Biomedical subjects

A P Alexenko

Publications and source records attributed to A P Alexenko.

13 recordsLinked to original sources

PubMed2001

Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental development, promoter sequences of genes and biological activity of protein products.

Multiple interferon (IFN)-tau genes exist in cattle, but it has remained unclear how many are expressed, the extent of their variation, and whether different genes exhibit similar patterns of expression and code for proteins with similar biological activities. A total of 118 complementary DNA (cDNA) were bi-directionally sequenced from reverse-transcribed bovine (bo) conceptus RNA over the period from blastocyst formation until day 25 of pregnancy. Fourteen different cDNAs, encoding eight different IFN-tau, were confirmed unique. All showed high sequence conservation (>98% nucleotide identity; >96% amino acid identity). The cDNA fell into three, recently evolved, phylogenetic groups (tau1, 2, and 3). Mean concentrations of IFN-tau messenger RNA were greater at day 17 and day 19 than at day 14 and day 25, with different genes showing comparable expression patterns, although there appeared to be a major bias in expression of two genes (for boIFN-tau1c and tau3a) in blastocysts. Genes representing members of the three boIFN-tau groups were cloned. Their promoter regions were conserved over regions considered important for transcriptional activation. Recombinant protein generated in Escherichia coli from representative genes in the three groups had similar but not identical antiviral activities. In summary, many IFN-tau genes, which are probably under similar transcriptional control, are expressed in bovine trophoblast during the peri-implantation period of development.

Amino Acid Sequence↗

PubMed2000

Intracellular regulation of endometrial PGF(2a) and PGE(2) production in dairy cows during early pregnancy and following treatment with recombinant interferon-tau.

Objectives were to examine how the conceptus and recombinant bovine interferon-tau (rbIFN-tau) regulate intracellular components of the PGF(2a) synthetic pathway and to determine if arachidonic acid (AA) is limiting in endometrial tissue of pregnant cows. In Experiment 1, uteri were collected from either cyclic or pregnant dairy cows on Day 17 post-estrus. Intercaruncular explants were dissected and incubated for 60 min to quantify PGF(2a) production in response to oxytocin (10(-6) M), A23187 (10(-5) M), melittin (10(-5) M), and phorbol 12, 13 dibutyrate (PDBu, 10(-6) M). Additional explants from the same cows were incubated for 24 h with and without AA. Oxytocin and A23187 did not stimulate PGF(2a) in explants from either cyclic or pregnant cows. Both PDBu, melittin, and A23187 + melittin stimulated PGF(2a) production in explants of cyclic cows, but not in explants of pregnant cows. The addition of AA to explant cultures for 24 hr did not increase PGF(2a) production during a subsequent 60-min incubation. In Experiment 2, explants were collected from cows that received intrauterine infusions of either BSA (1.9 mg/1.2 ml) or rbIFN-tau (0.2 mg rbIFN-tau + 1.7 mg BSA/1.2 ml) twice a day from Days 14 to 17 of the estrous cycle. Treatments of rbIFN-tau attenuated PGF(2a) secretion induced by in vitro PDBu and A23187 treatments. However, rbIFN-tau treatment in vivo had no effect on the in vitro induction of PGF(2a) secretion by melittin. IFN-tau may regulate the PGF(2a) synthetic pathway by reducing activity of PKC or PKC mediated events.

Animals↗

PubMed2000

A classification for the interferon-tau.

An attempt has been made to provide a rational organization for the many interferon-tau (IFN-tau) sequences entered in GenBank based on phylogenetic analysis and common amino acid substitutions, which might form the basis for a universal nomenclature scheme. Over the 13 years since these genes were first discovered, large numbers of cDNA and gene sequences have been reported, and there is reason to suspect that representatives of all the major ovine and bovine forms have now been described. The data are consistent with the presence of many genes and also allelic variants in sheep and cattle analogous to what has been observed for the IFN-alpha in the human. Future variants should be easily accommodated into the scheme outlined here. A flexible system of nomenclature, based on that used for HuIFN, is needed to provide a common base for comparison between research done in different laboratories and to assign relative biologic potencies to these molecules.

Animals↗

PubMed1999

Trophoblast interferons.

