30-kD Protein Research Articles

Low temperature acclimation mediated by ethanol production is essential for chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10 °C) for various length of time followed by a 48-h chilling temperature stress (2 °C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in fatty acid composition in rice roots under the present experimental condition. Alcohol dehydro¬genase (ADH) activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h, which indicates that LT-PT increased ethanol fermentation in the roots. 4-Methylpyrazole, a potent inhibitor of ADH, reduced the ethanol concentration and the chilling tolerance in the roots. This reduction of the chilling tolerance recovered with exogenously applied ethanol. Ethanol also induced 21- and 33-kD protein synthesis in the roots and these proteins may contribute the improvement of the tolerance. The present research suggests that LT-PT may increase chilling tolerance in rice roots owing to ethanol production, and ethanol may trigger a signal transduction cascade, which might lead to a decrease in membrane damage and injury.

Identification of ERGIC-53 as an intracellular transport receptor of α1-antitrypsin

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein–based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

The Native Cyclobutane Pyrimidine Dimer Photolyase of Rice Is Phosphorylated

The cyclobutane pyrimidine dimer (CPD) is a major type of DNA damage induced by ultraviolet B (UVB) radiation. CPD photolyase, which absorbs blue/UVA light as an energy source to monomerize dimers, is a crucial factor for determining the sensitivity of rice (Oryza sativa) to UVB radiation. Here, we purified native class II CPD photolyase from rice leaves. As the final purification step, CPD photolyase was bound to CPD-containing DNA conjugated to magnetic beads and then released by blue-light irradiation. The final purified fraction contained 54- and 56-kD proteins, whereas rice CPD photolyase expressed from Escherichia coli was a single 55-kD protein. Western-blot analysis using anti-rice CPD photolyase antiserum suggested that both the 54- and 56-kD proteins were the CPD photolyase. Treatment with protein phosphatase revealed that the 56-kD native rice CPD photolyase was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not. The purified native rice CPD photolyase also had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. According to the absorption, emission, and excitation spectra, the purified native rice CPD photolyase possesses both a pterin-like chromophore and an FAD chromophore. The binding activity of the native rice CPD photolyase to thymine dimers was higher than that of the E. coli-expressed CPD photolyase. These results suggest that the structure of the native rice CPD photolyase differs significantly from that of the E. coli-expressed rice CPD photolyase, and the structural modification of the native CPD photolyase leads to higher activity in rice.

Oligomerization of the potato virus X 25-kD movement protein

A 25-kD movement protein (25K protein) encoded by the first gene of the potexvirus Potato virus X triple gene block of transport genes is essential for the viral movement in infected plants. The 25K protein belongs to superfamily 1 of NTPase/helicases and exhibits in vitro RNA helicase, Mg2+-dependent NTPase, and RNA-binding activities. In the present work, the ability of 25K protein for homologous interactions was studied using the yeast two-hybrid system, protein chemical cross-linking in the presence of glutaraldehyde, far-Western blotting, and ultracentrifugation in sucrose density gradients. The 25K protein was shown to form homodimers and homooligomers. Sites of homologous protein-protein interactions were found in both the N- and C-terminal portions of the protein.

Protective Effects of Salicylic Acid and Vitamin C on Sulfur Dioxide-Induced Lipid Peroxidation in Mice

The antioxidant effects of exogenous salicylic acid (SA) and vitamin C (Vit C) on the oxidative stress induced by 56 mg/m3 of sulfur dioxide (SO2) in mouse livers and brains were investigated. The exposure of SO2 caused significant elevation of thiobarbituric acid-reactive substance (TBARS) levels and reduction of enzyme activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in brain and liver, accompanied by a decrease in relative growth rate, when compared with controls. Application of moderate concentrations of SA and Vit C markedly reduced the SO2-induced elevation of TBARS levels, with 5.5 mg/kg SA or 200 mg/kg Vit C being most effective. In contrast to the decrease of TBARS levels, the levels of SOD, POD, and CAT in liver and brain were significantly increased in comparison with controls. The polyacrylamide gel electrophoresis (PAGE) of total liver proteins showed that the SO2 inhalation caused a 30-kD protein band disappearance compared with the control. However, the band remained unchanged in the samples treated with 5.5 and 8.25 mg/kg SA or 100, 200, and 400 mg/kg Vit C. Therefore, this protein band may serve as a marker for the damage induced by SO2 and an additional basis for drug screening and selection.

