cENS-1/cERNI genes have been shown to be expressed very early during chicken embryonic development and as well as in pluripotent chicken embryonic stem (CES) cells. We have previously identified a promoter region, which is specifically active in CES cells compared to differentiated cells. In order to understand the molecular mechanisms which regulate the cENS-1/cERNI promoter, we analyzed the cis-acting elements of this promoter in CES and differentiated cells. We identified a short sequence, named the B region, 5'-CAAG TCCAGG CAAG-3', that exhibits a strong enhancer activity in CES and differentiated cells. Mutation of the B region in the whole cENS-1 promoter strongly decreases the promoter activity in CES cells, suggesting that this region is essential for activating the promoter. The B region is similar to the previously described response element for the transcription factor CP2 and we show by supershift experiments that a protein complex containing CP2 is bound to this B response element. All these results identify a nuclear factor belonging to the CP2 transcription factor family that is crucial for the activation of the cENS-1/cERNI promoter. The pattern of expression of cCP2 in early chicken embryo before gastrulation is very similar to that of cENS-1/cERNI which strongly suggests that cCP2 also plays an essential role in gene expression early in embryonic development.

To identify the complement control protein (CCP) module(s) of C2 that are required for C4b recognition, we constructed a panel of C2/factor B chimeras by substituting intact or partial factor B CCP modules for the corresponding ones of C2. Epitope mapping indicated that the anti-C2b mAb 3A3.3, which inhibits binding of C2 to C4b, reacts with the second CCP of C2 and similarly the anti-Ba mAb HA4-1A, which inhibits binding of factor B to C3b, reacts with the second CCP of factor B. The hemolytic activity of the chimeras CP1, CP2, and CP3a containing CCP1, CCP2, and a fragment of CCP3 of factor B, respectively, was substantially decreased compared with that of wild-type C2. The CP3 and CP1-3 chimeras, in which CCP3 and all three CCP modules of factor B, respectively, were substituted, had no hemolytic activity. Loss of activity could be attributed to the resistance of these two chimeras to C1s cleavage, which was probably due to conformational changes of the cleavage site. The combined results indicate that all three CCP modules of C2 contribute structural elements to the C4b-binding site of C2b. This site has been shown previously to be necessary for the initial binding of C2 to C4b which leads to the formation of the classical pathway C3 convertase.

CP2 is a member of a family of transcription factors that regulate genes involved in events from early development to terminal differentiation. In an effort to understand how it selects its target genes we carried out a database search, and located several CP2 binding motifs in the promoter region of bone morphogenetic protein-4 (BMP4). BMP4 is a key regulator of cell fate and body patterning throughout development. For the CP2 binding motifs in BMP4 promoter region to be relevant in vivo, CP2 and BMP4 should be expressed together. We found that CP2b and CP2c, two potent transcriptional activators, are expressed in a manner similar to BMP4 during osteoblast differentiation of C3H10T1/2 cells. In in vitro assays, the CP2 proteins bound to two CP2 binding motifs (-715 to -676 and -147 to -118) in the BMP4 promoter, and luciferase reporter assays indicated that this binding was essential for transcription of BMP4 during osteoblast differentiation. Taken together, our data indicate that CP2b and CP2c play important roles during bone development by activating BMP4 transcription.

We treated 12 patients with chronic myelogenous leukemia (CML) with a low-intensity preparative regimen followed by allogeneic stem cell transplantation in an attempt to confer a curative graft-versus-leukemia (GVL) effect with minimum morbidity. Seven patients in first chronic phase (CP1) and five in second chronic phase (CP2) (age 15-68 years) received a nonmyeloablative conditioning regimen of fludarabine and cyclophosphamide, followed by a G-CSF-mobilized peripheral blood stem cell (PBSC) transplant from an HLA-identical sibling. Cyclosporine (CsA) was used for graft-versus-host disease (GVHD) prophylaxis. Median follow-up was 384 days. Neutrophil recovery occurred at a median of 12 days. There was no transplant-related mortality. Of the seven CP1 patients transplanted, seven achieved a stable molecular remission; two with no post-transplant intervention, three after donor lymphocytes, imatinib and interferon, and two after a myeloablative stem cell transplant. Four of five CP2 patients died in blast crisis and one survived in molecular remission. Of the 12 patients with durable engraftment, six had Grades II-IV acute GVHD; six had limited chronic GVHD. These results suggest that cytoreduction is required to optimize the curative effect of allogeneic stem cell transplantation for CML.

