Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation. (C)) 2007 Elsevier Inc. All rights reserved.

On self-interacting oligoribonucleotides. I. Absorption and optical rotatory dispersion of 2′-5′- and 3′-5′-oligoguanylic acids

A series of 2′-5′-oligoguanylic acids are prepared by reacting G(cyclic)p with takadiastase T1 ribonuclease and separating the products chromatographically. The 3′-5′-oligoguanylic acids are obtained by separating the products of alkaline degradation of 3′-5′-poly(G). The optical rotatory dispersion and hypochromism of both 2′-5′- and 3′-5′-oligoguanylic acids are studied at two different pH. The optical rotatory dispersion spectrum of 2′-5′-GpG is significantly different from that of 3′-5′-GpG. The magnitude of rotation of the long-wavelength peak of 2′-5′-GpG is larger than that of 3′-5′-GpG. This finding contradicts the explanation that the extra stability and more intense circular dichroism band of other 3′-5′-dinucleoside monophosphates is due to H-bond formation between 2′-OH and either the base or the phosphate oxygen. The end phosphate group has a marked effect on the spectrum of GpG between 230 and 250 mμ. In addition the optical rotatory dispersion spectra of 2′-5′ exhibit strong pH, temperature, and solvent dependence between 230 and 250 mμ. ΔH and AS for order ⇌ disorder transition is estimated to be 9.7 kcal/mole and 35.2 eu, respectively. The optical rotatory dispersion spectra of guanine-rich oligoribonucleotides, GpGpC, GpGpU, GpGpGpC, and GpGpGpU are compared to the calculated optical rotatory dispersion from the semiempirical expression of Cantor and Tinoco, using measured optical rotatory dispersion of dimers. Contrary to previous studies, agreement is found not at all satisfactory. However, optical rotatory dispersion of 3′-5′-GpGpGpC and GpGpGpU can be estimated from the semiempirical expression, if a next-nearest interaction parameter is introduced empirically. Such interaction parameter can be calculated from the measured properties of trinucleotide sequences like GpGpG, GpGpC, and GpGpU, assuming that only the nearest-neighbor interaction is important. The optical rotatory dispersion of single-stranded poly(G) is also predicted. The importance of syn-anti equilibrium and next-nearest-neighbor interaction in oligoguanylic acids is suggested as a probable explanation.

Kansalaisvaikuttamista? Habermasilaisen julkisuusteorian anti suomalaiselle ympäristöliikkeelle

Tässä pro gradu -tutkielmassa analysoidaan suomalaista ympäristöliikettä Jürgen Habermasin julkisuuden käsitteen näkökulmasta. Työn tutkimusongelmana on tarkastella julkisuutta ympäristövaikuttamisen keinona. Ponnahduslautana tutkimuksessa käytetään 2000-luvun Lapin metsien käyttöä koskevia kiistoja, joiden ratkomisyrityksiin moni Suomessa toimiva ympäristöjärjestö on osallistunut. Pitkään ratkaisemattomina olleet kiistat ovat johtaneet tilanteeseen, jossa on perusteltua kysyä, mikä ongelmien selvittämisessä on mennyt pieleen. Olisiko ennen kaikkea ammattimaiseen ja institutionalisoituneeseen vaikuttamiseen nojanneet ympäristöjärjestöt voineet toimia kiistojen yhteydessä jollakin vaihtoehtoisella tavalla? Vaihtoehdoksi esitetään Jürgen Habermasin tarjoamaa julkisuuden mallia, jonka on nähty olevan keskeinen modernin yhteiskunnan mahdollisuuksia ja rajoitteita arvioitaessa. Käsitys julkisuudesta sisältää ajatuksen kriittiseen keskusteluun pohjautuvasta, kaikille avoimesta kansalaisvaikuttamisesta. Näin muodostuneesta yleisestä mielipiteestä tulee poliittisen päätöksenteon ohjenuora. Julkisuuden ihanteen rinnalla Habermasin teoria sisältää kuvauksen julkisuuden rakennemuutoksesta – kriittisen julkisuuden alennustilasta myöhäiskapitalistisessa kulutusyhteiskunnassa. Tässä tutkimuksessa käytän näitä kahta teoreettista näkökulmaa arvioidessani nykyaikaista ympäristövaikuttamista. Keskeisin lähdeteos on Habermasin Julkisuuden rakennemuutos ja sen teoreettista perinnettä jatkaneet tutkimukset. Aineistoni koostuu kuudesta teemahaastattelusta, jotka toteutettiin keväällä 2009 Greenpeacessa, Suomen luonnonsuojeluliitossa ja WWF:ssä. Tutkimusmenetelmänä käytän teemoittelua. Sen tuloksena esiin nousevista teemoista keskeisin kertoo ympäristöjärjestöjen ammattimaistumisesta. Tulosteni mukaan ympäristövaikuttamista leimaa institutionalisoituneisuus ja hallinnollisuus, jolloin julkisuusperiaatteen toimintaedellytykset ovat heikentyneet. Julkisuuden käsitteelle löytyy kuitenkin myös varovaista tilausta suomalaisten ympäristöjärjestöjen keskuudessa.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity. (C) 2010 Elsevier B.V. All rights reserved.

