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PubMed2006

Gene conjugation of molecular adjuvant C3d3 to hCGbeta increased the anti-hCGbeta Th2 and humoral immune response in DNA immunization.

Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGbeta-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGbeta following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGbeta-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGbeta and hCGbeta-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGbeta, pCMV4-hCGbeta, pcDNA3-hCGbeta-C3d3 and pCMV4-hCGbeta-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGbeta IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGbeta-C3d3-DNAs elicited the highest titer of hCGbeta-specific antibody among the serial doses and the immune response induced by pCMV4-hCGbeta-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGbeta-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGbeta at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGbeta-C3d3-DNAs and the ratio of IL-4/IFN-(gamma) showed a Th2 bias of immune response in the mice immunized with hCGbeta-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGbeta DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3.

Animals↗
PubMed1989

Analysis of the nag regulon from Escherichia coli K12 and Klebsiella pneumoniae and of its regulation.

Four genes, nagR, A, B and E, clustered in the nag locus of Escherichia coli K12 and Klebsiella pneumoniae, were cloned and physically mapped, and the corresponding gene products involved in amino sugar metabolism identified. Expression of the nag genes was also analysed using a series of lacZ fusions. In both bacteria, the genes are arranged in two divergent operons and controlled by a common NagR repressor. The corresponding gene nagR was found to map in the first operon together with the promoter proximal gene nagB, encoding the enzyme D-glucosamine isomerase (deaminase) (NagB) and the middle gene nagA, coding for N-acetyl-glucosamine deacetylase (NagA). Polar mutations in nagB and nagA prevent the efficient expression of nagR and cause constitutive expression of all nag genes. This includes the gene nagE encoding Enzyme IINag of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS), encoded in the second divergently transcribed operon. No further gene is found in this operon which in both organisms is directly adjacent to the gene glnS. It is interesting that the NagR repressor also affects the mannose PTS (genes manX, Y, Z), the second transport system involved in amino sugar uptake and phosphorylation.

Acetylglucosamine↗
PubMed2000

Molecular cloning and characterization of a transcriptional activator gene, amyR, involved in the amylolytic gene expression in Aspergillus oryzae.

A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the alpha-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including alpha-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A. oryzae. In addition, sequencing analysis found that amyR, agdA (encoding alpha-glucosidase), and amyA (encoding alpha-amylase), are clustered on a 12-kb DNA fragment of the largest chromosome in A. oryzae, and that amyR is about 1.5 kb upstream of agdA and transcribed in the opposite direction. Furthermore, transcriptional analysis revealed that the amyR gene was expressed in the presence of glucose comparable to the level in the presence of maltose, while the amylolytic genes were transcribed at high levels only in the presence of maltose.

Amino Acid Sequence↗
PubMed2000

[Expression of glycoprotein gene of the rabies virus 3aG strain by E-3 deleted adenovirus recombinant].

OBJECTIVE: To analyze Chinese rabies virus vaccine strain 3aG glycoprotein (GP) gene and further produce GP by E3-deleted human adenovirus recombinant. METHODS: Chinese rabies virus vaccine strain 3aG glycoprotein gene was cloned by RT-PCR and its sequence was determined by DNA sequencing. Cotransfection was performed to obtain adenovirus recombinant. The expressed glycoprotein was examined by ELISA and its immunogenicity was evaluated by testing neutralizing antibody level of mice inoculated with the recombinant virus. RESULTS: DNA sequencing showed that the open reading frame of GP gene contains 1,575 nucleotides and five of the deduced amino acids are different from the previous report. The recombinant adenovirus containing GP gene in E3 region was obtained by cotransfecting 293 cells and rounds of plaque purification. ELISA assay demonstrated that the GP gene can be efficiently expressed and rapid fluorescent focus inhibition test (RFFTT) showed it can also elicit GP specific neutralizing antibody. CONCLUSIONS: Chinese rabies virus vaccine strain 3aG glycoprotein was successfully expressed by E3-deleted human adenovirus recombinant and specific neutralizing antibody can be elicited in mice after immunization by the recombinant.