Soy sauce is a traditional fermented seasoning in Asian countries, that has high antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to identify the major antioxidants present, using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations in DSS. However, most of the antioxidant activity was present in colored fractions, two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their structural characteristics based on nuclear magnetic resonance (NMR) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydrate-containing pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS.

The parasitic protozoon Trichomonas vaginalis produces multiple forms of cysteine proteinase (CP). The molecular basis for this has now been examined by cloning DNA fragments encoding CPs. Using generic degenerate oligonucleotide primers based on two well-conserved regions within the central region of all eukaryotic CPs, several polymerase chain reaction fragments were isolated from T. vaginalis genomic DNA and shown to encode different CPs. One fragment with a well-represented sequence was used as a general probe to screen a T. vaginalis cDNA library at moderate stringency and five different cDNA clones were isolated. Preliminary sequencing showed that they encoded similar but distinct CPs. In the process of confirming the 5' end of one of these cDNA clones using RACE-PCR (rapid amplification of cDNA 5' ends-polymerase chain reaction), an additional sequence encoding a different CP was identified. The corresponding clone (TvCP3) and the three longest clones from the library screen (TvCP1, TvCP2 and TvCP4) were characterized further. TvCP1 and TvCP2 were full-length and TvCP3 and TvCP4 were apparently slightly less than full-length. Comparison of the predicted amino acid sequences of the four clones showed that TvCP1 and TvCP4 are related (72% identity). TvCP2 is closer to TvCP1 (60%) and TvCP4 (65%) than is TvCP3, which has 53%, 59% and 56% identity to TvCP1, TvCP2 and TvCP4, respectively. Comparison with the sequences of other known CPs indicated that the T. vaginalis gene products all belong to the cathepsin L/cathepsin H/papain branch of the papain superfamily. The TvCP1, TvCP2 and TvCP4 sequences are related (38-45% identity) to those of CP2 of Dictyostelium discoideum, human cathepsin L, three CPs from lobster and CPs from black gram, oilseed rape and rice (oryzains alpha and beta). TvCP3 shows less identity to the other eukaryotic CPs but is most similar to D. discoideum CP2 (38%). The four predicted amino acid sequences share some features distinct from the majority of CPs, which suggests they might have had a common evolutionary origin. The most striking feature of sequences TvCP1, TvCP2 and TvCP3 is the apparent lack of a pre-sequence (signal sequence) for TvCP1 and very short pre-sequences for TvCP2 and TvCP3. Southern analysis indicated that the organization of the genes corresponding to the TvCP cDNAs differs. The TvCP1, TvCP2 and TvCP3 genes are single-copy, whereas the TvCP4 gene appeared to be multiple-copy.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous reports have suggested that the repressor gene, c, of phiC31 is autoregulated and that likely operators are conserved inverted repeat sequences (CIRs1&2) located just upstream of the promoters, cp1 and cp2. Evidence is now presented that the CIRs 1&2 are indeed binding sites for one of the three inframe, N-terminally different protein isoforms of 42, 54 and 74 kDa produced by the c gene. A cp1-aphII fusion was repressed in a Streptomyces coelicolor A3(2) phiC31 lysogen and characterisation of an operator-constitutive (Oc) mutant showed a single mutation in CIR-1. CIR-1 containing fragments were retarded in electrophoresis gels by the 42 kDa repressor protein isoform and this retardation was inhibited by the addition of competing DNA fragments containing either CIR-1 or CIR-2. Using a combination of Southern blotting and analysis of available DNA sequence we also show that at least 18 copies of the CIRs are present throughout the phiC31 genome. Alignment of 9 CIR sequences showed that 8 contained a perfectly conserved 17 bp core whilst the exception had a single mismatch. The core includes a 16 bp inverted repeat (IR), and is usually part of a more extensive and less highly conserved palindrome. When superimposed on a previously derived transcription map of the early region, the CIRs lie in intergenic regions associated with transcription initiation and/or termination.