Early diagnosis of severe Mediterranean spotted fever cases by nested-PCR detecting spotted fever Rickettsiae 17-kD common antigen gene

We describe 3 cases of Mediterranean spotted fever (MSF) who presented with severe sepsis, in 2 of which the clinical diagnosis was unclear at presentation. In each case the diagnosis of MSF was made using a nested-PCR assay for Rickettsia conorii 17-kD protein gene. The nested-PCR based diagnosis preceded the serological results of MSF that were all negative at admission. The early diagnosis of MSF by specific PCR will facilitate an early institution of appropriate therapy, saving unnecessary tests and medications.

Molecular Detection and Characterization of Spotted Fever Group Rickettsiae in Taiwan

Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.

The possible interaction of CDA14 and protein elongation factor 1α

The CDA14, a 45kD protein, is currently annotated as PTX1-like protein or ERGIC 32. Over expressing CDA14 can slow PC11 cell proliferation rate. In HepG2 cells, it had been demonstrated that CDA14 is involved in protein transportation. The knowledge about the protein is very limited and not clarified. The CDA14 and its homologous proteins form a family and are restricted to eukaryotes. In the family, there are no homologous sequences with resolved three-dimensional structure and their functions are difficult to predict. Transcriptional expression of CDA14 in three hepatoma cell lines, Hu7, HCC and HepG2, was lower than normal liver tissue and liver carcinoma tissue. In this study, functional proteomic techniques were utilized in searching the interacting counterpart of CDA14. Several proteins involved in protein translation and folding were selectively precipitated with CDA14 and identified mass spectrometry. Interaction of CDA14 and elongation factor 1α was confirmed by Western blotting and confocal microscopy. Elongation factor 1α is a multiple function protein and involved in several biological mechanisms, including protein synthesis, cell proliferation, apoptosis and tumorigensis. Over-expression of CDA14 down regulated the proliferation of HepG2 cells. These results suggest that CDA14 participated in the elongation factor 1α regulated mechanisms.

Proteolytic Cleavage of Ostrich and Turkey Pancreatic Lipases

The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

Cytoplasmic Immunoreactivity of Thyroid Transcription Factor-1 (Clone 8G7G3/1) in Hepatocytes

The nuclear immunoreactivity for thyroid transcription factor-1 (TTF-1) is a useful marker for identification of carcinomas of thyroid and lung origin. Our aim was to determine whether cytoplasmic staining in the liver is a result of cross-reaction of anti-TTF-1 antibody (clone 8G7G3/1, DAKO, Carpinteria, CA) or true positivity resulting from aberrant expression of TTF-1 or products of the alternatively sliced TTF-1 gene. Fresh tissue samples from liver, thyroid, and lung were obtained for H&E-stained sections, TTF-1 immunostaining, and RNA and protein analyses. Western blot revealed an abundant band corresponding to an approximately 160-kd protein from liver but not either thyroid or lung tissue samples. By reverse transcriptase-polymerase chain reaction, messenger RNA of TTF-1 was not detectable in liver tissue. Our study demonstrates that TTF-1 immunoreactivity (clone 8G7G3/1) in the hepatocyte cytoplasm is due to an approximately 160-kd protein; this unique protein is not an alternative splicing product of TTF-1 and neither is it expressed in thyroid and lung tissues.

Regulation of apoptosis in fibroblast‐like synoviocytes by the hypoxia‐induced Bcl‐2 family member Bcl‐2/adenovirus E1B 19‐kd protein–interacting protein 3

