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PubMed1995

Purification and analysis of synthetic, triple-helical "minicollagens" by reversed-phase high-performance liquid chromatography.

To better study collagen-mediated cellular and enzymatic activities, a generally applicable solid-phase methodology has been developed by which aligned triple-helical peptides (designated THPs or "minicollagens") ranging from 79 to 124 residues can be assembled. Reversed-phase HPLC is typically the purification method of choice following chemical synthesis of small proteins of this size, as well as one of the analytical techniques used to verify product purity. We have thus compared the effects of different stationary phases (C18, C4, or diphenyl), organic modifiers (acetonitrile or isopropanol), support pore sizes (120 angstroms, 300 angstroms, or nonporous), and counterions for the reversed-phase HPLC analysis of THPs. Large pore C18 or C4 reversed-phase HPLC gave broad peaks, resulting in poor resolution of the desired THP from synthetic impurities. Broad peaks were presumably due to conformational instability of THPs to reversed-phase conditions and subsequent slow cis-trans isomerization of the peptide bonds. Peak sharpness was improved greatly by use large-pore diphenyl reversed-phase HPCL. We found that THPs can be best resolved from synthetic impurities by diphenyl or non-porous C18 reversed-phase HPLC using water-acetonitrile gradients. These results most likely reflect conditions which maintain the native conformation of collagen-like triple-helices.

Amino Acid Sequence↗
PubMed2001

FTIR study of horseradish peroxidase in reverse micelles.

Fourier transform infrared (FTIR) method was used to study the secondary structures of horseradish peroxidase (HRP) in aqueous solution and in reverse micelles for the first time. Results indicated that the structure of HRP in sodium bis(2-ethylhexy)sulfosuccinate (AOT) reverse micelles was close to that in aqueous solution. In cetyltrimethylammonium bromide (CTAB) and sodium dodecylfate (SDS) reverse micelles the position of some bands changed. Results indicated that the secondary structure had a close relationship with the surfactant species of the reverse micelles. Among the three types of reverse micelles, the system of AOT reverse micelles was probably the most beneficial reaction media to HRP.

Deuterium Oxide↗
PubMed1994

Analysis of the inductive effect of the genomic equivalent of HALF1 sequence in the reversion of rat dedifferentiated hepatoma cells.

HALF1 (human activator of liver function 1) is a closed DNA duplex implicated in reversion of rat dedifferentiated C2 hepatoma cells to a well-differentiated state. A copy of HALF1 is found in high-molecular-weight DNA in the human genome. The genomic equivalent of HALF1 and its flanking sequences [gH(5'-3') fragment] have previously been cloned and sequenced. To analyze the ability of the gH(5'-3') fragment to induce reversion process of C2 cells, two series of transfections were performed: (1) cotransfection of gH(5'-3') and plasmid pSVneo1 and (2) transfection of gH(5'-3') inserted into pSVneo1. The frequency of reversion was enhanced in transfected cells from the first experiment whereas no revertants were obtained from transfected cells in the second one. DNA analysis of the revertant clones revealed that reversion is associated with the transient presence of nonintegrated gH(5'-3') molecules. C2 cells were also transfected with gH(5'-3') cloned in pSV2dhfr. In the products of this transfection, the genesis of revertants correlated with amplification process of the gH(5'-3') sequence. We conclude that the presence of integrated copies of gH(5'-3'), even in high copy number, is not sufficient to induce the reversion process. We propose that extrachromosomal forms of gH(5'-3'), either given to cells or formed during amplification cycles, are involved in the reversion process of C2 cells.

Animals↗
PubMed2002

The reverse transcriptase of the R2 non-LTR retrotransposon: continuous synthesis of cDNA on non-continuous RNA templates.

R2 is a non-long terminal repeat (non-LTR) retrotransposon that inserts into the 28 S rRNA genes of arthropods. The element encodes two enzymatic activities: an endonuclease that specifically cleaves the 28 S gene target site, and a reverse transcriptase (RT) that can use the 3' end of the cleaved DNA to prime reverse transcription. R2 RT only utilizes RNA templates that contain the 3' untranslated region of the R2 element as templates in this target primed reverse transcription (TPRT) reaction. Here, detailed biochemical characterization of the R2 RT indicates that the enzyme is capable of making multiple, consecutive jumps between RNA templates. The terminal 3' nucleotide of the "acceptor" RNA and the 5' nucleotide of the "donor" RNA are frequently reverse transcribed in these jumps, indicating that the acceptor RNA does not anneal to the cDNA derived from the donor RNA template. These template jumps occur during TPRT as well as in non-specific extension reactions in which reverse transcription is primed by an oligonucleotide annealed to the RNA template. Analysis of these RT assays done in the absence of the target DNA also revealed that the R2 RT can initiate reverse transcription near the 3' end of any RNA molecule using the 3' end of a second RNA molecule as primer. Again there is no requirement for sequence complementarity between the RNA used as template and the RNA used as primer. These properties of the R2 RT differ substantially from those of retroviral RTs but have similarities to the RT of the Mauriceville retroplasmid of Neurospora crassa. We present a model which relates these unusual properties of the R2 RT to structural differences from retroviral RTs as well as correlates these properties to the likely retrotransposition mechanism of R2 and other non-LTR retrotransposons.

