An ab initio molecular orbital analysis of phosphorothioate mono-anion.

Ab initio calculations at the STO-3G level show that the energy of the highest occupied molecular orbital (HOMO) of the di-protonated phosphorothioate mono-anion is 0.0421 a.u. higher than that of the HOMO of the corresponding phosphate mono-anion. The electron density in the phosphorothioate HOMO is highly focused on the sulfur atom. The results of this ab initio molecular orbital analysis are consistent with the model of the phosphorothioate sulfur as a soft nucleophile. The ab initio molecular orbital analysis presented in this paper will be useful for predicting and rationalizing the probability of reaction of phosphorothioate anions with electrophiles and helps to provide a basis for the rational design of phosphorothioate-based oligonucleotide drugs.

IL-5: biology and potential therapeutic applications.

IL-5 is the predominant cytokine associated with antigen-induced eosinophilic inflammation in the lung. The activation of Th-2 cells leads to the production of IL-5. The pro-eosinophilic effects of IL-5 include: (1) enhanced replication and differentiation of eosinophilic myelocytes; (2) enhanced degranulation of eosinophils; (3) prolonged survival time of eosinophils: and (4) enhanced adhesion of eosinophils. The effects of IL-5 are mediated via the interaction of IL-5 with receptors (IL-5R) that are expressed on the eosinophil cell membrane. Intracellular signalling produced by occupation of the IL-5R by IL-5 occurs via the JAK-STAT system. IL-5 is a 45 kDa glycoprotein consisting of two identical polypeptide chains. The 5'-promoter region of the IL-5 gene contains elements that are down-regulated by glucocorticoids. Anti-IL-5 reagents have the potential to suppress IL-5 activity without the side effects of glucocorticoids. Studies using monoclonal antibodies (mAbs) against IL-5 have established the feasibility of suppressing eosinophilic inflammation by specifically blocking IL-5 activity. Studies with antisense IL-5 are beginning to provide the basis for non-glucocorticoid, sequence-specific oligonucleotide inhibitors of IL-5. Research has begun on the development of mAbs and antisense oligonucleotide inhibitors of IL-5 that can be inhaled and applied topically.

Transcription of 2'-deoxy-2'-fluoro-modified and phosphorothioate-modified RNA templates by HIV-1 reverse transcriptase.

RNA templates yield the corresponding DNA in the presence of human immunodeficiency virus reverse transcriptase (HIV-1 RT). The purpose of this study was to determine whether RNA that was modified with either 2'-deoxy-2'-fluoro analogs or with internucleotide phosphorothioate linkages could serve as templates for HIV-1 RT. Modified RNA that contained either 2'-deoxy 2'-fluoro pyrimidine nucleoside analogs or internucleotide phosphorothioate diester linkages 5'- to pyrimidine nucleosides were enzymatically synthesized and tested for template activity with recombinant HIV-1 RT. RNA that was modified with either 2'-deoxy-2'-fluorouridine or with internucleotide phosphorothioate linkages 5'- to pyrimidines yielded full length HIV-1 reverse transcription products, with complete fidelity in transcription. RNA that was modified with 2'-deoxy-2'-fluorocytidine, either alone or in combination with 2'-deoxy-2'-fluorouridine, did not function as templates for HIV-1 RT, under the conditions reported here. The ability of 2'-deoxy-2'-fluoro-modified and phosphorothioate-modified RNA to serve as template for the RNA-dependent DNA polymerase of HIV-1 RT has not hitherto been reported.

Interleukin-5: a proeosinophil cytokine mediator of inflammation in asthma and a target for antisense therapy.

Interleukin-5 (IL-5) is the predominant cytokine associated with antigen-induced eosinophilic inflammation in the lung. The activation of TH2 cells leads to the production of IL-5. The proeosinophilic effects of IL-5 include 1) enhanced replication and differentiation of eosinophilic myelocytes; 2) enhanced degranulation of eosinophils; 3) prolonged survival time of eosinophils; and 4) enhanced adhesion of eosinophils. The effects of IL-5 are mediated via the interaction of IL-5 with receptors (Il-5R) expressed on the eosinophil cell membrane. Intracellular signaling produced by occupation of the IL-5R by IL-5 occurs via the JAK-STAT system. IL-5 is a 45kD glycoprotein that consists of two identical polypeptide chains. The 5'-promoter region of the IL-5 gene contains elements that are down-regulated by glucocorticoids. A 16-mer deoxyoligonucleotide, antisense to IL-5 mRNA and with two phosphorothioate modifications, produced, at 20 micromolar concentration, complete inhibition of IL-5 secretion by human peripheral blood mononuclear cells. The targeted 16-mer sequence of the IL-5 mRNA did not display complete homology with any other known human gene sequences. These results suggest that the 16-mer phosphorothioate antisense IL-5 provides the basis for a non-glucocorticoid, sequence-specific inhibitor of IL-5.

A semi-empirical Hückel molecular orbital analysis of internucleotide phosphorothioate diesters.

