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PubMed2003

Phase-selective solubility of poly(N-alkylacrylamide)s.

The extent of the phase-selective solubility of poly(N-alkylacrylamide)s was studied by UV-vis and fluorescence spectroscopy using poly(N-isopropylacrylamide) and poly(N-octadecylacrylamide) as representative polar and nonpolar poly(N-alkylacrylamide)s in a mixture of polar and nonpolar thermomorphic solvents. Phase-selective solubilities of greater than 10000:1 were seen with each labeled polymer in polar and nonpolar solvents such as heptane and DMF or heptane and 90% EtOH-H(2)O. Using a poly(N-acryloxysuccinimide) as a common precursor, a pool-split synthesis was devised to prepare a library of poly(N-alkylacrylamide)s whose members varied only in the size of their N-alkyl substituent. The solubilities of these library members were measured in both the polar and nonpolar phases of a thermomorphic heptane/90% EtOH-H(2)O mixture at 25 degrees C. Such solvent mixtures are miscible hot (70 degrees C) and biphasic cold (25 degrees C). The results show that poly(N-pentylacrylamide) is selectively soluble (>99.5%) in the polar EtOH-rich phase at rest. Poly(N-alkylacrylamide)s with larger N-alkyl groups are predominantly (C(6), 85%; C(7), 95%) or exclusively (>C(8), >99.5%) in the heptane-rich phase at rest.

Journal Article↗
PubMed1992

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2005

Structure and relaxation dynamics of poly(amide urethane)s with bioactive transition metal acetyl acetonates in hard blocks.

Structural characteristics, thermal transitions and molecular dynamics of selected poly(amide urethane)s with transition metal acetyl acetonates Me(AcAc)(2) (Me = Sn(4+), Zn(2+), Cu(2+), Pb(2+)) as chain extenders, were comparatively investigated using small- and wide-angle X-ray scattering (SAXS, WAXS), differential scanning calorimetry (DSC), and dielectric techniques (dielectric relaxation spectroscopy, DRS; thermally stimulated currents, TSC). We studied the influence of metal chelates on the mixing of the soft-segment (SS) and hard-segment (HS) domains and the related degree of microphase separation (DMS). The reactivity of Me(AcAc)(2) with macrodiisocyanate was found to decrease in the order Sn(AcAc)(2)Cl(2) > Cu(AcAc)(2) > Zn(AcAc)(2) > Pb(AcAc)(2). While Pb(AcAc)(2) shows a higher tendency for crystallisation, both the dielectric and calorimetric results suggest that the corresponding polyurethane has comparatively low DMS. The type of the transition metal has moderate effect on the glass transition temperature and no influence on the shape of the dielectric alpha relaxation signal, indicating weak interactions between metal ions and SS domains. In contrast, structural parameters and the dielectric behaviour of the beta relaxation suggest preference for hydrogen-bonding interactions between Sn(4+) and Cu(2+) metal-chelates and HS domains. The temperature dependence of dc conductivity sigma(dc) is described by the Vogel-Tammann-Fulcher equation and signifies the coupling between the mobility of polymeric chains and charges' motion. It may be expected that the present combination of techniques and particular results with respect to DMS will contribute to the development and testing of novel biodegradation-resistant and antibacterial metal-polyurethanes for biotechnological and industrial applications.

Amides↗
PubMed2000

Selectivity equivalence of poly(ethylene glycol) stationary phases for gas chromatography.

The solvation parameter model is used to study differences in selectivity for poly(ethylene glycol) stationary phases for packed column (Carbowax 20M) and fused-silica, open-tubular column (HP-20M, AT-Wax, HP-INNOWax and DB-FFAP) gas chromatography. All phases are dipolar, strongly hydrogen-bond basic with no hydrogen-bond acidity and of moderate cohesion. No two phases are exactly alike, however, and selectivity differences identified with cavity formation and dispersion interactions, n- and pi-electron pair interactions, dipole-type interactions and hydrogen-bond interactions are quantified by differences in the system constants at a fixed temperature where retention occurs solely by gas-liquid partitioning. The system constants vary linearly with temperature over the range 60-140 degrees C (except for n- and pi-electron pair interactions which are temperature invariant) facilitating a general comparison of the importance of temperature on selectivity differences for compared phases. From a mechanistic point of view it is demonstrated that selectivity differences can result from chemical differences between the poly(ethylene glycol) stationary phases and from differences in the relative contribution of interfacial adsorption to the retention mechanism. The latter depends on both system properties and solute characteristics.