Rheumatoid arthritis (RA) synovial hyperplasia is related in part to a resistance to apoptosis exhibited by fibroblast-like synoviocytes (FLS). Since hypoxia is a regulator of apoptosis, and since RA synovium is hypoxic, we conducted this study to examine the effects of hypoxia on the Bcl-2 pathway and the role this may play in regulating apoptosis in FLS. Synovium samples from RA patients, osteoarthritis (OA) patients, and normal subjects were used for immunohistologic assessments and for generating FLS lines in vitro. FLS were stimulated under conditions of hypoxia (1% O(2)) and using 100 microM CoCl(2) to simulate the effects of severe hypoxia. Changes in the gene expression profile of FLS were evaluated using microarrays and were confirmed by quantitative polymerase chain reaction (PCR). Changes in protein expression were detected by Western blotting. The effect of transient transfection with a BNIP3 plasmid on the apoptosis of FLS was evaluated in the presence and absence of cytokines. Gene expression profiling demonstrated that BNIP3 was unique among the BCL2 family, in that it was induced by hypoxia in FLS. Quantitative PCR indicated a 2-3-fold induction of BNIP3 messenger RNA, and Western blotting showed a 3-5-fold increase in the 30-kd Bcl-2/adenovirus E1B 19-kd protein-interacting protein 3 (BNIP-3) monomer. BNIP-3 was widely expressed in RA synovium and was prominent in FLS from the lining layer. Overexpression of BNIP3 increased FLS apoptosis under hypoxic conditions, an effect that was inhibited by tumor necrosis factor alpha and interleukin-1beta. The proapoptotic protein BNIP-3 is induced in FLS by hypoxia and is widely expressed in RA synovium, but its proapoptotic effects may be inhibited in vivo by proinflammatory cytokines. Since overexpression of BNIP3 in FLS increases apoptosis, this may provide a novel approach for controlling synovial hyperplasia in RA.

Identification of leukocyte cationic proteins that interact with ceruloplasmin

Proteins from leukocytes were investigated for their ability to interact with ceruloplasmin (Cp), a copper-containing glycoprotein of human plasma. Extract from leukocytes was subjected to affinity chromatography on Cp-Sepharose, after which proteins were eluted from the resin with 0.5 M NaCl in Tris-HCl, pH 7.4. SDS-PAGE of the eluate revealed protein bands with molecular weights 78, 57, 40, 30, 16, and 12 kD. Among these, Western blotting detected myeloperoxidase (57, 40, and 12 kD) and lactoferrin (78 kD). Also, the 30-kD component had a sequence (1)I-(2)I/V-(3)G-(4)G-(5)R/H at the N-terminus that is likely to indicate the presence of neutrophilic elastase, cathepsin G, proteinase 3, and azurocidin (CAP 37) - all from the family of serprocidins. Mass spectrometry of tryptic fragments indicated the presence of the 16-kD eosinophilic cationic protein (seven peptides), 27-kD cathepsin G (eleven peptides), 27-kD azurocidin (eight peptides), 29-kD neutrophilic elastase (seven peptides), and 27-kD proteinase 3 (six peptides). Myeloperoxidase was represented by 57-, 40-, and 12-kD fragments (thirteen, ten, and four peptides, respectively). Thus, interaction with Cp of five cationic proteins, i.e. of eosinophilic cationic protein, cathepsin G, neutrophilic elastase, proteinase 3, and azurocidin is reported for the first time.

Characterization of Buckwheat 19-kD Allergen and Its Application for Diagnosing Clinical Reactivity

Background: The 19-kD protein of buckwheat (BW) has been suggested to be a major allergen, but its characteristics and clinical significance are poorly defined. Methods: cDNA of the 19-kD BW allergen was cloned and expressed in Escherichia coli. Allergenicity and cross-allergenicity were confirmed by inhibition immunoblotting or by ELISA inhibition. The recombinant (r19-kD) protein was assessed for clinical utility in the diagnosis of BW reactivity in 18 BW-allergic and 19 BW-asymptomatic sensitized subjects using receiver operating characteristic analysis. Results: The 19-kD BW allergen, which is composed of 135 amino acids, has a weak homology to the vicilin-like allergens of cashew (Ana o 1), English walnut (Jug r 2) and 7 S globulin from Sesamum indicum. The r19-kD protein can inhibit sIgE binding to native 19-kD BW allergen. The maximum percentage inhibition of sIgE binding to crude BW extract was 56%. About 83.3% of the BW allergy patients had sIgE bound to r19-kD protein, compared to only 1 of the 19 BW-asymptomatic sensitized subjects. The areas under the receiver operating characteristic curves for the skin prick tests [0.925 (95% confidence interval: 0.839–1.012), p < 0.001] as well as r19-kD protein sIgE ELISAs [0.860 (95% confidence interval: 0.725–0.995), p <0.001] were higher than that of BW sIgE coated allergen particle test results [0.803 (95% confidence interval: 0.661–0.945), p = 0.002]. Conclusions: The 19-kD BW allergen may be the major allergen from BW. For the diagnosis of clinical reactivity to BW, the r19-kD protein sIgE ELISA test was more discriminative than the coated allergen particle sIgE measurement using whole BW extract.