Anti-amyloidogenic property of leaf aqueous extract of Caesalpinia crista

Amyloid beta (A beta) is the major etiological factor implicated in Alzheimer's disease (AD). A beta(42) self-assembles to form oligomers and fibrils via multiple aggregation process. The recent studies aimed to decrease A beta levels or prevention of A beta aggregation which are the major targets for therapeutic intervention. Natural products as alternatives for AD drug discovery are a current trend. We evidenced that Caesalpinia crista leaf aqueous extract has anti-amyloidogenic potential. The studies on pharmacological properties of C. crista are very limited. Our study focused on ability of C. crista leaf aqueous extract on the prevention of (i) the formation of oligomers and aggregates from monomers (Phase I: A beta(42) + extract co-incubation); (ii) the formation of fibrils from oligomers (Phase II: extract added after oligomers formation); and (iii) dis-aggregation of pre-formedfibrils (Phase III: aqueous extract added to matured fibrils and incubated for 9 days). The aggregation kinetics was monitored using thioflavin-T assay and transmission electron microscopy (TEM). The results showed that C. crista aqueous extract could able to inhibit the A beta(42) aggregation from monomers and oligomers and also able todis-aggregate the pre-formed fibrils. The study provides an insight on finding new natural products for AD therapeutics. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Pre-eclampsia : The role of soluble VEGF receptor-1 and related anti-angiogenic factors beyond

Pre-eclampsia is a pregnancy complication that affects about 5% of all pregnancies. It is known to be associated with alterations in angiogenesis -related factors, such as vascular endothelial growth factor (VEGF). An excess of antiangiogenic substances, especially the soluble receptor-1 of VEGF (sVEGFR-1), has been observed in maternal circulation after the onset of the disease, probably reflecting their increased placental production. Smoking reduces circulating concentrations of sVEGFR-1 in non-pregnant women, and in pregnant women it reduces the risk of pre-eclampsia. Soluble VEGFR-1 acts as a natural antagonist of VEGF and placental growth factor (PlGF) in human circulation, holding a promise for potential therapeutic use. In fact, it has been used as a model to generate a fusion protein, VEGF Trap , which has been found effective in anti-angiogenic treatment of certain tumors and ocular diseases. In the present study, we evaluated the potential use of maternal serum sVEGFR-1, Angiopoietin-2 (Ang-2) and endostatin, three central anti-angiogenic markers, in early prediction of subsequent pre-eclampsia. We also studied whether smoking affects circulating sVEGFR-1 concentrations in pregnant women or their first trimester placental secretion and expression in vitro. Last, in order to allow future discussion on the potential therapy based on sVEGFR-1, we determined the biological half-life of endogenous sVEGFR-1 in human circulation, and measured the concomitant changes in free VEGF concentrations. Blood or placental samples were collected from a total of 268 pregnant women between the years 2001 2007 in Helsinki University Central Hospital for the purposes above. The biomarkers were measured using commercially available enzyme-linked immunosorbent assays (ELISA). For the analyses of sVEGFR-1, Ang-2 and endostatin, a total of 3 240 pregnant women in the Helsinki area were admitted to blood sample collection during two routine ultrasoundscreening visits at 13.7 ± 0.5 (mean ± SD) and 19.2 ± 0.6 weeks of gestation. Of them, 49 women later developing pre-eclampsia were included in the study. Their disease was further classified as mild in 29 and severe in 20 patients. Isolated early-onset intrauterine growth retardation (IUGR) was diagnosed in 16 women with otherwise normal medical histories and uncomplicated pregnancies. Fifty-nine women remaining normotensive, non-proteinuric and finally giving birth to normal-weight infants were picked to serve as the control population of the study. Maternal serum concentrations of Ang-2, endostatin and sVEGFR-1, were increased already at 16 20 weeks of pregnancy, about 13 weeks before the clinical manifestation of preeclampsia. In addition, these biomarkers could be used to identify women at risk with a moderate precision. However, larger patient series are needed to determine whether these markers could be applied for clinical use to predict preeclampsia. Intrauterine growth retardation (IUGR), especially if noted at early stages of pregnancy and not secondary to any other pregnancy complication, has been suggested to be a form of preeclampsia compromising only the placental sufficiency and the fetus, but not affecting the maternal endothelium. In fact, IUGR and preeclampsia have been proposed to share a common vascular etiology in which factors regulating early placental angiogenesis are likely to play a central role. Thus, these factors have been suggested to be involved in the pathogenesis of IUGR. However, circulating sVEGFR-1, Ang-2 and endostatin concentrations were unaffected by subsequent IUGR at early second trimester. Furthermore, smoking was not associated with alterations in maternal circulating sVEGFR-1 or its placental production. The elimination of endogenous sVEGFR-1 after pregnancy was calculated from serial samples of eight pregnant women undergoing elective Caesarean section. As typical for proteins in human compartments, the elimination of sVEGFR-1 was biphasic, containing a rapid halflife of 3.4 h and a slow one of 29 h. The decline in sVEGFR-1 concentrations after mid-trimester legal termination of pregnancy was accompanied with a simultaneous increase in the serum levels of free VEGF so that within a few days after pregnancy VEGF dominated in the maternal circulation. Our study provides novel information on the kinetics of endogenous sVEGFR-1, which serves as a potential tool in the development of new strategies against diseases associated with angiogenic imbalance and alterations in VEGF signaling.