Adenovirus E3 Proteins↗
PubMed2004

[Cloning and expression of flagellin gene from a Chinese Borrelia burgdorferi PD91 strain].

OBJECTIVE: To study the cloning and expression of flagellin gene from Chinese Borrelia burgdorferi, PD91 strain and to evaluate the feasibility of using recombinant protein as diagnostic antigen when comparing the gene sequence with flagellin gene from North American Borrelia burgdorferi B31. METHODS: The piece of genes coding flagellin from Chinese Borrelia burgdorferi PD91 by polymerase chain reaction (PCR) method was obtained, and constructed recombinant plasmid, before transformed into E. coli BL21 strain, and induced. The recombinant plasmid was identified with enzyme cutoff and gene sequence comparison. Efficient expression strain was selected and the expression product was analyzed with sodium amplified polymorphic-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot method. RESULTS: The recombinant protein (r-flagellin) expressed in host bacteria was successful. By means of western-blot assay, the immunological response showed the same antigenicity between r-flagellin and PD91 flagellin. The piece of genes coding flagellin of PD91 was 1011 bp, but when comparing with that of North American Borrelia burgdorferi it showed 94.70% homology. Homology between the sequence of amino acid of the r-flagellin and that of B31 flagellin was 95.85%. CONCLUSION: Flagellin gene of Borrelia garinii of Chinese Lyme disease spirochete was successfully cloned and expressed for the first time. It was proved that the immunoreactivity of r-flagellin was the same as the natural flagellin.

Amino Acid Sequence↗
PubMed1986

Structural and functional analysis of chicken U4 small nuclear RNA genes.

Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion.

Animals↗
PubMed1990

Compartmentalization of mammalian proteins produced in Escherichia coli.

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.

Amino Acid Sequence↗
PubMed1999

Introduction and expression of the cry1Ac gene of Bacillus thuringiensis in a cereal-associated bacterium, Bacillus polymyxa.

The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic.

Animals↗
PubMed1992

Transcription of the H19 gene in differentiating cytotrophoblasts from human placenta.

Placental differentiation is closely correlated with the appearance of specific proteins, yet factors regulating cytotrophoblast differentiation are unknown. One strategy employed to search for such factors makes use of differential screening of cDNA libraries. For this purpose, cytotrophoblasts were isolated from human term placentae and cultured for 24 and 120 hr. cDNA libraries were constructed from the cell's RNA, and differential screening resulted in the isolation of three identical clones highly expressed after 120 hr. A DNA sequencing of 139 bp at the 3' end of these clones and a search of the data bank revealed that the sequence was identical to the parallel domain in the human H19 gene. This highly conserved gene is unusual in that it may not encode a protein. In the mouse, its RNA was shown to accumulate to high levels in embryonic tissues of endodermal and mesodermal origin. Our present findings imply that, in humans, the H19 gene is efficiently expressed in placental tissue and differentiated cytotrophoblasts, which are of ectodermal origin. RNA blot hybridization revealed a unique bimodal pattern of expression for the H19 gene in cultured cytotrophoblasts. The modulation in expression of H19 during cytotrophoblast growth was not due to the increase in the number of multinuclear cells. Size fractionation of cytotrophoblasts by centrifugal elutriation revealed that H19 expression is correlated with the stage of cell differentiation. We therefore propose that H19 transcripts might play a regulatory role in the process of cytotrophoblast differentiation.

Animals↗
PubMed1990

Expression of functional factor VIII in primary human skin fibroblasts after retrovirus-mediated gene transfer.