The mechanisms responsible for prevention of corpus luteum regression during early pregnancy are diverse and appear to have arisen in concert with the evolutionary divergence of placental structure. That used by the sub-order Ruminantia is unique and involves the production of a Type I interferon (IFN), IFN-tau (tau). Although IFN-tau resembles other Type I IFNs (such as IFN-alpha, -beta, and -omega) in structure as well as in many of its biological properties, it is not virally inducible and is instead produced constitutively by embryonic trophectoderm during the period immediately prior to implantation. The transcription factor Ets-2 is a component of the regulatory mechanism involved in transcription of IFN-tau. These genes probably arose as the result of a duplication of an IFN-omega gene, 36 million years ago, at about the time the Ruminantia sub-order emerged. They have duplicated extensively since then and there may be 10 or more genes in some present-day species. The expression of different IFN-tau is unequal and they differ in biological potency. The rapid evolution of IFN-tau genes possibly reflects the placenta as a site of considerable genetic experimentation.

Animals↗

PubMed1999

The cross-species antiviral activities of different IFN-tau subtypes on bovine, murine, and human cells: contradictory evidence for therapeutic potential.

It is claimed that interferon-tau (IFN-tau) has broad cross-species reactivity and less cytotoxicity than other type I IFN when used at high concentration either in vitro or in living animals. It can also amelioriate the development of experimental allergic encephalomyelitis (EAE) without the usual side effects of IFN therapy in mice autoimmunized with myelin basic protein. For these reasons, IFN-tau may have therapeutic potential in humans. Here, the antiviral (AV) activities of eight different recombinant IFN-tau were compared with those of several bovine, human, and murine type I IFN on bovine MDBK cells, murine L929 cells, and human WISH cells. The data show that only one of the IFN-tau, OvIFN-tau4, has broad cross-species reactivity. It was comparable in this respect to HuIFN-omega1 and HuIFN-alpha1. The other IFN-tau, including the variant form (OvIFN-tau1mod) tested by others in cytotoxicity experiments and for its ability to protect mice against EAE, had relatively weak AV activity on mouse and human cells. It is possibly because this particular bioengineered form of IFN-tau binds the common type I receptor of these two species with such low affinity that it lacks cytotoxic effects. The basis for its potent anti-EAE activity is unclear, but it seems possible that it does not involve the type I IFN receptor.

Amino Acid Sequence↗

PubMed1999

Identification of the expressed forms of ovine interferon-tau in the periimplantation conceptus: sequence relationships and comparative biological activities.

Interferon-tau (IFN-tau) is secreted from trophectoderm of periimplantation ruminant conceptuses and is a critical component of pregnancy recognition. Multiple genes encode IFN-tau. The objectives of this study were to identify expressed forms of ovine IFN-tau and to compare their biological activities. Sequences analyzed after cloning 36 reverse transcription-polymerase chain reaction products of ovine conceptus RNA provided seven new cDNA that were similar in sequence to previously cloned forms (p3, p6, and p8 cDNA). Phylogenetic analysis of amino acid sequence for all new and previously reported forms showed that ovine IFN-tau forms can be divided into three main groups. Equivalent amounts of mRNA for p3, p6, and p8 forms were detected in conceptuses following RNase protection. Recombinant p3 and p8 protein had similar antiviral activity on ovine and bovine cells whereas p6 protein was less active. The p3 form was the most potent of the three in its ability to extend estrous cycle length in nonpregnant ewes. In summary, there appeared to be three main groups of ovine IFN-tau, each containing several variant forms. Antiviral activity was not particularly well correlated with ability to prevent luteolysis, suggesting that distinct intracellular mechanisms are used to exert the various actions of IFN-tau.

Amino Acid Sequence↗

PubMed1998

Different ovine interferon-tau genes are not expressed identically and their protein products display different activities.

Interferon tau (IFN-tau) proteins are secreted by the ovine conceptus for a few days before definitive attachment of the trophoblast to the uterine epithelium and act to prolong luteal life span. Multiple genes encode for IFN-tau in sheep, but it remains unclear which genes are expressed during early pregnancy and whether the proteins encoded by these genes are equipotent. Three distinct ovine (ov) IFN-tau gene variants, p3, p6, and s4, were examined to determine whether they differed in gene expression and whether the proteins displayed different biological activities. By using RNase protection assays, full-length protected fragments were detected for p3 and p6 in approximately similar proportions in conceptuses flushed from the uterus at Days 12-13, Days 15-16, and Days 18-19 of pregnancy, but the amount of full-length protected s4 transcripts was 10% to 20% of that for p3 and p6. Partially protected probe fragments were also evident, presumably from probe hybridization to related ovIFN-tau transcripts. Recombinant proteins were generated and exhibited 34.2 (p3), 8.4 (p6), and 11.9 (s4) units (x 10(-7)) of activity per milligram of protein when tested on Madin-Darby bovine kidney cells. Antiproliferative activity on human Daudi cells varied considerably between the interferons, with p3 being 2000-fold more potent than s4. The interferons were injected into the uterine lumen of ewes from 10 to 18 days postestrus. The functional life span of the corpus luteum (CL) was increased (p = 0.02) by either 300 microg/day p3 (31.7 +/- 7.8 days) or 300 microg/day p6 (25.5 +/- 4.2 days), but not by 300 microg/day s4 (19.2 +/- 2.9 days), when compared to controls (15.8 +/- 0.6 days). Injection of 1 mg/day s4 did, however, increase (p = 0.02) CL life span (23.5 +/- 4.1 days). These data suggest that IFN-tau genes are not equally expressed in trophectoderm and that ovIFN-tau genes encode for proteins with significantly different biological potency.