Hypertension, kardiotoxicitet och njursvikt i samband med tyrosinkinashämmaren sunitinib

Nybildning av blodkärl från tidigare existerande kärl, angiogenes, är ett väsentligt skede vid tumörtillväxt. Denna process regleras av bland annat tillväxtfaktorer, var av den vaskulära endoteliala tillväxtfaktorn har en central roll. Hämning av angiogenes kan ske antingen extracellulärt med hjälp av humaniserade monoklonala antikroppar eller intracellulärt med hjälp av småmolekylära hämmaren. Sunitinib är en småmolekylär multikinashämmare och inhiberar flera tyrosinkinasreceptorer som påverkar tumörtillväxten och metastasutvecklingen vid cancer. Sunitinibs främsta indikationer är gastrointestinala stromacellstumörer, metastaserad njurcellscancer och neuroendokrina tumörer i bukspottskörteln. Behandling med tyrosinkinashämmare orsakar biverkningar som hypertension, kardiotoxicitet och njursvikt, vilka antas bero på de hämmande effekterna på mål som inte är väsentliga för anti-cancer-aktiviteten (”off-target” biverkningar). Bland annat AMP-aktiverat proteinkinas (AMPK), ett kinas som upprätthåller metabolisk homeostas i hjärtat, inhiberas av sunitinib och antas framkalla kardiovaskulära biverkningar. För att reducera ”off-target” biverkningar strävar man till att hitta alternativ som minskar de skadliga effekterna utan att den terapeutiska aktiviteten försvagas. Bland annat ett begränsat kaloriintag har uppvisat skyddande effekt på hjärtat via mekanismer sammankopplade till ökad resistens mot oxidativ stress, inflammation och mitokondriell dysfunktion, samt avtagande apoptos och autofagi. Detta sker delvis genom aktivering av enzymet Sirt1. Syftet med den här studien var att undersöka ifall kaloribegränsning skyddar mot kardiovaskulära och renala biverkningar inducerade av sunitinib hos råttor. Dessutom studerades vilka signalkedjor i cellen som medverkar. I studien användes 40 spontant hypertensiva råttor samt 10 normotensiva Wistar-Kyoto råttor. Försöksdjuren delades in i fem grupper beroende på behandling; I WKY kontroll, II SHR kontroll, III SHR + kaloribegränsning 70 %, IV SHR + sunitinib 3 mg/kg och V SHR + sunitinib 3 mg/kg + kaloribegränsning 70 %. Behandlingsperioden var åtta veckor. Blodtrycket mättes varje vecka med svansmanchett, urinutsöndringen undersöktes vecka 4 och vecka 8 med metabolismburar, ultraljudsundersökning av hjärtat utfördes sista veckan och blodkärlens respons till acetylkolin och natriumnitroprussid studerades i samband med avlivning. Proteinerna Sirt1 och AMPK analyserades i hjärtat med Western blotting samt förekomsten av makrofagmarkören ED1 i njurarna med immunhistokemi. Studien visade att sunitinibdosen 3 mg/kg är mycket väl tolererbar hos råttor eftersom sunitinib inte orsakade högre blodtryck, kraftigare hypertrofi eller mer omfattande njurskada jämfört med obehandlade SHR- grupper. Utgående från resultaten kan man också konstatera att kaloribegränsningen har positiva kardiovaskulära effekter.

Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: Critical roles of IFN gamma

Mycobacterium indicus pranii (MIP) is approved for use as an adjuvant (Immuvac/Cadi-05) in the treatment of leprosy. In addition, its efficacy is being investigated in clinical trials on patients with tuberculosis and different tumors. To evaluate and delineate the mechanisms by which autoclaved MIP enhances anti-tumor responses, the growth of solid tumors consisting of Sp2/0 (myeloma) and EL4 (thymoma) cells was studied in BALB/c and C57BL/6 mice, respectively. Treatment of mice with a single intra-dermal (i.d.) injection of MIP 3 days after Sp2/0 implantation greatly suppresses tumor growth. MIP treatment of tumor bearing mice lowers Interleukin (IL)6 but increases IL12p70 and IFN? amounts in sera. Also, increase in CD8+ T cell mediated lysis of specific tumor targets and production of high amounts of IL2 and IFN? by CD4+ T cells upon stimulation with specific tumor antigens in MIP treated mice is observed. Furthermore, MIP is also effective in reducing the growth of EL4 tumors; however, this efficacy is reduced in Ifn?-/- mice. In fact, several MIP mediated anti-tumor responses are greatly abrogated in Ifn?-/- mice: increase in serum Interleukin (IL)12p70 amounts, induction of IL2 and lysis of EL4 targets by splenocytes upon stimulation with specific tumor antigens. Interestingly, tumor-induced increase in serum IL12p70 and IFN? and reduction in growth of Sp2/0 and EL4 tumors by MIP are not observed in nonobese diabetic severe combined immunodeficiency mice. Overall, our study clearly demonstrates the importance of a functional immune network, in particular endogenous CD4+ and CD8+ T cells and IFN?, in mediating the anti-tumor responses by MIP.

Conformational Polymorphism in a Non-steroidal Anti-inflammatory Drug, Mefenamic Acid

For several years, the non-steroidal anti-inflammatory drug mefenamic acid, MA, has been known to exist as dimorphs (I and II). We report a new metastable polymorph (III) of MA obtained during attempted co-crystallization experiments and establish its stability relationship with existing forms. At elevated temperatures I and III convert to II, as evident from DSC experiments. On the basis of the lattice energy calculations in conjunction with thermal analysis, the stability order is proposed to be I > II > III at ambient conditions, whereas at elevated temperature the order is II > I > III. In either condition III is a metastable form and hence transforms to I at ambient conditions and to II at higher temperatures. Also we report the structural studies of a DMF solvate and a cytosine complex.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Nanolipodendrosome-loaded glatiramer acetate and myogenic differentiation I as augmentation therapeutic strategy approaches in muscular dystrophy

Backgrond: Muscular dystrophies consist of a number of juvenile and adult forms of complex disorders which generally cause weakness or efficiency defects affecting skeletal muscles or, in some kinds, other types of tissues in all parts of the body are vastly affected. In previous studies, it was observed that along with muscular dystrophy, immune inflammation was caused by inflammatory cells invasion - like T lymphocyte markers (CD8+/CD4+). Inflammatory processes play a major part in muscular fibrosis in muscular dystrophy patients. Additionally, a significant decrease in amounts of two myogenic recovery factors (myogenic differentation 1 MyoD] and myogenin) in animal models was observed. The drug glatiramer acetate causes anti-inflammatory cytokines to increase and T helper (Th) cells to induce, in an as yet unknown mechanism. MyoD recovery activity in muscular cells justifies using it alongside this drug. Methods: In this study, a nanolipodendrosome carrier as a drug delivery system was designed. The purpose of the system was to maximize the delivery and efficiency of the two drug factors, MyoD and myogenin, and introduce them as novel therapeutic agents in muscular dystrophy phenotypic mice. The generation of new muscular cells was analyzed in SW1 mice. Then, immune system changes and probable side effects after injecting the nanodrug formulations were investigated. Results: The loaded lipodendrimer nanocarrier with the candidate drug, in comparison with the nandrolone control drug, caused a significant increase in muscular mass, a reduction in CD4+/CD8+ inflammation markers, and no significant toxicity was observed. The results support the hypothesis that the nanolipodendrimer containing the two candidate drugs will probably be an efficient means to ameliorate muscular degeneration, and warrants further investigation.

Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion

Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface alpha 5 beta 1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 x 10(8)) and Tomlinson J (Library size 1.37 x 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D₂0 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.