Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.

Recombinant human platelet factor-4 (rhPF4), purified from Escherichia coli, inhibited blood vessel proliferation in the chicken chorioallantoic membrane in a dose-dependent manner. Treatment of several cell types with rhPF4 in vitro suggested that the angiostatic effect was due to specific inhibition of growth factor-stimulated endothelial cell proliferation. The inhibitory activities were associated with the carboxyl-terminal, heparin-binding region of the molecule and could be abrogated by including heparin in the test samples, an indication that sulfated polysaccharides might modulate the angiostatic activity of platelet factor-4 in vivo. Understanding of the mechanisms of control of angiogenesis by endogenous proteins should facilitate the development of effective treatments for diseases of pathogenic neovascularization such as Kaposi's sarcoma, diabetic retinopathy, and malignant tumor growth.

Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities.

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain.

2'Fluoro-5-iodo-arabinosyl-cytosine: a new agent for herpes infections in the immunosuppressed patient.

Concurrent with our increased understanding of mechanisms of viral replication, new antiviral agents were developed with greater selectivity and sensitivity. Acyclovir was the first of these. We now present the initial compound of a series of 2'-Fluoro pyrimidine nucleosides with potent antiviral activity. This agent demonstrates both potency and sensitivity in vitro and in vivo against herpes simplex virus types I and II, and varicella zoster virus and is selective in vitro against cytomegalovirus. Initial clinical trials show the drug to be well tolerated and to be more effective than adenine arabinoside against varicella zoster in the immunosuppressed patient. Future developmental plans with the drug are outlined. FIAC (2'-Fluoro-5-iodo-aracytosine) is still at an early stage of clinical development and any comparison with acyclovir is premature; rather, therapy of severe viral infections in the future should consider combinations of such agents that show differential selectivity at multiple sites of action.

Role of the N- and C- terminal dimer interfaces of 434 repressor in recognizing sequence-dependent DNA structure.

Abstract The binding of proteins to specific DNA sequences plays a central role in the regulation of gene expression. Crucial to understanding how these proteins exert their effects is insight into the structure and flexibility of the protein-DNA complex. Over the past several years much has been learned about how the intimate contacts made between proteins and DNA enable proteins to recognize and bind with high specificity only to their cognate DNA binding sites. Studies conducted in our laboratory have shown that sequence-specific binding of DNA by proteins not only involves the close approach of amino acids and base pairs in the binding site, but also that base pairs not in contact with the protein affect binding and specificity through sequence-specific effects on DNA structure. The direct reading of the DNA sequence by proteins occurs by chemical complementarity between the interacting groups. Proper alignment of the interacting surfaces of functional groups on the protein and DNA molecules is crucial to the formation of stable and specific protein-DNA complexes. In many cases, the appropriate juxtaposition of the chemically complementary groups requires mutual adjustments in the structure of protein and DNA. Failure to do so can result in loss of affinity, loss of specificity or both. Together, the dimer interface and noncontacted bases within or adjacent to the binding site direct the structural complementarity between the functional groups on the protein and DNA.

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.

2'-Fluoro-5-iodoarabinosylcytosine, a new potent antiviral agent: efficacy in immunosuppressed individuals with herpes zoster.

2'-Fluoro-5-iodoarabinosylcytosine (FIAC) has potent antiviral activity in vivo against herpes simplex virus types 1 and 2 and cytomegalovirus. For examination of the clinical efficacy of FIAC, a randomized, double-blind study of FIAC versus adenine arabinoside (ara-A) was conducted in 34 immunosuppressed individuals with varicella-zoster virus infections. The median time to the appearance of the last new lesion was shorter in patients who received FIAC relative to those who received ara-A (two versus five days, respectively; P less than .001) FIAC also reduced pain and accelerated initial crusting within 72 hr in a significantly greater proportion of patients when compared with ara-A (P = .004 and P = .0009, respectively). FIAC caused few toxic reactions (mild nausea and transient elevation in activity of serum aspartate aminotransferase). Thus FIAC is therapeutically superior to ara-A for the treatment of varicella-zoster virus infections in immunosuppressed subjects.