Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two.

The tenth human fibronectin type three domain ((10)Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from (10)Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the biophysical properties of such antibody mimics, which will determine their suitability for in vitro and medical applications. We characterized target binding and biophysical properties of two related (10)Fn3-based antibody mimics that bind vascular endothelial growth factor receptor two (VEGF-R2). The first antibody mimic, which has a dissociation constant (K(d)) of 13 nM, is highly stable [melting temperature (T(m))=62 degrees C] and soluble, whereas the second, which binds VEGF-R2 with 40 x higher affinity, is less stable (T(m) < 40 degrees C) and relatively insoluble. We used our understanding of these two (10)Fn3 derivatives and of wild-type (10)Fn3 structure to engineer the next generation of antibody mimics, which have an improved combination of high affinity (K(d)=0.59 nM), stability (T(m)=53 degrees C) and solubility. Our findings illustrate that (10)Fn3-based antibody mimics can be engineered for favorable biophysical properties even when 20% of the wild-type (10)Fn3 sequence is mutated in order to satisfy target-binding requirements.

Expression of multiple neuregulin transcripts in postnatal rat brains.

The distribution of neuregulin transcripts in rat brains was studied by both RNA blotting and in situ hybridization. Our data demonstrate that multiple neuregulin transcripts are expressed in neurons of the basal forebrain, the hippocampus, the diencephalon, the cerebellum, the brainstem, and the spinal cord. Developmental changes in the distribution of neuregulin transcripts were observed only in the cerebellum and the hippocampus. The intense neuregulin hybridization signals in the brainstem motor and sensory nuclei and the spinal motor neurons are suggestive of a functional involvement of neuregulins in motor and sensory systems. The expression of neuregulins in other parts of the brain also indicates that these factors are involved in a variety of central nervous system functions.

Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system.

Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p185erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.

mRNAs for insulin-like growth factor-II (IGF-II) and variant IGF-II are co-expressed in human fetal ovary and uterus.

Insulin-like growth factor-II (IGF-II) is postulated to have autocrine and/or paracrine functions in developing fetal tissues, but has never been reported in human fetal reproductive organs. The forms of IGF-II found in normal human serum include a 67 amino acid form and a variant form resulting from alternate splicing of the mRNA such that Ser-29 is replaced by four other amino acid residues. We studied the expression of mRNA encoding IGF-II in human fetal ovaries and uteruses of 10, 15, 19 and 22 weeks of gestation. By reverse transcription followed by polymerase chain reaction (PCR), we identified the co-expression of two mRNAs encoding IGF-II in all developmental stages of fetal ovaries and uteruses tested. One of the PCR amplified fragments was 9 nucleotides larger than the other. The PCR amplified ovarian and uterine DNA fragments were mapped by digestion with the restriction endonucleases AvaII and PvuII and both the IGF-II fragment and the larger IGF-II fragment produced the anticipated DNA patterns by gel electrophoresis. The PCR amplified DNA fragments were cloned and sequenced to confirm that the expressed mRNAs encoded IGF-II and variant IGF-II. We conclude that IGF-II and variant IGF-II mRNA co-expression occurs in the human fetal female genital tract and that the two forms of the growth factors may have physiologic roles in reproductive tract development.

Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells.

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.