Lupine allergy: Not simply cross-reactivity with peanut or soy

Reports of lupine allergy are increasing as its use in food products increases. Lupine allergy might be the consequence of cross-reactivity after sensitization to peanut or other legumes or de novo sensitization. Lupine allergens have not been completely characterized. We sought to identify allergens associated with lupine allergy, evaluate potential cross-reactivity with peanut, and determine eliciting doses (EDs) for lupine allergy by using double-blind, placebo-controlled food challenges. Six patients with a history of allergic reactions to lupine flour were evaluated by using skin prick tests, CAP tests, and double-blind, placebo-controlled food challenges. Three of these patients were also allergic to peanut. Lupine allergens were characterized by means of IgE immunoblotting and peptide sequencing. In all 6 patients the ED for lupine flour was 3 mg or less for subjective symptoms and 300 mg or more for objective symptoms. The low ED and moderate-to-severe historical symptoms indicate significant allergenicity of lupine flour. Two patients allergic to lupine but not to peanut displayed IgE binding predominantly to approximately 66-kd proteins and weak binding to 14- and 24-kd proteins, whereas patients with peanut allergy and lupine allergy showed weak binding to lupine proteins of about 14 to 21 or 66 kd. Inhibition of binding was primarily species specific. Lupine allergy can occur either separately or together with peanut allergy, as demonstrated by 3 patients who are cosensitized to peanut and lupine. Lupine flour is allergenic and potentially cross-reactive with peanut allergen, thus posing some risk if used as a replacement for soy flour.

Sperm DNA Integrity From Sperm to Egg

Sperm DNA Integrity From Sperm to Egg

ACTIN BINDING PROTEIN29 fromLiliumPollen Plays an Important Role in Dynamic Actin Remodeling

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

Development of Lentiviral Vectors with Regulated Respiratory Epithelial Expression In Vivo

Development of gene transfer vectors with regulated, lung-specific expression will be a useful tool for studying lung biology and developing gene therapies. In this study we constructed a series of lentiviral vectors with regulatory elements predicted to produce lung-specific transgene expression: the surfactant protein C promoter (SPC) for alveolar epithelial type II cell (AECII) expression, the Clara cell 10-kD protein (CC10) for Clara cell expression in the airway, and the Jaagskiete sheep retrovirus (JSRV) promoter for expression in both cell types. Transgene expression from the SPC and CC10 vectors was restricted to AECII and Clara cell lines, respectively, while expression from the JSRV vector was observed in multiple respiratory and nonrespiratory cell types. After intratracheal delivery of lentivector supernatant to mice, transgene expression was observed in AECII from the SPC lentivector, and in Clara cells from the CC10-promoted lentivector. Transgene expression was not detected in nonrespiratory tissues after intravenous delivery of CC10 and SPC lentiviral vectors to murine recipients. In summary, incorporation of genomic regulatory elements from the SPC and CC10 genes resulted in respiratory specific transgene expression in vitro and in vivo. These vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders.

Clara cell 16-kd protein downregulates T H2 differentiation of human naive neonatal T cells

Levels of the Clara cell 16-kd protein (CC16) are lower in plasma and bronchoalveolar lavage fluid from adults with asthma relative to those seen in healthy control subjects, and CC16 inhibits the T(H)2 cytokine production from murine T cells. We sought to determine the plasma levels of CC16 in infants and to investigate whether CC16 might inhibit the T(H)2 cytokine production from neonatal T cells. Cord blood and blood samples at 4, 18, and 36 months of age were taken from 64 children prospectively, and CC16 levels were analyzed in plasma. Cord monocyte-derived dendritic cells (DCs) were pulsed with birch allergen extract alone or together with CC16 or prostaglandin D(2) receptor inhibitors, after which autologous naive CD4(+) T cells were added to the DCs. The production of IL-5, IL-13, and IFN-gamma was measured by means of ELISA and flow cytometry. The plasma levels of CC16 in children peaked at 4 months. CC16 did not directly affect the cytokine production from human T(H)2 cells. However, CC16 inhibited birch pollen extract-stimulated T(H)2 differentiation of naive T cells through the DC. Inhibition of the prostaglandin D(2) receptors did not consistently result in suppressed T(H)2 differentiation. The production of CC16 seems to peak early in life, and CC16 has an inhibitory effect on T(H)2 cell differentiation from human infants by affecting DCs. CC16 is an immunoregulatory protein, and its inhibitory effect on T(H)2 cell differentiation might be of importance in the pathogenesis of allergy in infants.

