine 12 (K12) in the spinal cord compared with control group. Notably, DMSO-CUR (100mg/kg) did not change the H4K12 acetylation level in the CNS. Our study suggests a novel approach to ameliorate the pharmacokinetics of CUR that allows a better permeation in the CNS.

Aim:Toxoplasma gondii infection during pregnancy poses a serious risk to the fetus, therefore timely and accurate diagnosis is essential. The aim of this study was to estimate the frequency of congenital infection via evaluating mother's immunological status and the possibility to improving the diagnostic and therapeutic approaches.Methods:Eighty five mothers with Toxoplasma seroconversion and their offspring were enrolled (among them, 2 spontaneous abortions were documented in the first trimester). Prenatal PCR diagnosis was carried out on 50 patients (60%), with 7 positive cases (14%). Morphological ultrasound scanning revealed anomalies in one fetus. Long-term follow-up included general physical examinations, serological status tested using Western blot, neuro-radiological, ophthalmologic and neurologic examinations, psychological and developmental tests, visual evoked potential tests and audiology tests, as well as anti-Toxoplasma treatment regimes.Results:Fourteen (17%) of the infants were infected at one-year serological follow-up. Chi-square for linear trend of vertical transmission from the first to the third trimester was significant (P=0.009). Western blot analysis showed IgM and IgA in half of the infected infants. In 69 uninfected infants, anti-Toxoplasma IgG immunoblot analysis excluded infection within the 3 months in 18 infants (26%) and in the others within 6 months of life. The most relevant instrumental findings are described.Conclusion:Western blot analysis may help to evaluate infection within the 6 months of life. The accuracy of ultrasound imaging to determine the brain damage in the fetus and newborns is doubtful, and should be combined with MR imaging. Multistep approaches can improve the timing of postnatal follow-up.

Cells belonging to the monocyte/macrophage lineage are in general highly resistant to peroxynitrite, a reactive nitrogen species extensively produced by these and other cell types under inflammatory conditions. Resistance is not dependent on the scavenging of peroxynitrite but is rather associated with the prompt activation of a survival signaling in response to various molecules largely available at the inflammatory sites, as arachidonic acid and products of the 5-lipoxygenase or cyclooxygenase pathways. We detected significant levels of Bad in the mitochondria of monocytes/macrophages and found that these signaling pathways converge in Bad phosphorylation, and thus in its cytosolic accumulation. Phosphorylation inhibits binding of Bad to Bcl-2, or BclXL, and promotes its translocation to the cytosol, thereby enabling Bcl-2 and BclXL to exert effects leading to prevention of mitochondrial permeability- transition (MPT). Upstream inhibition of the survival signaling indeed promotes the mitochondrial accumulation of Bad and the rapid onset of MPT-dependent toxicity. The above results contribute to the definition of the mechanism(s) whereby monocytes/macrophages survive to peroxynitrite in inflamed tissues.

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity of taking into account not only ability of flavonoids to bind to Ah receptor (induction factor) but also to concentrate on their distribution and metabolism (including colon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than μM. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.

In prion-related encephalopathies, microglial activation occurs early and is dependent on accumulation of disease-specific forms of the prion protein (PrPSc) and may play a role in nerve cell death. Previously, we found that different types of PrPSc (i.e. type 1 and type 2) coexisted in approximately 25% of patients with sporadic Creutzfeldt-Jakob disease (CJD); and a close relationship was detected between PrPSc type, the pattern of PrP immunoreactivity, and extent of spongiform degeneration. To investigate whether microglial reaction is related to the biochemical type and deposition pattern of PrPSc, we carried out a neuropathologic and biochemical study on 26 patients with sporadic CJD, including all possible genotypes at codon 129 of the prion protein gene. By quantitative analysis, we demonstrated that strong microglial activation was associated with type 1 PrPSc and diffuse PrP immunoreactivity, whereas type 2 PrPSc and focal PrP deposits were accompanied by mild microglia reaction. These findings support the view that the phenotypic heterogeneity of sporadic CJD is largely determined by the physicochemical properties of distinct PrPSc conformers.

As our group has shown, the NO-synthase inhibitor L-NNA decreased 2–3 times heat shock-induced synthesis of the heat shock protein HSP70 (FEBS Lett. 370 (1995) 159–162). It was suggested that NO is involved in such induction. In the present study, it was found that (1) injection of the NO donor dinitrosyl iron complex (DNIC) into rats results in accumulation of HSP70 in the heart; (2) heat shock is accompanied by increased generation of NO (EPR assay) and HSP70 accumulation in cultured cells; (3) DNIC induces HSP70 accumulation in cultured cells not exposed to heat shock.