Chromatography, Gas↗
PubMed1975

Determination of mRNA half-life in HeLa cultures by a poly(A)-independent direct analysis of specific radioactivity of mRNA.

mRNA specific radioactivity in HeLa cultures exposed to (3H)uridine (10 muM) was determined directly by a highly selective poly(A)-independent method which we have described previously. Neither uridine in mRNA nor UTP approached the specific radioactivity of the exogenous (3H)uridine, but attained steady-state specific radioactivities which remained a third below the value of the added precursor. Using the labeling data for the evaluation of mRNA turnover, previously described by Greenberg, mRNA half-life in exponentially growing HeLa cultures was found to be 0.87 times the cell doubling time. Decay curves of mRNA in prelabeled cultures were in accordance with these values (half-life equals 0.79 times the cell doubling time) when corrected for growth and also for "reutilization" which was accomplished by relating uridine labeling in mRNA to UTP specific radioactivity. The experiments showed that an exact evaluation of mRNA turnover is possible only when the following points are taken into account. a) A constant supply of exogenous labeled uridine must be provided to guarantee a constant specific radioactivity of UTP. b) Labeling of CTP and of cytidine in RNA are delayed when compared with UTP and uridine in RNA. Corrections for cytidine labeling in RNA are therefore required. c) As rRNA approached a definitely lower steady-state specific radioactivity than mRNA, mRNA specific radioactivities must be determined directly (i.e. by radioactivity and absorbance at 260 nm in isolated mRNA fractions) in order to evaluate true turnover of this RNA species.

Cell Count↗
PubMed1988

Splice site selection dominates over poly(A) site choice in RNA production from complex adenovirus transcription units.

The adenovirus late genes are organized in a complex transcription unit in which the production of multiple RNAs is controlled by specific RNA processing events. Adding to the complexity is the fact that the early E3 transcription unit is wholly contained within the late transcription unit. Within this overlapping region there are splicing events and poly(A) site choices specific to each transcription unit. Early in infection, there is near exclusive use of the E3 splice sites with little L4 poly(A) site use whereas the reverse is true late in infection. Using a plasmid containing an intact E3 transcription unit, we demonstrate that the L4 poly(A) site, located in the first E3 intron, is not used either in a mock-infected cell or a late infected cell; nor is it used if the same transcription unit is driven by the major late promoter. In addition, the nature of the poly(A) site appeared to be unimportant since the E3 poly(A) site was also not used in the intron if inserted in place of the L4 poly(A) site. Thus, splice site selection appears to dominate over poly(A) site choice. This conclusion was confirmed by the observation that deletion of either the E3 splice donor (5'ss) or the E3 splice acceptor (3'ss) allowed the use of the L4 poly(A) site. Finally, the L4 poly(A) site within the E3 intron was also used when additional sequence, including splicing signals from the L4 region and the tripartite leader, was inserted between the promoter and the E3 processing sites. It appears that splicing is the dominant event in governing production of E3/L4 RNAs.

Adenoviruses, Human↗
PubMed1990

In the beginning is the end: regulation of poly(A) addition and removal during early development.

The addition of poly(A) tails to nearly all mRNAs within the nucleus was reviewed in the July issue of TIBS. Here we shift focus to the fate of poly(A) tails during early development. At specific times during oogenesis and embryogenesis, the poly(A) tails of certain maternal mRNAs are lengthened, while the tails of a number of other mRNAs are removed. The selective poly(A) addition reactions are regulated by a short, U-rich sequence in the 3' untranslated region, while the removal of poly(A) from specific mRNAs is a 'default state', requiring no specific sequence. These regulated changes in poly(A) length are likely to play a major role in translational regulation in the egg and early embryo.

Animals↗
PubMed1991

Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation.

