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Mutations in the expression vector which encodes the lipase-solubilized bovine liver microsomal cytochrome b5 facilitate the development of a recombinant human erythrocyte cytochrome b5 and its expression in Escherichia coli, for defining its functional and physical features. The human cytochrome b5 protein consists of two extra His residues than the bovine cytochrome b5 protein. The midpoint reduction potential of the human recombinant form is -9 plus or minus 2 mv and depends on the value of pH. One-dimensional H NMR spectroscopy of the ferricytochrome form of the erythrocyte reveals the occurrence of 90% of major orientation and 10% of minor orientation binding between the protein and heme.

Megasphaera elsdenii reveals a short-chain acyl-CoA dehydrogenase (SCAD), which exhibits a molecular mechanism. The functioning is controlled by cloning and ordering the gene coding for SCAD. The suggested functioning needs an amino acid residue that is recognized by site-directed mutagenesis. Identification of the amino acid order of the 50 N-terminal residues of SCAD is required to clone the gene. Two groups of varying oligonucleotide primers are prepared by the order of the data, and a 120-bp specific probe from Megasphaera elsdenii DNA is produced by a polymerase chain reaction process.

Researchers assembled the coding sequence for human serum transferrin from restriction fragments developed from a complete cDNA clone isolated from a human liver cDNA library. The assembled clone was introduced into the expression vector pNUT and stably transfected into altered baby hamster kidney (BHK) cells, resulting in secretion of around 125 mg/L recombinant protein into the tissue culture medium.

Gene expression is governed by combinatorial processes that are complex and adequately flexible. The use of the polymerase chain reaction and degenerate oligonucleotides in the study of regulatory processes improved the knowledge of protein-protein interactions in the transcription factor. The approach is called Cyclic Amplification and Selection of Targets and is useful in the identification of binding sites. These technique is limited to the complexes that show affinity to DNA but is very valuable in the study of the control of gene expression.

A study was done on the cyclic amplification and selection of targets for multicomponent complexes. Results show that casting for multicomponent complexes indicate that myogenin can cooperatively interact, directly or indirectly with many factors including one member of the NF1 family, MEF2, COMP1 and as many as five other bHLH factors or complexes. The interactions increase the affinity of myogenin binding as well as the transcription of reporter constructs expressed in differentiating myotubules.

Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving β-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases. [ABSTRACT FROM AUTHOR]

The R-spondin (Rspo) protein family is a recently described group of four distinct human secreted proteins. Reported activities for Rspo proteins include essential roles in vertebrate development and their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt signaling result in the activation of beta-catenin. In a general functional screen for human secreted proteins using transgenic mouse models, we identified human R-spondin1 (hRspo1) protein as a potent and specific mitogen for the gastrointestinal epithelium and demonstrated a potential therapeutic application for the protein in mouse models of cancer therapy-induced mucositis. In contrast to previous studies, our data indicated only partial overlap between Wnt and Rspo ligand activities, suggesting that there may be independent receptor/signaling pathways for Rspo proteins that intersect those of Wnt at the level of beta-catenin. Here we summarize the current reported data on the Rspo family and discuss these results in terms of alternate mechanisms of action. We have extended our observations on the potential therapeutic application of Rspo proteins by showing that all four human Rspo family members are capable of inducing epithelial proliferation and report the first non-vertebrate Rspo family member.