Despite improvements in HLA typing, graft-versus-host disease (GVHD) continues to impair the results after volunteer unrelated donor bone marrow transplantation (VUD-BMT) in adult patients compared with matched sibling BMT. Here, the outcome after VUD-BMT using a specific regimen with high-dose anti-T-lymphocyte globulin (ATG) was analysed. Fifty-five adult patients, median age 34 years (range 17-55 years), with acute or chronic leukaemia or myelodysplastic syndrome (MDS) were transplanted in first complete remission (CR1)/first chronic phase (CP1) (early disease) (n = 21) or in advanced (CR2/CP2, no remission) disease (n = 34) from an unrelated marrow donor. GVHD prophylaxis consisted of ATG-S (Fresenius) 60-90 mg/kg b.w. prior to transplantation, in addition to cyclosporin A and short-course methotrexate. Graft failure did not occur and white blood cell count (WBC)>> 1.0 x 10(9)/l was reached at median day +16. The cumulative incidence of acute (a)GVHD grade II-IV was 15% [95% CI (8%, 28%)] and of chronic GVHD was 51% [95% CI (38%, 68%)]. The cumulative incidence of relapse within 1 year was 0% [95% CI (0%, 19%)] and 21% [95% CI (11%, 40%)] for patients with early and advanced disease respectively. With a median follow-up of 28 months (range 16-45 months), 2-year disease-free and overall survival for patients transplanted in CR1/CP1 was 81% and 81% [95% CI (64%, 98%)], respectively, and for patients with advanced disease was 33% [95% CI (17%, 50%)] and 40% [95% CI (23%, 57%)] respectively. Complete and persistent donor chimaerism was seen in 77.5% of 40 patients evaluated. All 14 chronic myeloid leukaemia (CML)-CP1 patients became bcr-abl negative within 250 d. High-dose ATG pretransplant results in a low incidence of severe aGVHD without compromising donor chimaerism or elimination of minimal residual disease. Our results are similar to data obtained after matched sibling donor transplantation.

Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range. The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign. The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center. The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus. The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated. Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances. These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster. A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences. A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site. These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Dictyostelium discoideum is a simple eukaryote that lives as an amoeba until starvation triggers aggregation. The aggregate forms a slug which then develops into a fruiting body with two main cell types, stalk and spore cells. Two mechanisms have been proposed to explain cell-type differentiation. Studies using expression of the ecmA gene as a prestalk cell marker indicated that gradients of morphogens determine cell fate in the slug. However, studies using dyes or the cysteine proteinase 2 (CP2) gene product as a prestalk cell marker indicated that cell autonomous factors such as cell-cycle phase at the time of starvation cause an initial choice of cell fate. To help resolve these differences, we have used transformed cells containing the promoter of the prestalk gene ecmA fused to beta-galactosidase (Jermyn and Williams, 1991) to study the differentiation of Dictyostelium cells at low cell density, at which cell-to-cell interactions and morphogen gradients are minimal. We find that under all conditions of low cell density in which express ion the ecmA fusion gene occurs, it is invariably detected in less than 25% of the cells from a clonal population. This suggests that a cell-autonomous mechanism is involved in ecmA expression. We then used double-labeled immunofluorescence to examine the ontogeny of the CP2-positive and the ecmA-positive cells. In developing aggregates, 9 to 12% of the cells are CP2-positive from 12 to 24 hr of development. The ecmA-positive cells are first detected at 16 hr as a subset of the CP2-positive cells and then increase in number. At approximately 20 hr, the CP2-positive cells and the ecmA-positive cells are almost completely overlapping sets. By late development, all of the CP2-positive cells are ecmA-positive and an additional 10% of the CP2-negative cells are also ecmA-positive. This indicates that up to 20 hr development, ecmA is expressed only in CP2-positive cells. The data thus suggest that cell-cycle phase at the time of starvation causes an initial choice of cell type and that during later development other factors influence cell fate.

We have previously purified and characterized a rat liver protein C'BP-1 that is either identical or closely related to C/EBPdelta (Aniskovitch and Jacob, 1997). The mouse metallothionein-I (MT-I) promoter contains two C'BP-1 binding sites, one of which includes the MRE-c' region (-135 to -110). The C'BP-1 binding activity was detected by EMSA as a major activity for MRE-c' in nonproliferating adult liver cells but not in rat hepatoma cells. In this study, we purified and characterized a factor, C'BP-2, which had a dominant binding activity for MRE-c' in Morris hepatoma 3924A, a poorly differentiated, fast-growing tissue. C'BP-2 is a 28 kDa protein which exists in solution as a monomer. As observed for C'BP-1, affinity-purified C'BP-2 stimulated transcription from the mMT-I gene promoter. DNase I footprinting revealed two C'BP-2 binding sites in the regions that overlap with the CCAAT homologies of the C'BP-1 binding sites on the mMT-I promoter. C'BP-2 made essential contacts with the CCAAT homology and in the region upstream of this sequence. Competition electrophoretic mobility shift assay and methylation interference analysis revealed that C'BP-2 is a protein closely related, but not identical, to CP2. These data suggest that C'BP-1 and C'BP-2 may play a role in hepatocyte proliferation and/or differentiation.

Antigens of the Schistosoma mansoni digestive tract are recognized early in the infective process. Two immunogenic components of the excretory/secretory products are proteolytic enzymes that degrade host hemoglobin in the lumen of the parasite gut. These enzymes, CP1 and CP2, belong to the class of cysteine proteinases. In this study, a preparation containing both proteinases has been used to detect proteinase antibodies in the sera of individuals living in Burundi. Of 133 individuals tested, 92% were excreting schistosome eggs. All patients with documented infections had positive anti-proteinase IgG titers (mean = 1:614), while 82% had positive IgM titers (mean = 1:267). Six weeks following praziquantel treatment, patients were assessed for egg excretion and antibody titer. Anti-proteinase IgG titers were significantly lower (mean = 1:259) than pre-treatment titers. Patients who were infected with S. japonicum or S. haematobium typically showed a cross-reactive IgG response. Patients from non-endemic regions yielded negative titers, and those with non-trematode parasites were negative (79%) or weakly positive. S. mansoni cysteine proteinases may be used for the detection of schistosome infections.

Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na(+)-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (-1551 to +117) was cloned and characterized. The ASIC2 promoter lacked a canonical TATA box, but contained one putative CCAAT box. Nucleotide sequencing of the promoter revealed the presence of a number of transcription factor-binding sites and a 404 bp CpG island upstream the transcription start site. Nested deletion mutants and transfection results showed that the construct between -133 and +117 base pairs conferred basal transcription specific activity. Mutation of Sp1 and CP2 sites in this region resulted in a 70 and 95% decrease in promoter activity, respectively. Gel shift assays demonstrated the existence of specific protein binding to the SP1 and CP2 elements. There was no mutation in the CpG island in six glioma cell lines, but methylation-specific PCR showed methylation in some of glioma cell lines and tumor tissues, and treatment with the methylation inhibitor 5-Aza-2'-deoxycytidine could partially restore ASIC2 expression in cell lines, suggesting that epigenetic mechanisms may contribute to dysregulated ASIC2 expression.

Three thylakoid complexes were isolated by deoxycholate preparative electrophoresis. The protein composition of each fraction was analyzed by SDS analytical electrophoresis. No protein of the PS 1 enriched fraction (fraction 1) was found in the PS 2 enriched fraction (fraction 2) and inversely. The antenna complex (fraction 3) did not have any contamination by proteins of fraction 1 or fraction 2. Fraction 1 was mainly composed of the CP1, the reaction center complex of the PS1, and by low molecular weight proteins, previously found in other PS 1 preparations. Tentative assignments of these proteins are presented; among them are iron sulfur proteins. After analytical SDS electrophoresis of fraction 2, the reaction center complex was dissociated. Nevertheless three proteins of 50 kD, 42 kD and 35 kD were assigned to this complex. Fraction 2 contained also the three cytochromes of the thylakoid membranes: cyt f, cyt b6, cyt b559. Fraction 3 was exclusively composed of one protein pigment complex, CP2.