Interleukin‐6 and chemokines in the neuropsychiatric manifestations of systemic lupus erythematosus

To define the cytokine and chemokine profile in cerebrospinal fluid (CSF) from patients with neuropsychiatric systemic lupus erythematosus (NPSLE). Forty-two SLE patients who had been hospitalized because of NP manifestations were studied. Patients were evaluated at hospitalization and 6 months later; a CSF sample was obtained at each evaluation. As controls, CSF from 6 SLE patients with septic meningitis, 16 SLE patients with no history of NP manifestations (non-NPSLE), and 25 patients with nonautoimmune diseases were also studied. Soluble molecules, including cytokines (interleukin-2 [IL-2], IL-4, IL-6, IL-10, tumor necrosis factor alpha [TNFalpha], and interferon-gamma [IFNgamma]) and chemokines (monocyte chemotactic protein 1 [MCP-1], RANTES, IL-8, monokine induced by IFNgamma [MIG], and interferon-gamma-inducible 10-kd protein [IP-10]), were measured with the use of cytometric bead array kits. CSF levels of the following molecules were significantly increased in NPSLE patients as compared with non-NPSLE and nonautoimmune diseases control patients, respectively: IL-6 (32.7 versus 3.0 and 2.96 pg/ml), IL-8 (102.8 versus 29.97 and 19.7 pg/ml), IP-10 (888.2 versus 329.7 [P not significant] and 133.6 pg/ml), RANTES (3.8 versus 2.5 and 2.2 pg/ml), MCP-1 (401.7 versus 257.9 [P not significant] and 136.9 pg/ml), and MIG (35.4 versus 11.4 and 3.5 pg/ml). Low levels of IL-2, IL-4, IL-10, TNFalpha, and IFNgamma were found in all groups. All cytokines and chemokines, except TNFalpha, were significantly higher among the SLE patients with septic meningitis than among the NPSLE patients. Six months later and in the absence of NP manifestations, all elevated molecule levels, except RANTES, in patients with NPSLE had decreased significantly, and no differences were noted between the NPSLE and non-NPSLE groups. A central nervous system response composed of IL-6 and chemokines, but not Th1/Th2 cytokines, is associated with NP manifestations in SLE patients.

P2009 Diagnostic value of neopterin in patients with active pulmonary tuberculosis and relation with CRP

Search for novel specific antigens are urgently needed for the detection of tuberculosis (TB). In this study, we evaluated the diagnostic potential of a novel Mycobacterium tuberculosis (M.tb)-specific candidate antigen (Rv2645) from DNA segment region of differentiation (RD) 13 of M.tb and investigated T-cell recognition during natural infection in humans and experimental mice. Rv2645-specific IFN-γ levels were much higher in the peripheral blood mononuclear cells (PBMCs) of TB patients than that in healthy donors (HDs) (including Bacille Calmette-Guerin (BCG)-vaccinated donors). The enzyme-linked immunospot (ELISPOT) assay with Rv2645 had a high overall agreement (98.0%) with the results from the clinical T-SPOT.TB with 10-kD culture filtrate protein (CFP10) and 6-kD early secreted antigenic target (ESAT6) peptides. The combination of Rv2645 and CFP10-ESAT6 was better than the individual protein, with increased sensitivity and a similar specificity of 96.0% and 98.2%, respectively. Rv2654 also induced M.tb-specific skin-test responses in heat-inactivated M.tb H37Rv immunized mice. Epitope mapping revealed that Rv264530–44 and Rv2645136–143 may be the dominant T-cell and B-cell epitopes, respectively, of Rv2645. This is the first report demonstrating the Rv2654 is a strongly recognized T-cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent.