Active variants of human parathyroid hormone (1-34) with multiple amino acid substitutions.

We used site-directed mutagenesis to construct 55 single-site variants of rhPTH, a recombinantly-expressed form of human parathyroid hormone (1-34) containing three amino acid changes compared to the natural sequence (ML8, ML18 and FY34). We identified several mutations, at residues Lys(13), Glu(19), Val(21), Glu(22), Lys(27) and Asp(30), that increase biological activity by up to 2. 5-fold, as measured by stimulation of adenylate cyclase activity in rat UMR-106 cells. We constructed a series of 15 variants in which two to eight substitutions at these positions were combined, and found that the mutations behaved additively, leading to peptides with significantly enhanced potency. The most active combination variant, with six substitutions (KS13, ES19, VQ21, ES22, KQ27 and DN30), is 15 times more active than the parent molecule. However, the extent to which such combinations increase the activity of the peptide depends critically on the identity of the residues at positions 8 and 18. We constructed two of the combination variants in a variety of sequence backgrounds containing different combinations of leucine, methionine and norleucine at positions 8 and 18. Enhancements in potency were significantly reduced when Met or Nle was present at either of these positions, both in UMR-106 cells and human SaOS-2 cells. A corresponding non-additivity was observed in direct measurements of receptor binding affinity on UMR-106 cells. These results suggest that interactions, either direct or indirect, between certain PTH side chains prevent these mutations from behaving in an additive manner.

MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening.

The goal of this work was to investigate the use of MDCK (Madin-Darby canine kidney) cells as a possible tool for assessing the membrane permeability properties of early drug discovery compounds. Apparent permeability (Papp) values of 55 compounds with known human absorption values were determined using MDCK cell monolayers. For comparison, Papp values of the same compounds were also determined using Caco-2 cells, a well-characterized in vitro model of intestinal drug absorption. Monolayers were grown on 0. 4-microm Transwell-COL membrane culture inserts. MDCK cells were seeded at high density and cultured for 3 days, and Caco-2 cells were cultured under standard conditions for 21 to 25 days. Compounds were tested using 100 microM donor solutions in transport medium (pH 7.4) containing 1% DMSO. The Papp values in MDCK cells correlated well with those in Caco-2 cells (r2 = 0.79). Spearman's rank correlation coefficient for MDCK Papp and human absorption was 0.58 compared with 0.54 for Caco-2 Papp and human absorption. These results indicate that MDCK cells may be a useful tool for rapid membrane permeability screening.

Destabilization of bacteriophage T4 mRNAs by a mutation of gene 61.5.

We identified a novel gene of bacteriophage T4, gene 61.5, which appears to be involved in protein synthesis late in infection. Northern blot analysis revealed that a mutant of 61.5 accumulated truncated transcripts of representative late genes. Using a double mutant of genes 61.5 and 55, which prevents transcription of late genes, we demonstrate that even transcripts of middle genes, while full-length when initially expressed, are similarly truncated at later stages of infection. These results indicate that the abnormality in transcript length occurs late in infection, regardless of whether the transcript derives from a middle or a late gene. Primer-extension analysis revealed that the 5' ends of the late gene 23 transcripts that accumulated in gene 61.5 mutant-infected cells were located at internal discrete sites as well as at the expected transcription start site. Moreover, the decay rates of full-length transcripts from genes uvsY or 45 were more than twofold faster in the absence of a functional gene 61.5. These results suggest that mutation of gene 61.5 activates endonucleolytic cleavage of middle and late transcripts, probably by RNase M.

Identification of residues critical to the activity of human granulocyte colony-stimulating factor.

Alanine scanning mutagenesis of human granulocyte colony-stimulating factor (G-CSF) was used to identify residues critical for the cell-proliferative activity of the protein. Fifty-eight residues, most of them on the protein surface, were independently mutated to alanine. Most of the variants retained full biological activity; however, 15 mutants were significantly impaired in their ability to stimulate bone marrow cell proliferation in vitro. Four of these variants contain mutations at buried residues and two have substitutions at side chains involved in intramolecular hydrogen bonds. The remaining nine down mutations identify two regions on the surface of the molecule important for biological activity. Consistent with these observations, measurements of binding to NFS-60 cells indicate that the residues most important for receptor binding are Lys40 and Phe144 in site 1 and Glu19 in site 2. In addition to these residues, Val48 and Leu49 in site 1 and Leu15, Asp112, and Leu124 in site 2 are also important for biological activity. These results suggest the presence of two binding sites on the cytokine surface required for dimerization of the G-CSF receptor.

Iontophoretic delivery of oligonucleotides across full thickness hairless mouse skin.

In recent years there has been an increased interest in the use of oligonucleotides as therapeutic agents. Oligonucleotide therapeutics may have significant potential over traditional drugs due to their high degree of specificity and increased affinity. The major drawbacks to the use of oligonucleotide therapeutics are the problems associated with their delivery and their relative instability in serum. The serum instability problem has been partially overcome through the use of oligonucleotides with modified backbones. Transdermal electrotransport may be used to overcome the problems associated with delivery. Here we report the use of transdermal electrotransport in the delivery of oligonucleotides across hairless mouse skin. The effects of pH, salt concentration, current density, and oligonucleotide concentration, structure, and length have been investigated.

A method for the high-level expression of a parathyroid hormone analog in Escherichia coli.

A recombinant parathyroid hormone analog, rPTH(1-34*), was obtained from Escherichia coli using a gene polymerization strategy. The PTH gene polymer contains up to 8 copies of the gene, each separated by a cleavable linker. The polymer was expressed at very high levels and formed inclusion bodies which could be easily isolated by low-speed centrifugation. A polyhistidine leader peptide allows rapid purification via nickel chelation chromatography of the PTH polymer solubilized from the inclusion bodies. Yields of greater than 500 mg/liter have been obtained. After isolating the polymer, monomeric rPTH(1-34*) is released from the polymer by chemical cleavage with cyanogen bromide. Following cyanogen bromide cleavage and high-performance liquid chromatography purification, highly purified, biologically active rPTH(1-34*) is obtained at a yield of approximately 300 mg/liter. This is a general strategy for the high-level production of a variety of peptides and small proteins.

Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6.

In bacteriophage T4, there is a strong tendency for genes that encode interacting proteins to be clustered on the chromosome. There is 1.6 kb of DNA between the DNA helicase (gene 41) and the DNA primase (gene 61) genes of this virus. The DNA sequence of this region suggests that it contains five genes, designated as open reading frames (ORFs) 61.1 to 61.5, predicted to encode proteins ranging in size from 5.94 to 22.88 kDa. Are these ORFs actually genes? As one test, we compared the DNA sequence of this region in bacteriophages T2, T4, and T6 and found that ORFs 61.1, 61.3, 61.4, and 61.5 are highly conserved among the three closely related viruses. In contrast, ORF 61.2 is conserved between phages T4 and T6 yet is absent from phage T2, where it is replaced by another ORF, T2 ORF 61.2, which is not found in the T4 and T6 genomes. As a second, independent test for coding sequences, we calculated the codon base position preferences for all ORFs in this region that could encode proteins that contain at least 30 amino acids. Both the T4/T6 and T2 versions of ORF 61.2, as well as the other ORFs, have codon base position preferences that are indistinguishable from those of known T4 genes (coefficients of 0.81 to 0.94); the six other possible ORFs of at least 90 bp in this region are ruled out as genes by this test (coefficients less than zero). Thus, both evolutionary conservation and codon usage patterns lead us to conclude that ORFs 61.1 to 61.5 represent important protein-coding sequences for this family of bacteriophages. Because they are located between the genes that encode the two interacting proteins of the T4 primosome (DNA helicase plus DNA primase), one or more may function in DNA replication by modulating primosome function.

A humanized antibody that binds to the interleukin 2 receptor.

The anti-Tac monoclonal antibody is known to bind to the p55 chain of the human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementarity-determining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 10(9) M-1, about 1/3 that of murine anti-Tac.

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome.

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.

Inhibition of two homeotic mutants of Drosophila by 5-bromodeoxyuridine and fluorouracil.

Nasobemia (Ns) and spineless-aristapedia (ssa40a) are dominant and recessive homeotic mutants of Drosophila which convert parts of the antenna to leg structures. Exposure of Ns and ssa40a larvae to half-lethal concentrations of 5-bromodeoxyuridine (BUdR) and flourouracil (FU) together or separately during the presumptive time of gene action suppresses the expressivity and penetrance of the mutants.