Oligonucleotides that contain internucleotide phosphorothioate diesters have potential as anticancer therapeutic agents through sequence-specific antisense mechanisms. Although the chemical reactivity of internucleotide phosphorothioate diesters has been utilized to form phosphorothioate oligonucleotide conjugates, the theoretical basis for this chemical reactivity needs further elucidation. Calculations reported here, performed by the Huckel molecular orbital method, show that the energy of the highest occupied molecular orbital (HOMO) of the internucleotide phosphorothioate diester is 15.9 kcal/mole higher than that of the corresponding HOMO of the internucleotide phosphate diester. The electron density in the phosphorothioate HOMO is almost completely centered on the sulfur atom. The results of this parametricized Huckel molecular orbital analysis are consistent with the model of the internucleotide phosphorothioate sulfur as a soft nucleophile.

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.

Internalization of monoclonal antibodies selected for immunotoxin activity against small-cell lung cancer.

Two hybridomas producing MOABs with anti-SCLC activity were selected for immunotoxin activity by an indirect screen and were twice cloned. Binding activity of the MOABs to SCLC cells was demonstrated by immunoperoxidase activity, which could be blocked by streptavidin. The MOABs mediated the internalization of a biotinylated Fab' anti-mouse Ig marker at 37 degrees C. Internalization of the biotinylated marker by the SCLC target cells resulted in protection of the marker from streptavidin-blocking. These results show that MOABs selected for immunotoxin activity against SCLC can mediate internalization of an antibody fragment with a mass about 50% greater than that of the toxin. MOABs selected for immunotoxin activity may be useful for delivering agents other than toxins to the inside of SCLC cells.

Laboratory tests for total and allergen-specific immunoglobulin E.

The principles of measurement of IgE by enzyme-immunoassay are given. A Bayesian approach is taken for the clinical interpretation of serum total IgE. Application of Bayes' Theorem to serum total IgE can significantly influence the probability of a clinical diagnosis of allergy. There is a correlation between allergen-specific IgE measured by enzyme-immunoassay, by radioallergosorbent test and by skin tests for immediate hypersensitivity. However, there is a need to standardize the reagents used for allergy skin testing, as well as the reagents and methods used for the laboratory measurement of allergen-specific IgE.

Rapid screening with indirect immunotoxin for monoclonal antibodies against human small cell lung cancer.

For the first time, a screening procedure for antitumor monoclonal antibodies (MOABs) has been developed in which the ability of MOABs to mediate the indirect action of another immunotoxin is the primary criterion for selection of hybridomas for expansion and cloning. Use of the indirect immunotoxin makes it possible to screen MOABs for cytotoxic potential without the necessity of covalently coupling toxin to each individual MOAB. Hybridomas derived from spleens of immunized mice were screened for the synthesis of monoclonal antibodies able to participate in the delivery of a pharmacologically active, indirect immunotoxin conjugate to H69 lung cancer cells. The indirect immunotoxin conjugate was goat anti-mouse immunoglobulin disulfide linked to pokeweed antiviral protein. There was no correlation between the results of the screening with the indirect immunotoxin and an enzyme-linked immunosorbent assay screen for binding of MOAB to tumor cell membranes. An indirect immunotoxin prepared from Fab' fragments of goat anti-mouse immunoglobulin was also effective as an indirect immunotoxin conjugate. Each screening of more than 300 hybridomas was performed in 3 days. The indirect immunotoxin screen detected antitumor MOABs which were not detected by the conventional enzyme-linked immunosorbent assay method. A MOAB selected by the indirect immunotoxin screen was conjugated to pokeweed antiviral protein; the conjugate mediated immunocytotoxicity in a standard direct toxicity assay.

Diffusion control of the binding of carcinoembryonic antigen (CEA) with insoluble anti-CEA antibody.

Analysis of the kinetics of CEA binding to rabbit anti-CEA antibody insolubilized on filter paper discs provided six lines of evidence indicating diffusion control of this reaction: 1) The observed forward rate constant, k1obs, decreased when solvent viscosity was increased with sucrose or Ficoll. 2) k1obs per receptor site increased from 1.2 x 10(4) M-1 sec-1 to 2.6 x 10(4) M-1 sec-1 as receptor site density on immunosorbent discs decreased from 5.2 x 10(-13) to 2.1 x 10(-13) moles/disc. 3) Stirring the reacting mixture of CEA and insoluble anti-CEA increased k1obs from 1.2 x 10(4) M-1 sec-1 to 3.4 x 10(4) M-1 sec-1. 4) The activation energy for CEA-insoluble anti-CEA binding was indistinguishable (p greater than 0.1 by t-test) from 4.1 Kcal/mole expected for diffusion controlled reactions. 5) Bimolecular reaction theory predicted a diffusion limited forward rate constant, k1max, for CEA binding to insoluble anti-CEA, which was consistent with our observed k1 of 1.2 x 10(4) M-1 sec-1. 6) k1obs for CEA binding to soluble rabbit anti-CEA antibody was 6.4 x 10(4) M-1 sec-1, 5 times faster than the k1obs of the heterogeneous phase reaction for receptor site density of 5.2 x 10(-13) moles/disc. Diffusion control of ligand binding to insoluble receptors, as exemplified by the CEA-insoluble anti-CEA model system, may be a very general biologic phenomenon.