We have developed a retroviral-vector system for the transfer and expression of a cloned blood clotting factor VIII cDNA. Since inclusion of the complete cDNA into existing vectors is precluded by its large size, we deleted most codons for the B-domain, which is also excised during in vivo maturation of factor VIII. When inserted into the retroviral vector M5-neoR (Laker, C., Stocking, C., Bergholtz, V., Hess, N., DeLamarter, J. F., and Ostertag, W. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8458-8462), the sequence was shown to be efficiently expressed in murine fibroblast cell lines, as well as in primary human skin fibroblasts. Upon infection of murine fibroblast cell lines, clones containing only a single copy of the integrated vector-provirus secreted up to 125 milliunits of factor VIII antigen/10(6) cells/day. Equivalent amounts were found in a factor VIII activity assay, which signifies that the factor VIII protein secreted by the infected fibroblasts is fully functional. Primary human skin fibroblasts infected with the vector virus secreted up to 30 milliunits/10(6) cells/day.

Animals↗
PubMed1999

CCR2-64I polymorphism is not associated with altered CCR5 expression or coreceptor function.

A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3' to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4(+) T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels.

Blood Donors↗
PubMed1995

Expression of genomic and cDNA transgenes after co-integration in transgenic mice.

In general, genomic transgenes are expressed efficiently in mice, while their cDNA-based transgenes are frequently silent. Clark et al. (1992) have shown that silent cDNA transgenes under the control of the sheep beta-lactoglobulin promoter can be activated after co-injecting them with a genomic sheep beta-lactoglobulin transgene. We have tested the general utility of this concept using mouse whey acidic protein (WAP) transgenes. Here we show that WAP cDNA transgenes are virtually silent in transgenic mice. In contrast, WAP transgenes containing all introns are expressed in approximately 50% of the lines at levels ranging from 1% to more than 100% of the endogenous RNA (McKnight et al., 1992). When a WAP-genomic transgene was co-injected with a WAP-cDNA, basal activation of the cDNA was possible. However, the activity of the WAP-cDNA transgene did not exceed 1% of the WAP-genomic transgene. This suggests that a permissive integration site capable of supporting basal level transcription can be established, but further events are required for full activation of the transgene.

Animals↗
PubMed2000

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations.

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.

Chromatography, Liquid↗
PubMed2001

Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy.

A long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Ligand-directed tumor targeting of cationic liposome-DNA complexes (lipoplexes) is showing promise for targeted gene delivery and systemic gene therapy. Lipoplexes directed by ligands such as folate, transferrin or anti-transferrin receptor scFv, showed tumor-targeted gene delivery and expression in human breast, prostate, head and neck cancers. The two elements, ligand/receptor and liposome composition, work together to realize the goal of functional tumor targeting of gene therapeutics. The tumor suppressor gene, p53, has been shown to be involved in the control of DNA damage-induced apoptosis. Loss or malfunction of this p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy or radiation. The systemically delivered ligand-liposome-p53 gene therapeutics resulted in efficient expression of functional wild-type p53, sensitizing the tumors to chemotherapy and radiotherapy. This is a novel strategy combining current molecular medicine with conventional chemotherapy and radiotherapy for the treatment of cancer. The systemic delivery of normal tumor suppressor gene p53 by a non-viral, tumor-targeted delivery system as a new therapeutic intervention has the potential to critically impact the clinical management of cancer.

Animals↗
PubMed2000

DRAL is a p53-responsive gene whose four and a half LIM domain protein product induces apoptosis.

DRAL is a four and a half LIM domain protein identified because of its differential expression between normal human myoblasts and the malignant counterparts, rhabdomyosarcoma cells. In the current study, we demonstrate that transcription of the DRAL gene can be stimulated by p53, since transient expression of functional p53 in rhabdomyosarcoma cells as well as stimulation of endogenous p53 by ionizing radiation in wild-type cells enhances DRAL mRNA levels. In support of these observations, five potential p53 target sites could be identified in the promoter region of the human DRAL gene. To obtain insight into the possible functions of DRAL, ectopic expression experiments were performed. Interestingly, DRAL expression efficiently triggered apoptosis in three cell lines of different origin to the extent that no cells could be generated that stably overexpressed this protein. However, transient transfection experiments as well as immunofluorescence staining of the endogenous protein allowed for the localization of DRAL in different cellular compartments, namely cytoplasm, nucleus, focal contacts, as well as Z-discs and to a lesser extent the M-bands in cardiac myofibrils. These data suggest that downregulation of DRAL might be involved in tumor development. Furthermore, DRAL expression might be important for heart function.

Animals↗
PubMed2005

A biologic role of HIF-1 in the renal medulla.

BACKGROUND: Activation of hypoxia-inducible factor-1 (HIF-1) is the primary defensive mechanism against hypoxia. HIF-1 activation generally occurs in pathologic disruption of tissue oxygenation. However, a biologic role of HIF-1 in the medulla of the kidney, which is considered perpetually hypoxic under physiologic conditions due to its unique circulation, remains to be elucidated. METHODS: The expression of HIF-1alpha was detected by immunohistochemical analysis. Functional studies of HIF in medulla were carried out by gene transfer of various plasmids by retrograde injection via ureter. RESULTS: Our immunohistochemical analysis detected HIF-1alpha in the inner stripe and the inner medulla of normal rats. Water deprivation increased the number of HIF-1alpha-positive cells, which may be mediated by an increase in medullar workload and a decrease in local blood flow. To perform functional studies, we performed gene transfer. Efficient expression of the transgene was confirmed using an enhanced green fluorescent protein (E-GFP) expressing vector. Our histologic and immunoblotting analysis detected the transgene product at the inner medulla and the inner stripe 48 hours after injection. Administration of negative-dominant HIF induced severe damage in the medulla of normal rats. In contrast, gene transfer of constitutively active HIF (HIF/VP16) induced expression of various HIF-regulated genes and protected the medulla against ischemic insults. CONCLUSION: Our studies demonstrated a crucial role of HIF in the renal medulla under normal and hypoxic circumstances.

Animals↗
PubMed2001

ATP production and efficiency of human skeletal muscle during intense exercise: effect of previous exercise.

The aim of the present study was to examine whether ATP production increases and mechanical efficiency decreases during intense exercise and to evaluate how previous exercise affects ATP turnover during intense exercise. Six subjects performed two (EX1 and EX2) 3-min one-legged knee-extensor exercise bouts [66.2 +/- 3.9 and 66.1 +/- 3.9 (+/-SE) W] separated by a 6-min rest period. Anaerobic ATP production, estimated from net changes in and release of metabolites from the active muscle, was 3.5 +/- 1.2, 2.4 +/- 0.6, and 1.4 +/- 0.2 mmol ATP x kg dry wt(-1) x s(-1) during the first 5, next 10, and remaining 165 s of EX1, respectively. The corresponding aerobic ATP production, determined from muscle oxygen uptake, was 0.7 +/- 0.1, 1.4 +/- 0.2, and 4.7 +/- 0.4 mmol ATP x kg dry wt(-1) x s(-1), respectively. The mean rate of ATP production during the first 5 s and next 10 s was lower (P < 0.05) than during the rest of the exercise (4.2 +/- 1.2 and 3.8 +/- 0.7 vs. 6.1 +/- 0.3 mmol ATP x kg dry wt(-1) x s(-1)). Thus mechanical efficiency, expressed as work per ATP produced, was lowered (P < 0.05) in the last phase of exercise (39.6 +/- 6.1 and 40.7 +/- 5.8 vs. 25.0 +/- 1.3 J/mmol ATP). The anaerobic ATP production was lower (P < 0.05) in EX2 than in EX1, but the aerobic ATP turnover was higher (P < 0.05) in EX2 than in EX1, resulting in the same muscle ATP production in EX1 and EX2. The present data suggest that the rate of ATP turnover increases during intense exercise at a constant work rate. Thus mechanical efficiency declines as intense exercise is continued. Furthermore, when intense exercise is repeated, there is a shift toward greater aerobic energy contribution, but the total ATP turnover is not significantly altered.

Adenosine Triphosphate↗
PubMed2005

[Immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans].

OBJECTIVE: To investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans. METHODS: Ni-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA. RESULTS: Under the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h. CONCLUSION: The expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.

Bacterial Outer Membrane Proteins↗