In retroviral proviruses, the poly(A) site is present in both long terminal repeats (LTRs) but used only in the 3' position. One mechanism to account for this selective poly(A) site usage is that LTR U3 sequences, transcribed only from the 3' poly(A) site, are required in the RNA for efficient processing. To test this possibility, mutations were made in the human immunodeficiency virus type 1 (HIV-1) U3 region and the mutated LTRs were inserted into simple and complex transcription units. HIV-1 poly(A) site usage was then quantitated by S1 nuclease analysis following transfection of each construct into human 293 cells. The results showed that U3 sequences confined to the transcription control region were required for efficient usage of the HIV-1 poly(A) site, even when it was placed 1.5 kb from the promoter. Although the roles of U3 in processing and transcription activation were separable, optimal 3' end formation was partly dependent on HIV-1 enhancer and SP1 binding site sequences.

Cloning, Molecular↗
PubMed1992

Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity. Increased specificity toward src peptides.

A tyrosine protein kinase (TPK-I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella-Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123-126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c-src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 microM versus 670 microM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N-terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position -2 or glutamic acid at position -1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl. These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.

Amino Acid Sequence↗
PubMed2006

Thiocyanate ion-selective PVC membrane electrode based on N,N'-ethylene-bis(4-methylsalicylidineiminato)nickel(II).

A highly selective poly(vinyl chloride) (PVC) membrane electrode based on an N,N'-ethylene-bis(4-methyl-salicylidineiminato) nickel(II) [Ni(EBMSI)] complex as a carrier for a thiocyanate-selective electrode is reported. The influences of the membrane composition, pH and possible interfering anions were investigated based on the response properties of the electrode. The electrode exhibited a good Nernstian slope of -58.9 +/- 0.7 mV decade(-1), over a wide pH range of 3.5 - 8.5 and a linear range of 1.0 x 10(-6) - 1.0 x 10(-1) M for thiocyanate. The detection limit of electrode was 3.1 x 10(-7) M SCN(-). The selectivity coefficients determined by a fixed interference method (FIM) indicate that a good discriminating ability towards the SCN- ion compared to other anions. The proposed sensor had a fast response time of about 5 - 15 s and could be used for at least 3 months without any considerable divergence in the potential. It was applied as an indicator electrode in the titration of thiocyanate with Ag+ and in the potentiometric determination of thiocyanate in saliva and urine samples.

Journal Article↗
PubMed1993

Coupling of poly(A) site selection and trans-splicing in Leishmania.

Intergenic regions of polycistronic pre-mRNAs of trypanosomatid protozoans are the sites of two processing reactions: polyadenylation of the upstream gene and trans-splicing of the capped miniexon to the downstream gene. Their close proximity and the lack of consensus motifs at poly(A) sites led us to test whether poly(A) site selection is governed by the location of the downstream splice acceptor in the DHFR-TS locus of Leishmania major. Whenever the position of the downstream splice site was altered, the poly(A) site was shifted 400-500 nucleotides upstream of the new splice site. In contrast, when the wild-type poly(A) site was eliminated, the downstream splice site was unaffected, and polyadenylation was maintained 200-500 nucleotides upstream of the splice site. In a second set of experiments, T7 RNA polymerase expressed in Leishmania was used to direct the synthesis of artificial pre-RNAs in vivo whose expression was found to require the presence of a downstream splice acceptor. We conclude that poly(A) site selection in Leishmania is specified by the position of the downstream splice acceptor and propose a scanning model for poly(A) site selection after splice site recognition.

Animals↗
PubMed1991

Determination of ethaverine and papaverine using ion-selective electrodes.

Ion-selective poly(vinyl chloride) membrane electrodes for the opium alkaloids papaverine and ethaverine are presented. The electrode membranes contain ion pairs of the alkaloids with the anionic counter ion tetraphenylborate. The detection limits for all electrodes were approximately 2 x 10(-6) mol dm-3 at pH 5.0 in 100 mmol dm-3 buffered solutions and the measured slopes were close to the values theoretically expected. The selectivity coefficient observed for the ethaverine-tetraphenylborate electrode is 10(-1.1) with respect to papaverine. The suitability of the membranes for single-use electrodes is discussed.

Electrochemistry↗
PubMed2004

A copper ion-selective electrode with high selectivity prepared by sol-gel and coated wire techniques.