Animals↗

PubMed1998

Loss of the signature six carboxyl amino acid tail from ovine interferon-tau does not affect biological activity.

Interferon-tau (IFN-tau) is a type I IFN that is secreted from conceptuses of Bovidae (sheep, cattle, and related ruminant ungulates) for a few days during early pregnancy. It acts to prolong the life span of the corpus luteum. All secreted forms of IFN-tau, like the related IFN-omega, are 172 amino acids in length and differ from IFN-alpha and -beta by the presence of six additional amino acids at their carboxyl termini. The aim of this study was to determine whether this carboxyl tail was important for biological activity of IFN-tau, particularly for its antiluteolytic function in ewes. Full-length ovine IFN-tau (p3) and a mutated form truncated by six amino acids at its carboxyl terminal (p3Trn6, 166 amino acids) were produced in Escherichia coli. Both proteins had similar antiviral activities (2.12 +/- 0.92 x 10(8) IU/mg for p3; 1.96 +/- 0.58 x 10(8) IU/mg for p3Trn6) when tested on Madin-Darby bovine kidney (MDBK) cells. Antiproliferative activity, as measured on human Daudi cells by determining the protein concentration required to inhibit growth by 50%, was slightly higher (p < 0.05) for p3Trn6 (7.36 +/- 0.46 pM) than for p3 (13.99 +/- 0.85 pM). Most importantly, p3 and p3Trn6 were equally capable of prolonging the life span of the corpus luteum of nonpregnant ewes when the proteins were administered at doses of either 60 or 300 microg/day into the uterine lumen through indwelling uterine cannulae from Day 10 to Day 18 postestrus. Therefore, the carboxyl-terminal amino acid extension for IFN-tau does not appear to serve a functional role in the action of these proteins.

Animals↗

PubMed1997

The antiproliferative and antiviral activities of IFN-tau variants in human cells.

The IFN-tau are type I IFN expressed by the early trophoblast of cattle and sheep but have activity on human cells and have been predicted to have potential therapeutic value. We have compared a series of mutant bovine and ovine IFN-tau with regard to their ability to inhibit the proliferation of Daudi cells and to evoke an antiviral (AV) response in WISH cells. Whereas Daudi cell growth was inhibited by Bo-IFN-tau1 in the 1 nM range, WISH cells were much less responsive, requiring exposure to 150 nM for protection against vesicular stomatitis virus. Replacement of lysines at positions 34, 107, 121, and 132 in Bo-IFN-tau, which are in regions predicted to interact with the type I receptor, led to modest but significant alterations in antiproliferative (AP) and AV activities. Replacement of the lysine residues at 160 and 164 had marked effects on biopotency, with K160 being particularly important. The different IFN-tau were able to activate the transcription factors ISGF3 and AAF (GAF) in Daudi cells at concentrations that correlated reasonably well with their AP potencies. Stat activation occurred in WISH cells in response to approximately 2 nM Bo-IFN-tau1, but ISGF3 formation could not be demonstrated even at the 100-fold higher IFN-tau concentrations that gave viral protection. Pretreatment of WISH cells with Hu-IFN-gamma allowed ISGF3 formation to be observed in response to subsequent treatment with Bo-IFN-tau1 or type I human IFN but did not increase the AV responsiveness of the cells. No evidence was found that IFN-tau elicit uniquely different responses on human cells than type I Hu-IFN, except they are much less potent. The data emphasize the importance of a region near the carboxyl terminus for the functional activity of type I IFN, and that although ISFG3 formation may be necessary, its mere presence is not sufficient to provide an antiviral response.

Amino Acid Sequence↗

PubMed1995

A bifunctional vector suitable for both site-directed mutagenesis and recombinant expression of interferon-tau in Escherichia coli.

