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Results for “Protein expression”

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PubMed2003

Cloning, mapping and protein expression of wheat thaumatin protein gene (TaTLP1).

To understand wheat powdery mildew resistance mechanism, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA library screening were performed to isolate the full-length cDNA of wheat thaumatin protein gene from wheat-Haynaldia villosa 6VS/6AL translocation line. The putative amino acid sequence of this gene consists of 173 amino acid residues, and is an acid polypeptide. It was highly homologous to thaumatin proteins isolated from other plants, so it is designated as TaTLP1 (GenBank accession number: AF384146). Northern blot analysis of TaTLP1 showed that the transcription difference obviously existed between resistant wheat-Haynaldia villosa 6VS/6AL translocation line and susceptible "Yangmai 5". The result of western blot showed that the protein expression product of TaTLP1 gene in wheat seedling leaf was a soluble cytoplasm protein, whose expression was induced by fungus Erysiph graminis and apparently related to disease resistance of the 6VS/6AL translocation line. Southern blot indicated that the TaTLP1 gene had 1-2 copies in wheat genome, and had been localized on the specific region of 7B and 7D chromosomes in wheat.

Amino Acid Sequence↗

PubMed2003

A new tool for routine testing of cellular protein expression: integration of cell staining and analysis of protein expression on a microfluidic chip-based system.

The key benefits of Lab-on-a-Chip technology are substantial time savings via an automation of lab processes, and a reduction in sample and reagent volumes required to perform analysis. In this article we present a new implementation of cell assays on disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow in microfluidic channels and two-color fluorescence detection of single cells. This new technology allows for simple flow cytometric studies of cells in a microfluidic chip-based system. In addition, we developed staining procedures that work "on-chip," thus eliminating time-consuming washing steps. Cells and staining-reagents are loaded directly onto the microfluidic chip and analysis can start after a short incubation time. These procedures require only a fraction of the staining reagents generally needed for flow cytometry and only 30,000 cells per sample, demonstrating the advantages of microfluidic technology. The specific advantage of an on-chip staining reaction is the amount of time, cells, and reagents saved, which is of great importance when working with limited numbers of cells, e.g., primary cells or when needing to perform routine tests of cell cultures as a quality control step. Applications of this technology are antibody staining of proteins and determination of cell transfection efficiency by GFP expression. Results obtained with microfluidic chips, using standard cell lines and primary cells, show good correlation with data obtained using a conventional flow cytometer.

Antibodies↗

PubMed2002

[Effect of naoyi'an on basic fibroblastic growth factor mRNA expression and tumor necrosis factor protein expression in brain of rats following intracerebral hemorrhage].

OBJECTIVE: To investigate the effect of Naoyi'an granule (NYAG) on basic fibroblastic growth factor (bFGF) mRNA expression and tumor necrosis factor (TNF) protein expression following intracerebral hemorrhage and provide the theoretical evidence of NYAG in treating intracerebral hemorrhage and promoting the rehabilitation of neural function. METHODS: Model rats of intracerebral hemorrhage induced by infusion of collagenase VII into the caudate-putamen were used to determine the related parameters of behavior scores (BS), Northern blot, Western blot assay and optical density (OD) scanning in the model and the model treated with NYAG. And the data got from the two groups were compared. RESULTS: BS in the model group began to lower 24 hrs after modeling and a significant decrease was shown 7 days later, while in the NYAG group, it decreased significantly three days after modeling, the difference between the two groups was significant (P < 0.05). Levels of bFGF mRNA expression and TNF protein expression increased after modeling, it reached the peak in three days and began to decrease gradually in seven days in both groups. However, the level in the NYAG group was higher than that in the model group in various times of the experimental process. CONCLUSION: NYAG could enhance the bFGF expression and suppress the TNF expression so as to improve the behavior deficit in treating intracerebral hemorrhage, which may be one of the main mechanisms of NYAG for promoting the rehabilitation of neural function.

Animals↗

PubMed1999

Constitutive protein expression of monocyte chemotactic protein-1 (MCP-1) by myelomonocytic cell lines and regulation of the secretion by anti- and proinflammatory stimuli.

We have investigated the protein expression of the chemokine monocyte chemotactic/chemoattractant protein-1 (MCP-1) in various human myelomonocytic leukemia cell lines. Applying specific ELISA, we demonstrated that this chemokine is produced constitutively by the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 440 and 1400 pg/ml MCP-1 per million cells. In the culture medium of two other unstimulated cell lines, MONO-MAC-1 and THP-1, almost no MCP-1 was detected. Stimulation of HL-60 and MONO-MAC-6 with lipopolysaccharide (LPS), and stimulation of ML-2 and MUTZ-3 with 12-tetradecanoyl phorbol 13-acetate (TPA) dramatically increased the MCP-1 level in the culture medium. The highest amount of MCP-1 (> 80 ng/ml within 24 h) was achieved by TPA stimulation of MUTZ-3 cells. Out of 15 cytokines tested for induction or enhancement of MCP-1 secretion, interleukin-3 (IL-3), IL-6, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and tumor necrosis factor (TNFalpha) were able to augment (twofold to 12-fold) the MCP-1 level in the culture medium of MONO-MAC-6 cells. While the antinflammatory cytokines IL-4, IL-10 and IL-13 failed to suppress MCP-1 secretion, the glucocorticoid dexamethasone strongly inhibited the MCP-1 production of unstimulated and stimulated MONO-MAC-6 cells. Thus, several regulatory elements are involved in MCP-1 secretion. Despite the quantitative differences of MCP-1 production among the cell lines analyzed, our results demonstrated a constitutive secretion in differentiation-arrested myelomonocytic leukemia cell lines and emphasize the usefulness of these malignant cell lines as models to study MCP-1 secretion and regulation.

Anti-Inflammatory Agents↗

PubMed1996

Expression of heat shock proteins HSP70 and HSP90 in endometrial carcinomas. Correlation with clinicopathology, sex steroid receptor status, and p53 protein expression.

BACKGROUND: It has been suggested that heat shock proteins HSP70 and HSP90 are involved in the functional modulation of sex steroid receptors and are expressed in normal endometrium. However, little is known about the expression of HSP70 and HSP90 in endometrial carcinomas. METHODS: The immunohistochemical reactivity of monoclonal antibodies against HSP70 and HSP90 was examined in 42 endometrial carcinomas, and the presence or absence of correlation with the clinicopathologic features, sex steroid receptor status, and p53 protein expression was analyzed. RESULTS: Expression of HSP70 was found in 52% of endometrial carcinomas and was correlated with nonendometrioid histology (P < 0.05), a poorly differentiated state (P < 0.01), p53 protein expression (P < 0.01), and absence of sex steroid receptors (P < 0.001) in the tumor. By contrast, strong expression of HSP90 was observed in 29% of endometrial carcinomas, and occurred more frequently in well-differentiated carcinomas that were positive for sex steroid receptors. CONCLUSIONS: Both HSP70 and HSP90 are significantly correlated with the histology and the sex steroid receptor status of endometrial carcinomas. Expression of HSP70 may be associated with a loss of sex steroid receptors in either nonendometrioid or poorly differentiated carcinoma of the endometrium, which frequently exhibits p53 protein expression. Conversely, strong expression of HSP90 may indicate high levels of sex steroid receptors in the tumor cells.

Adult↗

PubMedDate not supplied

A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells.

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.

Animals↗

PubMed2000

[The protein expression of insulin receptor substrate-1 and protein tyrosine phosphatase with two src-homology II in the adipose tissue from type II diabetic patients].

OBJECTIVE: To investigate the protein expression of insulin receptor substrate-1 (IRS-1), protein tyrosine phosphatase with two src-homology 2 (SH-PTP(2)) in adipose tissues of type II diabetic patients, and explore molecular mechanisms of insulin resistance from adipose tissues of type II diabetic individuals. METHODS: The levels of protein expression of IRS-1 and SH-PTP(2) in adipose tissues from type II diabetic individuals were measured by Western blot analysis; meanwhile compared the level of protein expression of one's own abdominal subcutaneous and omentum majus adipose tissue from patients with type II diabetes mellitus. RESULTS: The expression of IRS-1 protein in abdominal subcutaneous (A) 2.14 +/- 0.67 and omentum majus 3.25 +/- 0.70 adipose tissues from patients with type II diabetes mellitus was significantly lower than that of control group [subcutaneous: 4.33 +/- 0.57 (P < 0.001), omentum majus: 8.65 +/- 2.85 (P < 0.05)]. The expression of SH-PTP(2) protein had no difference compared with control group (P > 0.05). The protein expression of IRS-1 was significantly lower in abdominal subcutaneous adipose tissue (1.09 +/- 0.13) than that in omentum majus adipose tissue (2.10 +/- 0.22) from the type II diabetic patients (P < 0.05), but the protein expression of SH-PTP(2) was significantly higher in subcutaneous adipose tissue (70.75 +/- 2.18) than that in omentum majus (43.69 +/- 11.07, P < 0.05). CONCLUSION: The abnormal changes in expression of IRS-1 and SH-PTP(2) protein in adipose tissues of the patients with type II diabetic mellitus may be one of the mechanisms of leading to insulin resistance. The abdominal subcutaneous adipose tissue of type II diabetic patients, as visceral adipose tissues, contributes to their insulin resistance.

Adipose Tissue↗

PubMedDate not supplied

Expression of Mad1 protein inhibits proliferation of cancer cells and inversely correlated with Myc protein expression in primary gastric cancer.

We performed immunohistochemical assay for Mad1 and Myc protein in 76 gastric cancer tissues. Cancers with reduced Mad1 expression had high proliferative index (S and G2/M-phase) and there was inverse correlation between Mad1 and Myc expression in the same cancer cells. There was no significant correlation between expression of Mad1 and cell differentiation or extent of the disease. There was a trend that reduced expression of Mad1 was associated with poor survival of the patients. Mad1 may influence biologic behavior of cancer cell by inhibiting transcriptional activity of Myc and reduced levels of Mad1 expression seem to be associated with carcinogenesis in human gastric cancer.

Adult↗

PubMed2002

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells.

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.

Animals↗

PubMedDate not supplied

Comparative analysis of the tear protein expression in blepharitis patients using two-dimensional electrophoresis.

Change in the expression of body fluid proteins is caused by many diseases or environmental disturbances. The changes in tear proteins are also associated with various pathological eye conditions. Especially, chronic blepharitis is one of the most common conditions seen in the ophthalmologist's office. However, there are no specific clinical diagnostic tests for blepharitis, and it is difficult to treat effectively. Therefore, the aim of this study was to screen prognostic or diagnostic marker tear proteins for blepharitis and investigate pathogenesis of this disease using proteomics techniques. The tear proteins expressed in patients suffering from blepharitis (patient, n=19) and healthy volunteers (control, n=27) were analyzed using the two-dimensional electrophoresis (2-DE) technique. The differentially expressed proteins in patients were identified with ESI-Q-TOF (electrospray-quadrupole-time-of-flight) mass spectrometry and confirmed with western blotting. Nine proteins in patient were down regulated about 50% compared to those of the control: serum albumin precursor, alpha-1 antitrypsin, lacritin precursor, lysozyme, Ig-kappa chain VIII, prolactin inducible protein (PIP/GCDFP-15), cystatin-SA III, pyruvate kinase, and an unnamed protein. The use of the two-dimensional eletrophoretic technique could give more insight into the disease-related protein expression changes in tear fluids. Our findings reveal that the composition of tear proteins in blepharitis patients is different from that of healthy subjects and may provide further insights into the pathogenesis of blepharitis.

Aged↗

PubMed2006

Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis.

Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted rickettsial pathogens that cause human and canine monocytic ehrlichiosis respectively. We tested the hypothesis that these pathogens express unique proteins in response to their growth in vertebrate and tick host cells and that this differential expression is similar in closely related Ehrlichia species. Evaluation of nine E. chaffeensis isolates and one E. canis isolate demonstrated that protein expression was host cell-dependent. The differentially expressed proteins included those from the p28/30-Omp multigene locus. E. chaffeensis and E. canis proteins expressed in infected macrophages were primarily the products of the p28-Omp 19 and 20 genes or their orthologues. In cultured tick cells, E. canis expressed only the p30-10 protein, an orthologue of the E. chaffeensis p28-Omp 14 protein which is the only protein expressed by E. chaffeensis propagated in cultured tick cells. The expressed Omp proteins were post-translationally modified to generate multiple molecular forms. E. chaffeensis gene expression from the p28/30-Omp locus was similar in tick cell lines derived from both vector (Amblyomma americanum) and non-vector (Ixodes scapularis) ticks. Differential expression of proteins within the p28/p30-Omp locus may therefore be vital for adaptation of Ehrlichia species to their dual host life cycle.

Amino Acid Sequence↗

PubMed2003

Various factors (allergen nature, mouse strain, CpG/recombinant protein expressed) influence the immune response elicited by genetic immunization.

BACKGROUND: Genetic immunization is a very promising therapeutic approach for allergy treatment. In the present study we investigate the influence of the nature of the allergen, the mouse strain, and the relative amount of CpG to expressed recombinant protein on immune responses using two major peanut allergens, Ara h 1 and Ara h 4. METHODS: The cDNA of Ara h 1 and of an isoform of Ara h 4 were cloned and inserted in pcDNA3. Antigen specific IgG1, IgG2a and IgE were followed after genetic immunization with 100 microg of these clones in mouse strain SKH-Hr1 or BALB/c and with 1 microg of the clones+99 blank plasmid in SKH-Hr1. RESULTS: Genetic immunization in SKH-Hr1 with Ara h 1 elicited a classical Th1 type response, but Ara h 4 elicited a mixed Th1/Th2 response with high IgG1 and even IgE in some mice. In BALB/c both plasmids produced a high IgG1 level. Decreasing the amount of plasmid injected did not change the immune response profile. However, increasing the amount of CpG administered relative to the recombinant Ara h 4 protein expressed reversed the Th1/Th2 response pattern in SKH-Hr1 mice. CONCLUSIONS: Immune responses after genetic immunization are strongly influenced by the nature of the allergen, the mouse strain, and the ratio of CpG to recombinant protein expressed.

Allergens↗

PubMed2003

Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase.

OBJECTIVE: The cardiac ankyrin repeat protein (CARP), a nuclear transcription co-factor that negatively regulates cardiac gene expression, is increased in human heart failure and in animal models of cardiac hypertrophy. The mechanism by which CARP expression is regulated and the consequences of CARP overexpression on cardiac contractility are unknown. METHODS AND RESULTS: Compared to vehicle treated controls, 4-day treatment of male Wistar rats with the beta-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) induced hypertrophy and significantly increased CARP mRNA and CARP protein levels in left ventricles. The signalling pathways were investigated in more detail in isolated neonatal rat cardiomyocytes. Treatment of cells with isoprenaline (1 micromol/l) caused a significant increase in CARP mRNA and protein by approximately 50%. Combined beta(1)- and beta(2)-adrenoceptor blockade, inhibition of protein kinase A (PKA; Rp-cAMPS, 100 micromol/l), and inhibition of calmodulin-dependent protein kinases (CaMK; KN-62, 10 micromol/l) completely reversed the effects of isoprenaline. To examine the consequences of CARP overexpression on contractile function, an adenovirus encoding human CARP as well as a control virus were constructed. Although the basal force of contraction was not different, contractile response to Ca(2+) and isoprenaline was significantly diminished in engineered heart tissue infected with the recombinant adenovirus that carries the CARP gene (Ad.CARP). CONCLUSIONS: Our study provides the first evidence that overexpression of CARP, which is thought to act as a transcriptional co-repressor, may deteriorate contractile function of the heart tissue. Furthermore, beta-adrenoceptor stimulation and activation of PKA and CaMK have been identified as mechanisms that induce expression of CARP in cardiomyocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2004

Oxytocin stimulates prostaglandin F2alpha secretion and prostaglandin F synthase protein expression in porcine myometrial tissue.