Animals↗
PubMed1994

Reversal of low-molecular-weight heparin anticoagulation by synthetic protamine analogues.

Protamine reversal of unfractionated and low-molecular-weight heparin (LMWH) causes hypotension, bradycardia, pulmonary artery hypertension, and declines in oxygen consumption. Furthermore, protamine incompletely reverses the anti-Xa activity of LMWH. The present study assesses the efficacy and toxicity of three protamine variants having +16 and +18 charges in reversal of LMWH (Logiparin, LHN-1): [+16] P(AK2A2K2)4, [+18] PK(K2A2K2A)3K2AK3, and [+18B] acetyl-PA(K2A2K2A)4K2-amide. The [+18B] compound was made by acetylating and amidating the [+18] to decrease in vivo degradation and to increase the alpha-helix forming propensity. Variants were examined in a canine model (n = 7, each variant) and compared to controls (n = 7, each variant) and compared to controls (n = 7) reversed with standard protamine with a +21 charge. Animals were anesthetized, anticoagulated with LMWH (150 IU factor Xa activity/kg), and reversed with protamine variants (1.5 mg/kg with 100 IU/mg). Blood pressure (BP), heart rate (HR), cardiac output (CO), pulmonary artery pressures, oxygen saturations, and oxygen consumption (VO2) were continuously monitored. Comparisons were undertaken at baseline, after heparin, before variant administration, and for 30 min thereafter. A total toxicity score (TTS) was calculated for each variant, accounting for maximal declines in BP, HR, CO, and VO2 during the first 5 min after reversal. Protamine [+21] was most toxic, TTS -7.6, with the variants being less toxic (P < 0.01, ANOVA): TTS = [+16] -2.8, [+18] -1.3, and [+18B] -4.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

The RNA/protein symmetry hypothesis: experimental support for reverse translation of primitive proteins.

Although the "RNA-world" theory, or the RNA-first theory is renowned for a promising theory of biogenesis, it is also possible that both RNAs and proteins have coevolved forming a stable metabolic complex from the very beginning. I investigated this possibility assuming that the genetic information flowed symmetrically in the era of the origin of life, i.e. the primitive translation machinery worked in both directions (from RNA to protein and from protein to RNA). According to this RNA/protein symmetry theory, the genetic information would have come from existing cellular proteins via reverse translation. This process would have been completed in a short period of time without searching an enormous RNA sequence space. Furthermore, reverse translation would have ensured biological continuity; proteins that were essential for cellular metabolism would have been utilized in the same way as before the protein sequence information would have been transferred into the RNA sequences. I also propose a possible mechanism for the process of reverse translation. The reverse translation would proceed in the 3' to 5' direction using a set of at least 20 reverse transfer RNAs (rtRNAs) that can recognize their specific amino acid residue and carry their corresponding codon. A source of genetic information would be a primary sequence of a protein molecule. Several basic steps of reverse translation were demonstrated using rtRNA(Arg).

3' Untranslated Regions↗
PubMed1987

Different scalp localization of pattern onset and reversal visual evoked potentials.

Visual evoked potentials were recorded monopolarly from seven electrodes on a horizontal line from T3 to T4 and bipolarly in one channel from left of the midline to right of the midline. Stimulation of the right and left half-fields was performed with a checkerboard generator of 10 degrees x 13.5 degrees and checks of various sizes at high contrast. The results may be summarized as follows: 1. Source-sink analysis for lateral half-field pattern onset with small checks shows C1, peaking about 80 msec, to have a source on the scalp contralateral to the half-field stimulated with a sink on the midline. 2. To half-field pattern reversal, bipolar recording across the scalp midline indicates an early phase reversal peaking about 80 msec followed by another phase reversal peaking at about P100 latency. Source-sink analysis indicates the first reversal has a contralateral source, while the second has a source on the scalp midline or ipsilateral to the field stimulated. 3. Pattern reversal results vary with check size, with a smaller check size increasing the amplitude of the first phase reversal and decreasing the amplitude of the second.

Evoked Potentials, Visual↗
PubMed1984

Inherited XX sex reversal in the cocker spaniel dog.

