Effects of various cooking conditions on acrylamide formation in rolled patty.

In this study on acrylamide formation, the effects of the type of frying oil, frying period and covering with egg during frying of a rolled patty (a traditional Turkish carbohydrate-rich food) were investigated. The differences between frying periods were statistically significant for each oil (p < 0.01). For comparable frying periods, the maximum acrylamide content was found in the rolled patties fried with sunflower oil, and the minimum acrylamide content was found in the rolled patties fried with corn oil. A decrease of 39-65% in acrylamide formation in the rolled patties covered with egg was found for each of the three types of oil. In addition, a high linear correlation (R > 0.90) was found between L (light) values and acrylamide amounts.

Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection.

Primary T cell activation requires B7-CD28 and CD40-CD154 costimulation, but effector T cell functions are considered to be largely independent of these costimulatory pathways. Although blockade of costimulation with cytolytic T lymphocyte-associated antigen 4-immunoglobulin (CTLA-4-Ig) or monoclonal antibody (mAb) to CD154 prolongs allograft survival, chronic rejection follows, which suggests that additional key costimulatory pathways are active in vivo. We found that both antibody to inducible costimulator (anti-ICOS) and an ICOS-Ig fusion protein suppressed intragraft T cell activation and cytokine expression and prolonged allograft survival in a manner similar to that in ICOS-/- allograft recipients. The combination of anti-ICOS therapy and cyclosporin A led to permanent engraftment. In addition, ICOS-B7RP-1 costimulation was required for the development of chronic rejection after CD40-CD154 blockade. These data demonstrate a key role for the ICOS-B7RP-1 pathway in acute and chronic rejection and highlight the benefits of targeting this pathway in combination with the use of conventional immunosuppressive agent.

The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE.

The inducible costimulatory molecule (ICOS) is expressed on activated T cells and participates in a variety of important immunoregulatory functions. After the induction of experimental allergic encephalomyelitis in SJL mice with proteolipid protein (PLP), brain ICOS mRNA and protein were up-regulated on infiltrating CD3+ T cells before disease onset. ICOS blockade during the efferent immune response (9-20 days after immunization) abrogated disease, but blockade during antigen priming (1-10 days after immunization) exacerbated disease. Upon culture with PLP and compared with immunized controls, splenocytes produced either decreased interferon-gamma (IFN-gamma, in efferent blockade) or excessive IFN-gamma (in priming blockade). PLP-specific immunoglobulin G1 was decreased in animals treated with anti-ICOS during antigen priming, but not in other groups.

The 5' flanking region of the human bone morphogenetic protein-7 gene.

We describe here the cloning and characterization of the 5' flanking region of the human Bone Morphogenetic Protein-7 (BMP-7) gene from a 3.3 kb genomic DNA fragment. Functional analysis by transient transfection using the luciferase reporter gene indicated that this region had a low basal promoter activity in human Wilm's tumor derived renal (G401) and rat osteoblast (ROS 17/2.8) cell lines. Sequential deletion analysis of the promoter revealed sequences whose presence correlated with decreased expression of the reporter gene. Coexpression of transcription factors involved in epithelial/mesenchymal interactions during kidney and eye development dramatically stimulated the expression of the reporter gene from the putative BMP-7 promoter. Finally, a subset of agents that upregulated the expression of the reporter gene from the cloned promoter were also shown to increase the expression of the endogenous BMP-7 in G401 and ROS cell lines in vitro.

Osteogenic protein-1 mRNA in the uterine endometrium.

OP-1, a bone morphogenetic protein (BMP) in the TGF-beta superfamily, is expressed at high levels in the kidney and in the endometrium of the uterus of non-pregnant mice. During pregnancy the OP-1 mRNA in the endometrium rapidly declined at 4 dpc. Thereafter, OP-1 transcripts were detected in the trophoblastic giant cells of the placenta and the fetal tissues. The uterine OP-1 mRNA downregulation could be mimicked by administration of 17 beta-estradiol but not by progesterone to non-pregnant animals. In contrast, OP-1 mRNA expression in kidneys and ovaries was not affected by pregnancy or estrogen treatment. The selective effect of estrogen on OP-1 mRNA in the uterus suggests that OP-1 expression is regulated by tissue specific mechanisms.

Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development.

Osteogenic protein-1 (OP-1; BMP-7) is a member of the bone morphogenetic protein subfamily. Because members of the TGF-beta superfamily have a role in tissue development, the distribution of OP-1 expression in developing human embryos (5-8 gestational weeks) and fetuses (8-14 gestational weeks) and mouse (9.5-17.5 gestational days) fetuses was examined. Northern hybridization with specific OP-1 probes revealed two mRNA species of 4 and 2.2 KB. Highest levels of OP-1 mRNA were found in human fetal kidney and heart between 12-14 weeks of gestation. By in situ hybridization, the OP-1 transcripts were found in various tissues, i.e., the ectodermal epithelium of the mouse fore- and hindlimbs, heart, teeth, intestinal epithelium, perichondrium, hypertrophic chondrocytes, and periosteum/osteoblast layer of developing human bones. In kidneys, transcripts were first detected in the epithelium of the branching uretheric buds, whereas at later stages glomeruli were the major site of OP-1 mRNA accumulation. These data suggest that, although OP-1 has been isolated from bone matrix, it may have additional regulatory roles in the morphogenesis and/or function of the kidney, limb bud, tooth, heart, and intestine.

Osteogenic protein-2. A new member of the transforming growth factor-beta superfamily expressed early in embryogenesis.

Osteogenic protein-2, OP-2, a new member of the transforming growth factor-beta (TGF-beta) superfamily, closely related to the osteogenic/bone morphogenetic proteins, was discovered in mouse embryo and human hippocampus cDNA libraries. The TGF-beta domain of OP-2 shows 74% identity to OP-1, 75% to Vgr-1, and 76% to BMP-5, hence OP-2 may also have bone inductive activity. The genomic locus of OP-2 has seven exons, like OP-1, and spans more than 27 kilobases (kb). In the C-terminal TGF-beta domain, OP-2 has a unique additional cysteine. Mouse embryos express relatively high levels of OP-2 mRNA at 8 days, two species of 3 and 5 kb. A careful study of mRNA expression of the osteogenic proteins in specific organs revealed discrete mRNA species for BMP-3, BMP-4, BMP-5, and BMP-6/Vgr-1 in lung or liver of young and adult mice. OP-1 is expressed in kidney; however, OP-2 and BMP-2 mRNAs were not detected in any organs studied, suggesting an early developmental role.

Murine osteogenic protein (OP-1): high levels of mRNA in kidney.

The murine OP-1 gene (EMBL accession No. X56906) encoding the homolog of human osteogenic protein-1 was isolated from cDNA and genomic libraries using human OP-1 cDNA as probe. The deduced murine OP-1 amino acid sequence revealed 11 amino acids changes, three of them in the mature protein. Murine OP-1 probes were used for analysis of OP-1 mRNA in mouse embryo and organ tissues. Northern blot hybridization revealed multiple mRNA species: the major species of 2.2 kb, minor species of 1.8 and 2.4 kb and a large 4 kb species, which may represent alternative splices. Tissue specific expression was studied in brain, lung, heart, liver, spleen, kidney, adrenal and bladder tissue. Maximal levels of OP-1 mRNA were found in kidney which may explain the phenomenon of epithelial osteogenesis, first described by Huggins in 1931 using epithelium from the urinary tract. Moreover, our data suggest that kidneys may be the main site of OP-1 synthesis, even though it is distant from its physiological site of action, skeletal bone.

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily.

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.

OP-1 cDNA encodes an osteogenic protein in the TGF-beta family.

Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.

The yeast ubiquitin genes: a family of natural gene fusions.

Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.

The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses.

Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI1, UBI2, and UBI3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, UBI4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UBI4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4/UBI4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rapidly, apparently a novel phenotype in yeast. Furthermore, ubi4/ubi4 diploids are sporulation-defective. ubi4 mutants are also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the UBI4 gene is similarly induced by either heat stress or starvation. These results indicate that UBI4 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.

The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein.

Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly-Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.

Isolation of protein uH2A using a one step preparative gel electrophoresis.

A one step electrophoretic procedure for the isolation of protein uH2A has been devised which may improve the overall yield. The improvement involves elimination of intermediate steps which might result in the decrease of the yield. The method may serve as an alternate to the conventional methods and can also be used successfully for the isolation of several different proteins.