The possibility of PGF(2)alpha production and presence of prostaglandin F synthase (PGFS; PGD(2) 11-ketoreductase) was studied in control and oxytocin (OT)-stimulated myometrial slices isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows. Oxytocin (10(-7) M) stimulated (p<0.01) PGF(2)alpha production in both cycling and early pregnant myometrial slices. Prostaglandin F(2)alpha release was higher (p<0.01) in control as well as OT-treated myometrium of early pregnant sows in comparison to cycling myometrium. Prostaglandin F synthase expression at protein level was evident in myometrial slices of cyclic as well as early pregnant sows. The signals of PGFS was stronger (p<0.05) in cycling myometrium exposed to OT compared to that of control. There were no significant differences (p>0.05) in PGFS protein expression between control and OT-stimulated myometrial tissue of early-pregnant sows. The results of this study indicate the local PGF(2)alpha synthesis and the presence of PGFS in porcine cycling and early pregnant myometrial tissue. In addition, OT increased PGD(2) 11-ketoreductase protein expression in myometrium harvested during the porcine estrous cycle. However, the OT-stimulated PGF(2)alpha myometrial secretion was observed in both, cycling and pregnant gilts.

Animals↗

PubMed2005

Absence of prognostic significance of p21(WAF1/CIP1) protein expression in non-small cell lung cancer.

Prognostic value of p21WAF1/CIP1 expression in non-small-cell lung cancer patients (NSCLC) remains unclear. In this study the authors investigated the clinical significance of p21WAF1/CIP1 expression in a group of 117 NSCLC patients, who underwent curative pulmonary resection. Expression of p21WAF1/CIP1 protein was assessed immunohistochemically and samples showing>5% of positive tumor cells were considered positive. Seventy-six samples (65%) showed positive nuclear p21WAF1/CIP1 protein expression. There was no relationship between the expression of p21WAF1/CIP1 protein and major clinico-pathological factors, and neither there was an impact of p21WAF1/CIP1 protein expression on disease-free and overall survival. p21WAF1/CIP1 protein occurrence was not correlated with previously determined p53 protein expression and there was also no relationship between all possible p21WAF1/CIP1/p53 phenotypes and survival. In uni- and multivariate analysis only stage of disease was independent prognostic factors. These results suggest the lack of prognostic relevance of p21WAF1/CIP1 expression (analyzed separately or jointly with p53 protein) in surgically treated NSCLC patients.

Aged↗

PubMed1999

Immunological characterization of the sheep prion protein expressed as fusion proteins in Escherichia coli.

The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot.

Animals↗

PubMed2001

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins.

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.

Base Sequence↗

PubMed2000

[p27 protein expression and its significance in human laryngeal carcinoma].

OBJECTIVE: To determine clinical significance and expression of p27 protein in human laryngeal carcinoma tissue. METHOD: To measure p27 protein expression in 50 cases laryngeal carcinoma tissue by immunohistochemical LSAB (labelled streptavidin biotin) method, 15 cases vocal cord polyps used as control. RESULT: There was significant difference between p27 protein expression in laryngeal carcinoma and in vocal cord polyps, among pathological staging of laryngeal carcinoma (G1-G2 and G3), between the survived and the dead laryngeal carcinoma patients 3 years after operation, but no significant difference among clinical staging and between the patients with and without lymph node metastases. There was significant difference between the survival rate curves (Kaplan-meier curve) in p27 protein expression positive group and that in p27 protein expression negative group. CONCLUSION: p27 Protein expression is related to malignant degree of laryngeal carcinoma, so it can be an important marker of identifying the malignancy and prognosis of laryngeal carcinoma.

Adult↗