Hydrogels containing alpha-amino acid residues (L-phenylalanine, L-histidine) were used to complex the chemotherapeutic agent cisplatin. The release of the drug in phosphate buffer solution showed an initial burst effect, followed by a near zero-order release phase over the seven days of reported period. Unlike the nonreleasing pattern of the hydrogel poly(N-acryloyl-L-phenylalanine-co-N-isopropylacrylamide) (CP2), the homopolymer poly(N-acryloyl-L-phenylalanine) (P9) hydrogel showed a released amount of cisplatin loaded from a water/DMSO mixture that was three times greater than that loaded from simple water. The hydrogel P9 formed with cisplatinum(II) complex species of well-defined stoichiometry; the drug species was released by a chemically controlled process. The Pt(II)/L (L is the monomeric unit of the polymer) stoichiometric molar ratio of 0.5, corresponding to two close carboxylate groups per Pt(II), was found by the viscometric data on the soluble polymer analogue. The platinum species released from cisplatin-loaded (from water) hydrogel retained its cytotoxic activity toward Me665/2/21 human melanoma cell line, in the same manner shown by the native cisplatin. On the contrary, the platinum species released from cisplatin-loaded (from water/DMSO) hydrogel was devoid of any cytotoxic effect.

Cerebral peptide 2 (CP2), a 41 amino acid neuropeptide, was identified because it was transported from the cerebral ganglia of Aplysia to other central ganglia. Immunocytology indicates that CP2 is distributed widely in the CNS and peripheral tissues of Aplysia. Most CP2-immunoreactive neurons were found in the cerebral ganglia and extensively overlap with the distribution of cerebral peptide 1 (CP1). HPLC analyses confirm that individual cerebral neurons synthesize both CP1 and CP2. In other ganglia, CP1 and CP2 are localized predominantly to different neurons. CP2-immunoreactive fibers and varicosities are present in the neuropil of all ganglia but were found surrounding cell bodies and axon hillocks most often in the buccal and abdominal ganglia. Thus, the effects of CP2 on neurons in these ganglia were determined using intracellular recording. In the buccal ganglia, CP2 evokes rhythmic activity in many motor neurons that seems similar to that observed during ingestion; however, only one identified neuron was found to be depolarized directly. By contrast, in the abdominal ganglion, many neurons are depolarized directly by CP2. A number of these have been shown to be part of the circuit that regulates respiratory pumping. Injection of CP2 into freely behaving Aplysia increases the rate of respiratory pumping and causes other changes in behavior. CP2 is stable in hemolymph, which raises the possibility that it may act as a hormone. Thus, CP2 is a bioactive neuropeptide that is present in many neurons and likely functions as a transmitter or a hormone.

Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.

Upon starvation, cells of the simple eukaryote Dictyostelium discoideum aggregate and differentiate into several cell types. Two main cell types are prestalk and prespore, which later usually become stalk and spore cells. The differentiation is plastic, and several factors can alter cell-type ratios. Two mechanisms have been proposed to regulate the initial cell type. We and others have proposed that cell type is initially determined by cell-cycle phase at the time of starvation: prestalk cells are derived from cells which, are the time of starvation, happen to be in a roughly 2-hr-long sector of the cell cycle which overlaps S and early G2 and that certain extracellular factors are then used to maintain the proper prestalk:prespore ratio and to control later stages of development such as the prestalk-to-stalk conversion. To examine the relationship between initial cell-type choice and the cell cycle, and how this 2-hr-long sector is generated, we increased the length of S phase by mild treatments of cells with DNA-synthesis inhibitors. When the fraction of the cell cycle occupied by S phase is increased and the cells are then starved, the prestalk:prespore ratio increases. This increase was observed using two markers for prestalk cells, CP2 and ecmA::lacZ. In addition, there is a close correlation between the fraction of the cell cycle occupied by S phase and the prestalk:prespore ratio, irrespective of total cell-cycle length. These results validate the hypothesis that the initial choice of cell type is determined by cell-cycle phase at the time of starvation, and indicate that the cell-type choice mechanism monitors the cell cycle rather than using an independent 2-hr-long timer started at the beginning of S phase.

Due to evolutionary divergence, cattle (taurine, and indicine) and buffalo are speculated to have different responses to heat stress condition. Variation in candidate genes associated with a heat-shock response may provide an insight into the dissimilarity and suggest targets for intervention. The present work was undertaken to characterize one of the inducible heat shock protein genes promoter and coding regions in diverse breeds of Indian zebu cattle and buffaloes. The genomic DNA from a panel of 117 unrelated animals representing 14 diversified native cattle breeds and 6 buffalo breeds were utilized to determine the complete sequence and gene diversity of HSP70.1 gene. The coding region of HSP70.1 gene in Indian zebu cattle, Bos taurus and buffalo was similar in length (1,926 bp) encoding a HSP70 protein of 641 amino acids with a calculated molecular weight (Mw) of 70.26 kDa. However buffalo had a longer 5' and 3' untranslated region (UTR) of 204 and 293 nucleotides respectively, in comparison to Indian zebu cattle and Bos taurus wherein length of 5' and 3'-UTR was 172 and 286 nucleotides, respectively. The increased length of buffalo HSP70.1 gene compared to indicine and taurine gene was due to two insertions each in 5' and 3'-UTR. Comparative sequence analysis of cattle (taurine and indicine) and buffalo HSP70.1 gene revealed a total of 54 gene variations (50 SNPs and 4 INDELs) among the three species in the HSP70.1 gene. The minor allele frequencies of these nucleotide variations varied from 0.03 to 0.5 with an average of 0.26. Among the 14 B. indicus cattle breeds studied, a total of 19 polymorphic sites were identified: 4 in the 5'-UTR and 15 in the coding region (of these 2 were non-synonymous). Analysis among buffalo breeds revealed 15 SNPs throughout the gene: 6 at the 5' flanking region and 9 in the coding region. In bubaline 5'-UTR, 2 additional putative transcription factor binding sites (Elk-1 and C-Re1) were identified, other than three common sites (CP2, HSE and Pax-4) observed across all the analyzed animals. No polymorphism was found within the 3'-UTR of Indian cattle or buffalo as it was found to be monomorphic. The promoter sequences generated in 117 individuals showed a rich array of sequence elements known to be involved in transcription regulation. A total of 11 nucleotide changes were observed in the promoter sequence across the analyzed species, 3 of these changes were located within the potential transcription factor binding domains. We also identified 4 microsatellite markers within the buffalo HSP70.1 gene and 3 microsatellites within bovine HSP70.1. The present study identified several distinct changes across indicine, taurine and bubaline HSP70.1 genes that could further be evaluated as molecular markers for thermotolerance.

The TFCP2/Grainyhead family of transcription factors is divided into two distinct subfamilies, one of which includes the Grainyhead-like 1-3 (GRHL1-3) proteins and the other consists of TFCP2 (synonyms: CP2, LSF, LBP-1c), TFCP2L1 (synonyms: CRTR-1, LBP-9) and UBP1 (synonyms: LBP-1a, NF2d9). Transcription factors from the TFCP2/TFCP2L1/UBP1 subfamily are involved in various aspects of cancer development. TFCP2 is a pro-oncogenic factor in hepatocellular carcinoma, pancreatic cancer and breast cancer, may be important in cervical carcinogenesis and in colorectal cancer. TFCP2 can also act as a tumor suppressor, for example, it inhibits melanoma growth. Furthermore, TFCP2 is involved in epithelial-mesenchymal transition and enhances angiogenesis. TFCP2L1 maintains pluripotency and self-renewal of embryonic stem cells and was implicated in a wide variety of cancers, including clear cell renal cell carcinoma, breast cancer and thyroid cancer. Here we present a systematic review of current knowledge of this protein subfamily in the context of cancer. We also discuss potential challenges in investigating this family of transcription factors. These challenges include redundancies between these factors as well as their interactions with each other and their ability to modulate each other's activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the pig industry worldwide. Currently available commercial vaccines provide limited protection due to delayed and weak cell-mediated immunity and neutralizing antibody production, thus the immunomodulators should be considered in order to improve the efficacy of PRRSV vaccines. Heat shock protein gp96 may be used as a modulator to enhance both innate and adaptive immune responses. In the present study, two multi-epitope subunit vaccines, named as Cp1 and Cp2, were designed based on the conserved B cell epitopes of viral proteins with the N-terminal 22-370 amino acids (aa) of porcine gp96 (Gp96N) chosen as the adjuvant. Immune responses elicited by the different combinations of Cp1/Cp2 and Gp96N were examined in mice and piglets. The results indicated that the group of Cp1/Cp2-Gp96N (CG) combination induced 3-4-fold higher titers of Cp1/Cp2-ELISA antibodies and neutralizing antibodies (NAs) in mice than the groups which received Cp1/Cp2 immunization alone or with Freund's adjuvant. Additionally, Gp96N significantly enhanced the levels of lymphocyte proliferative responses of splenocytes or peripheral blood mononuclear cells from vaccinated mice or piglets. The production of IFN-γ in mice splenocytes, TNF-α, IFN-γ, and IL-12 in sera of piglets were also remarkably increased with the treatment of Gp96N, while IL-4 was reduced by half and IL-10 was decreased to an undetectable level. These results suggest that the porcine Gp96N could effectively enhance the innate and adaptive immune responses of Cp1/Cp2 with a Th1-type bias. Therefore, the multi-epitope subunit vaccine Cp1/Cp2 co-administered with porcine Gp96N might potentially be a promising candidate vaccine for the prevention and control of PRRSV in pigs.