An analysis of the role of IgE in intolerance to aspirin and tartrazine.

Total serum IgE and eosinophil count were determined for 30 patients with intolerance to aspirin. Total IgE levels in the aspirin intolerant patients were similar to those expected in a non-atopic population. In contrast, total eosinophil count (TEC) tended to be elevated in the aspirin intolerant group. Elevated TEC was observed both in bronchospastic (57%) and in urticarial (25%) aspirin intolerance. Specific anti-aspiryl and anti-tartrazyl antibodies of the IgE class were assayed by the galactosidase immunosorbent test (GIST). IgE anti-aspiryl antibodies were possibly detected in one patient, but did not correlate with clinical intolerance to aspirin. It is unlikely that the clinical symptoms and the eosinophilia of intolerance to aspirin and tartrazine are mediated by antibodies of the IgE class.

A metallurgical examination of fractured stainless-steel ASIF tibial plates.

Between 1970 and 1973 99 tibial fractures were treated by rigid internal fixation with ASIF plates. The fractures were all regarded as sufficiently stable for exercise without weight bearing, thus needing no additional external support during the healing period. Four of the plates broke late in the healing period, after the onset of weight bearing. These fractures had some degree of delayed union with slight resorption of the bone ends, resulting in cyclical bending of the plate. Examination of 2 of the fractured plates by scanning electron microscopy, electron microprobe analysis and optical metallography revealed that the primary cause of plate fracture was fatigue. There was no evidence that corrosion fatigue or inclusion content were factors leading to plate fracture.

A galactosidase immunosorbent test for carcinoembryonic antigen.

A galactosidase immunosorbent test for carcinoembryonic antigen (CEA) is described in which the amount of galactosidase adsorbed to a cellulose disc is a hyperbolic function of CEA concentration. Thus, molecules with CEA-like activity can be characterized by mathematical analysis of data obtained from the galactosidase immunosorbent test. By such analysis, CEA-reactive molecules in normal human plasma were distinguished from normal cross-reacting antigen and from authentic CEA. Variation of the amount of antibody-enzyme conjugate used in the galactosidase immunosorbent test permitted CEA-reactive material in plasma of a patient with rectal carcinoma to be antigenically distinguished from the CEA-reactive material in urine of a patient with bladder carcinoma. The galactosidase immunosorbent test is a useful tool for analysis of CEA-reactive molecules.

Application of a galactosidase immunosorbent test to carcinoembryonic antigen in plasma.

The galactosidase immunosorbent test for carcinoembryonic antigen is simple to perform, uses stable reagents, does not require radioactive reagents, and is adaptable to large numbers of samples. Concentration of carcinoembryonic antigen in sera or plasma was determined by the galactosidase immunosorbent test and by the Egan-Todd double antibody assay (93% agreement), indirect Z-gel (83% agreement), and direct Z-gel assay (ps = 0.97). The galactosidase immunosorbent test has potential as a clinically useful nonisotopic assay for carcinoembryonic antigen.

Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells.

Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20-24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [3H]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients. Synthesis and secretion of light-chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after motosis the cells which then entered the G1 phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level.

Preparation and characterization of fluorescent N-(3-pyrene)maleimide adducts of myosin.

N-(3-pyrene)maleimide adducts of myosin (PM-myosin) are fluorescent and possess actin-activated Mg2+ ATPase activity. Addition of ATP to PM-myosin produces a reversible decrease of 10% in fluorescence intensity of the pyrene fluorophore in the presence of actin. Analogues of ATP which are poor substrates for myosin ATPase or which merely dissociate actomyosin produce less decrease in fluorescence of PM-myosin than does ATP. Since fluorescence of acto-PM-myosin is sensitive to environmental changes associated with ATP hydrolysis, and/or with fluorophore-actin interactions. PM-myosin may be a useful analysis of molecular aspects of muscle contraction.

Relatedness among contractile and membrane proteins: evidence for evolution from common ancestral genes.

A statistical method for quantifying the relatedness among proteins was used to perform 2926 paired comparisons of amino-acid composition among 77 contractile and membrane-associated proteins from diverse species and sources. Relatedness of amino-acid compositions correlates with homology of amino-acid sequence. A high degree of relatedness was detected among K(+)-dependent membrane ATPase of Streptococcus faecalis, coupling factors F(1) and CF(1) from mitochondria and chloroplasts, outer fiber protein of cilia, ciliary dynein, tubulin, various actins, and myosin subfragment S-1. Heavy meromyosin and tropomyosin were related to each other but not to the first group of proteins. Differences in the degree of methylation may account for some differences in physiological function. Because of their diverse sources, the high degree of relatedness among these proteins is more compatible with evolution from common ancestral genes than with convergent evolution. Squid axon filarin, molluscan paramyosin, and bacterial flagellins appear to be unrelated either to each other or to any of the other proteins studied. Existence of persistent homologies among so many diverse proteins implies conservation of genetic information during evolution by utilization of codons for preferred amino-acid sequences in various proteins.