A sol-gel electrode and a coated wire ion-selective poly(vinyl chloride) membrane, based on thiosemicarbazone as a neutral carrier, were successfully developed for the detection of Cu (II) in aqueous solutions. The sol-gel electrode and coated electrode exhibited linear response with Nernstian slopes of 29.2 and 28.1 mV per decade respectively, within the copper ion concentration ranges 1.0 x 10(-5) - 1.0 x 10(-1) M and 6.0 x 10(-6) - 1.0 x 10(-1) M for coated and sol-gel sensors. The coated and sol-gel electrodes show detection limits of 3.0 x 10(-6) and 6.0 x 10(-6) M respectively. The electrodes exhibited good selectivities for a number of alkali, alkaline earth, transition and heavy metal ions. The proposed electrodes have response times ranging from 10-50 s to achieve a 95% steady potential for Cu2+ concentration. The electrodes are suitable for use in aqueous solutions over a wide pH range (4-7.5). Applications of these electrodes for the determination of copper in real samples, and as an indicator electrode for potentiometric titration of Cu2+ ion using EDTA, are reported. The lifetimes of the electrodes were tested over a period of six months to investigate their stability. No significant change in the performance of the sol-gel electrode was observed over this period, but after two months the coated wire copper-selective electrode exhibited a gradual decrease in the slope. The selectivity of the sol-gel electrode was found to be better than that of the coated wire copper-selective electrode. Based on these results, a novel sol-gel copper-selective electrode is proposed for the determination of copper, and applied to real sample assays.

Journal Article↗
PubMed1987

Cloning of cDNA for maize superoxide dismutase 2 (SOD2).

A cDNA clone encoding maize cytosolic superoxide dismutase 2 (SOD2) was isolated from a cDNA library constructed in pUC9 plasmids from size-selected poly(A)+ RNA. The library was screened with mixed synthetic oligode-oxynucleotide probes. The sequence of the probes was derived from the amino acid sequence from a region of the protein near the NH2 terminus. One positive clone contained an insertion of a 612-base-pair fragment. The amino acid sequence, deduced from the nucleotide sequence of the cDNA, revealed that the clone contained the coding region for all but the first of the 151 amino acids of the SOD2 protein. Additional 5' and 3' flanking sequences, absent on the pUC9 Sod2 clone, were obtained from a lambda gt11 clone isolated from a maize leaf library probed with the pUC9 Sod2 insert. Hybrid-selection translation assays also demonstrate that the cDNA clone contains Sod2 sequences.

Amino Acid Sequence↗
PubMed2006

Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs.

Alternative polyadenylation leads to mRNAs with variable 3' ends. Since a 3'-untranslated region (3'-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3'-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3'-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3'-UTR. CFIm is a heterodimeric 3' end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3'-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.

3' Untranslated Regions↗
PubMed1994

Primary structure and functional properties of an epithelial K channel.

Expression cloning in Xenopus oocytes was used to identify a clone for a renal K channel. The clone, named ROMK2, was obtained from a cDNA library constructed in the plasmid vector pSPORT using size-selected poly(A)+ RNA from whole rat kidney. ROMK2 consists of 1,837 nucleotides, with an open reading frame of 1,116 bases predicted to code for a 372-amino acid peptide. The clone appears to be a splice variant of a recently reported K channel (ROMK1) from rat renal outer medulla (Ho, K.H., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362: 31-37, 1993). Northern blot analysis indicates that ROMK2 is expressed in renal cortex, medulla, and papilla. Expression in other tissues appears to be much lower. The functional properties of the channel as measured in Xenopus oocytes indicate its close relationship to ROMK1 and more distant relationship to the inward rectifier K channel (IRK1) (Kubo, Y, T.J. Baldwin, Y. N. Jan, and L. Y. Jan. Nature Lond. 362: 127-133, 1993). The inward conductance of the channel is a saturable function of external K, with a half-maximal conductance at <5 mM. The selectivity sequence for ion permeability based on reversal potential measurements was K > Rb > NH4 > Na, Li. The conductance to Rb was only one-half that to K. Extracellular Ba2+ and Cs+ blocked the channel in a voltage-dependent manner. The high sensitivity of Cs+ block to voltage is consistent with the channel's operating as a multi-ion pore. The channel was blocked by high concentrations (100 microM) of glibenclamide. It did not appear to be blocked by extracellular Na+ or tetraethyl-ammonium ion. Patch-clamp measurements indicated a single-channel conductance of 30 pS in the presence of 110 mM K and high open probability that was weakly dependent on voltage. This channel may be involved in maintaining the membrane potential of renal cells and/or mediating renal K secretion.

Amino Acid Sequence↗
PubMed1981

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments.

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.

Chemical Phenomena↗