In cycling, critical power (CP) and work above CP (W') can be estimated through linear and nonlinear models. Despite the concept of CP representing the upper boundary of sustainable exercise, overestimations may be made as the models possess inherent limitations and the protocol design is not always appropriate.

OBJECTIVE

To measure and compare CP and W' through the exponential (CPexp), 3-parameter hyperbolic (CP3-hyp), 2-parameter hyperbolic (CP2-hyp), linear (CPlinear), and linear 1/time (CP1/time) models, using different combinations of TTE trials of different durations (approximately 1-20min).

METHODS

Repeated measures.

METHODS

Thirteen healthy young cyclists (26±3years; 69.0±9.2kg; 174±10cm; 60.4±5.9mLkg-1min-1) performed five TTE trials on separate days. CP and W' were modeled using two, three, four, and/or five trials. All models were compared against a criterion method (CP3-hyp with five trials; confirmed using the leaving-one-out cross-validation analysis) using smallest worthwhile change (SWC) and concordance correlation coefficient (CCC) analyses.

RESULTS

CP was considerably overestimated when only trials lasting less than 10min were included, independent of the mathematical model used. Following CCC analysis, a number of alternative methods were able to predict our criterion method with almost a perfect agreement. However, the application of other common approaches resulted in an overestimation of CP and underestimation of W', typically these methods only included TTE trials lasting less than 12min.

CONCLUSIONS

Estimations from CP3-hyp were found to be the most accurate, independently of TTE range. Models that include two trials between 12 and 20min provide good agreement with the criterion method (for both CP and W').

BACKGROUND

Effective therapies have increased life expectancy of human immunodeficiency virus (HIV)-infected pediatric patients. We investigated the underlying causes of death, mortality, and acquired immune deficiency syndrome (AIDS) rates in HIV-infected pediatric patients in Madrid, Spain.

METHODS

We studied a multicenter cohort of 478 HIV-infected pediatric patients in Madrid. Mortality and AIDS incidence rates, causes of death, CD4 T-cell, and HIV RNA were analyzed during calendar periods (CPs): pre-HAART (highly active antiretroviral therapy) (CP1: 1982-1996) and post-HAART era (CP2: 1997-2009).

RESULTS

During 5690 person-years of follow-up 157 (32.8%) deaths occurred. Median age at death increased (CP1: 3.2 years [1.0-6.3] vs. CP2: 7.7 years [3.1-11.4]; P < 0.01). Mortality and AIDS rates decreased 10.6-fold (95% confidence intervals [CI]: 6.9-16.7) and 6.9-fold (95% CI: 5.0-9.6), respectively, between CPs. Nevertheless, mortality was 10.4-fold (95% CI: 5.8-18.8; P < 0.001) higher than in age-similar general population in late-CP2. In all, 169 causes of death were reported. Multiple causes were reported in 16 of 151 (10.6%) patients. In 81.1% (137/169), the causes were AIDS-defining, 11.8% (20/169) HIV-related, and 7.1% (12/169) non-HIV-related. Infections were the leading causes (60.8%, 101/166); from 1999 to 2007 the risk of death from infections was 115.9 times (95% CI: 42.0-265.8; P < 0.001) higher than in the age-similar general population. Comorbidity was reported in 66.9% (101/151) of patients. Median HIV-1 RNA at death decreased (CP1: 5.9 [5.0-6.3]; CP2: 5.3 [4.2-5.8]; P < 0.01).

CONCLUSIONS

Despite decline in mortality and AIDS rates, it is important to monitor all causes of death as prolonged survival might allow underlying comorbidity to become more clinically relevant.