In order to produce workable quantities of a large number of mutant forms of recombinant ovine and bovine interferon-t (IFN-t), a bifunctional vector, pME-2, was developed, which combines a mutant selection system and a strong promoter providing controlled expression. An EcoRI/KpnI fragment containing the complete Trp promoter, a Shine-Dalgarno sequence, and an AT-rich region from the pTrp-2 expression vector was inserted into the large fragment of EcoRI/KpnI-digested pALTER-1 plasmid, which had been modified by eliminating a ClaI site. The pALTER-1 phagemid provides a highly efficient, antibiotic-dependent system for selection of mutant plaques. The existing T7 promoter was then eliminated from the recombinant phagemid to create the pME-2 vector. Ovine and bovine IFN-t genes lacking the coding region for the signal peptide, but with an ATG codon ahead of the open reading frame, were inserted into the multicloning site of pME-2. Following site-directed mutagenesis designed to produce elongations, truncations, and single and multiple amino acid replacements in the protein products, mutant genes were selected in Escherichia coli BMH 71-18 and efficiently expressed in E. coli JM-101 in response to the inducer of the Trp promoter indole acetic acid. The recombinant IFN were solubilized from washed inclusion bodies in guanidinium-HCl and 2-mercaptoethanol and allowed to refold in aerated buffer. The procedure provides high yields of fully active, homogeneous IFN-t and can be accomplished within 1 week.

Amino Acid Sequence↗

PubMed1991

Mapping of an epitope of human leukocyte alpha interferon A which is recognized by the murine monoclonal antibody NK2.

An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays. The results from these assays suggested that the epitope is within the sequence 112-121. Analysis of a hybrid protein IFN-A(1-92)/F(93-166) revealed that the N-terminal region of IFN-A played no significant role in NK2 binding. Three residues of IFN-F (Asn113, Val114, and Lys121) were substituted for the corresponding residues from IFN-A (Lys113, Glu114, and Arg121) by site-directed mutagenesis of the gene encoding IFN-F. NK2 was able to bind the mutated protein, IFN-F(A 113, 114, 121), as well as unmodified IFN-A. The data show that the epitope recognized by NK2 is located within the C-terminal region of IFN-A (residues 112-121). This epitope consists of the essential residues 114 and 116, and residues 112, 113, 115, 117, and 121 presumably contribute the configuration of the epitope.

Amino Acid Sequence↗

PubMed1990

Reconstruction of an epitope capable of binding murine monoclonal antibodies NK2 within the sequence of human leukocyte interferon alpha F by site-directed mutagenesis.

Using site-directed mutagenesis, an epitope of human leukocyte IFN-A capable of binding murine monoclonal antibodies NK2 has been reconstructed within the sequence of IFN-F, which lacked ability to bind these particular type of antibodies. It was found by the analysis of the recombinant IFN-A1-92/F93-100 encoding by a hybrid gene that N-terminal part of IFN-A played no significant role in NK2 binding. Then by site-directed mutagenesis of IFN-F gene three residues of IFN-F polypeptide chain (Asn113, Val114 and Lys121) were substituted for the residues of IFN-A sequence (Lys113, Glu114 and Arg121), and it was shown that the mutated IFN-F(A 113, 114) and IFN-F(A 113, 114, 121) restored the partial and full ability, respectively, to bind NK2 as compared with unmodified IFN-A, while IFN-F(A 121) lacked NK2 binding completely, as well as parental IFN-F. It shows that the residues Lys113 and Glu114 are crucial for NK2 binding, whereas Arg121, presumably, supports the necessary spatial arrangement of the epitope, being, nevertheless, essential for the binding of NK2.

Antibodies, Monoclonal↗

PubMed1984

The biochemical study of rat T-cell growth factor (interleukin 2).

T-cell growth factor (interleukin 2) was partially purified from the conditioned medium from Con A-stimulated rat splenocytes. It had the molecular weight of 23,000 according to gel filtration and in the range of 17,000-18,000 daltons according to PAGE in the presence of SDS. The molecular forms of IL 2 exhibited isoelectric points (pI) of 5.4-5.8 (major peak), 4.3-4.5 and 6.5-6.8 (minor peaks). T-cell growth-promoting activity was remarkably stable to temperature, pH, SDS and 2-ME treatments. Rat IL 2 did not bind to immobilized Con A, WGA, RC 1, and fucose-binding protein. The conditioned medium also contained the inhibitor of IL 2-dependent T-cell proliferation.

Animals↗