An enzyme-linked immunosorbent assay and a Western blot analysis were developed to study the antibody response toPneumocystis cariniiin serum and bronchoalveolar lavage fluid from 27 human immunodeficiency virus 27 (HIV)-infected patients withP. cariniipneumonia (Pcp), 32 patients without Pcp, and 51 HIV-negative controls. Urea was used for the correct dilution of epithelial lining fluid, and albumin was used to evaluate transudation from plasma for the assessment of local production of antibodies toP. carinii. By contrast with those of immunoglobulin G (IgG), IgA responses toP. cariniiwere increased in serum from HIV-positive patients compared to negative controls. Local production of antibodies toP. carinii, especially IgA, was decreased in patients with Pcp. In a study of 10 patients of each group, IgG and IgA responses to gp116 fromP. cariniiwere lower in patients with Pcp than in other groups. These results suggest that, in addition to alveolar macrophages, local antibodies may play a role in host defense againstP. carinii.

BACKGROUND Macrophage migration inhibitory factor (MIF) is a lymphokine originally identified for its capacity to inhibit the random migration of macrophages. Recent data have further extended knowledge of the physiological role of this protein, showing that MIF is produced by several human organs and tissues. The present study was intended to evaluate the expression and tissutal localization of MIF in the human prostate. METHODS Prostate tissues were obtained from patients undergoing surgical adenomectomy for benign prostatic hyperplasia and were analyzed by Western blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and immunoelectron microscopy. RESULTS The presence of both MIF protein and mRNA was demonstrated in the prostate. Immunocytochemical studies localized MIF protein in the secretory luminal epithelial and basal layer cells. CONCLUSIONS The present study demonstrated that the human prostate is a site of MIF synthesis. Macrophages populate the human prostate and represent an important mechanism of defense of integrity and functionality of the gland. It is speculated that MIF might play a role in preserving prostate physiological activity by maintaining its macrophage population. Prostate 39:159–165, 1999. © 1999 Wiley-Liss, Inc.

The characteristics of a temperature sensitive feline infectious peritonitis virus (TS-FIPV) were examined. TS-FIPV, unlike its parent strain, DF2 wild type FIPV (WT-FIPV), propagated at 31 degrees C (permissive temperature) but not at 39 degrees C (nonpermissive temperature). This temperature preference of TS-FIPV was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures (38-39 degrees C) prevail. Viral structural proteins and RNA were synthesized at 39 degrees C but some undefined maturational defect prevented the formation of infectious TS-FIPV at its nonpermissive temperature. TS-FIPV was more thermolabile than WT-FIPV which indicated alterations in the structural proteins of TS-FIPV, and a difference in the envelope protein of the two viruses was revealed by Western blot analysis. Plaque assay characterization showed that TS-FIPV produced small plaques in comparison to the large plaques of WT-FIPV. These unique characteristics possessed by TS-FIPV may account for its nonvirulent nature and ability to stimulate protective immune responses in cats.

Heat shock potentiated the nitric oxide production (EPR assay) in the liver, kidney, heart, spleen, intestine, and brain. The heat shock-induced sharp transient increase in the rate of nitric oxide production preceded the accumulation of heat shock proteins (HSP70) (Western blot analysis) as measured in the heart and liver. In all organs the nitric oxide formation was completely blocked by the NO-synthase inhibitor Nω-nitro-l-arginine (L-NNA). L-NNA also markedly attenuated the heat shock-induced accumulation of HSP70. The results suggests that nitric oxide is involved in the heat shock-induced activation of HSP70 synthesis.

Background To examine the expression of SMAD4 at gene and protein levels in glioma samples with different WHO grades and its association with survival. Methods Two hundreds fifty-two glioma specimens and 42 normal control tissues were collected. Immunochemistry assay, quantitative real-time PCR and Western blot analysis were carried out to investigate the expression of SMAD4. Kaplan-Meier method and Cox's proportional hazards model were used in survival analysis. Results Immunohistochemistry showed that SMAD4 expression was decreased in glioma. SMAD4 mRNA and protein levels were both lower in glioma compared to control on real-time PCR and Western blot analysis (both P < 0.001). In addition, its expression levels decrease from grade I to grade IV glioma according to the results of real-time PCR, immunohistochemistry analysis and Western blot. Moreover, the survival rate of SMAD4-positive patients was higher than that of SMAD4-negative patients. We further confirmed that the loss of SMAD4 was a significant and independent prognostic indicator in glioma by multivariate analysis. Conclusions Our data provides convincing evidence for the first time that the reduced expression of SMAD4 at gene and protein levels is correlated with poor outcome in patients with glioma. SMAD4 may play an inhibitive role during the development of glioma and may be a potential prognosis predictor of glioma.