Nine XX true hermaphrodites and two XX males were discovered in a family of American cocker spaniels. The true hermaphrodites were partially-masculinized females with ovotestes; the XX males had malformed male external genitalia and cryptorchid aspermatogenic testes. Wolffian and Mullerian duct derivatives were present in both true hermaphrodites and XX males. All four sires of sex-reversed dogs were normal XY males; five of the dams were anatomically normal females and one was an XX true hermaphrodite. A second true hermaphrodite reproduced as a female, producing anatomically normal offspring. All matings that produced sex-reversed offspring were consanguineous. Matings of the parents of sex-reversed cocker spaniels to normal beagles with no family history of intersexuality produced only normal offspring. Examination of G-banded karyotypes of the affected animals, their parents, and siblings, revealed no structural anomalies of the chromosomes that were consistently associated with sex-reversal. In assays for serologically-detectable H-Y antigen, the group of XX true hermaphrodites and the group of XX males had mean levels of the antigen not significantly different from that in normal male controls. Female parents of sex-reversed dogs and some of their female siblings were typed H-Y antigen positive, but the mean level of the antigen in this group was less than that of normal male controls. It is proposed that XX sex reversal in cocker spaniels is due to a mutant gene which when homozygous in females, results in a level of H-Y antigen similar to that found in normal males and the gonads develop as ovotestes or testes. When the gene is heterozygous in females, the level of serologically-detectable H-Y antigen is lower than that found in normal males and the gonads develop as normal ovaries. The persistence of Mullerian structures in the presence of testicular tissue suggests that Mullerian inhibiting substance is deficient or ineffective in its action in this condition.

Animals↗
PubMed1985

Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli.

The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvr A6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp+ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp+ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7-2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvr A6 background.

Dose-Response Relationship, Radiation↗
PubMed1981

Differential reversal of various dopamine antagonists by anticholinergics in Sidman avoidance: possible relationship to adrenergic blockade.

Various dopamine receptor antagonists have divergent clinical and neurochemical properties. The relative ability of anticholinergics (benztropine and scopolamine) to reverse these drugs was assessed in squirrel monkeys and rats performing a Sidman avoidance task. In monkeys, benztropine markedly attenuated the effects of oxiperomide, metoclopramide, halopemide, tiapride and mezilamine as well as haloperidol. Chlorpromazine and fluphenazine were antagonized to a moderate extent; thioridazine and perlapine were not antagonized; and clozapine was actually potentiated by benztropine. In the rat, benztropine antagonized haloperidol strongly but reversed fluphenazine, thioridazine or clozapine only weakly or not at all. The overall effects of scopolamine in both species were similar to those of benztropine. The dopamine receptor antagonists that were most completely reversed by benztropine were found to inhibit 3H-spiroperidol more strongly than 3H-WB-4101 binding in calf caudate, while the reverse was true for drugs that were antagonized only moderately or not at all by benztropine. These results support a previous suggestion that anticholinergic reversal is less marked against dopamine antagonists with alpha-adrenergic blocking properties. Benztropine reversal of experimental dopamine receptor antagonists in the squirrel monkey Sidman avoidance test may contribute to their preclinical characterization.

Adrenergic alpha-Antagonists↗
PubMed1995

In vitro activity of S 9788 on a multidrug-resistant leukemic cell line and on normal hematopoietic cells-reversal of multidrug resistance by sera from phase I-treated patients.

The triazinoaminopiperidine derivative S 9788 is a new multidrug-resistance modulator that is currently being evaluated in phase I clinical trials. In this study, the reversal effect of S 9788 in comparison with verapamil was shown in vitro in human T-leukemic CCRF-CEM/VLB cells expressing the multidrug-resistance (MDR) phenotype. S 9788 increased in a dose-dependent manner the cytotoxic activity of doxorubicin or vinblastine, with complete reversal of resistance occurring at 2 microM for a concomitant continuous exposure (96 h) to the cytotoxic drugs. At respective concentrations equivalent to the IC10 value (the concentration inhibiting 10% of cell growth), S 9788 was 44 times more potent than verapamil in CCRF-CEM/VLB cells. S 9788 at 2 microM did not enhance the in vitro toxicity of doxorubicin or vinblastine in the human normal bone-marrow erythroid (BFU-E) and myeloid (CFU-GM) progenitors. The effect of exposure duration and concentrations on the synergistic action of modulator and cytotoxic agent closely depended on the cytotoxic agent studied. Post-incubations with S 9788 alone after a 1-h coadministration with vinblastine and S 9788 dramatically increased the reversal effect (4-41 times) in proportion to both the duration of postincubation and the concentration of S 9788. In contrast, for doxorubicin resistance, post-incubation with S 9788 alone induced a maximal 2-fold increase in the reversal effect that was not proportional to the post-incubation duration. In patients treated with S 9788 as a 30-min intravenous infusion during phase I trials, a good correlation was found between the serum levels of S 9788 and the ability to reverse MDR in CCRF-CEM/VLB cells. The reversal effect was dose-dependent and was effective beginning at a plasma concentration of 0.25 microM. These data form a basis for the design of phase II trials using a combination of a loading dose of S 9788 given before vinblastine or doxorubicin administration followed by a maintenance infusion of S 9788 alone for a period of 2-24 h.

Antineoplastic Agents↗
PubMed1996

Membrane interactions of some catamphiphilic drugs and relation to their multidrug-resistance-reversing ability.

The multidrug-resistance (MDR)-reversing ability of the catamphiphilic drugs could be mediated through their interaction with the membrane phospholipids. This could lead directly (through changes in membrane permeability and fluidity) and/or indirectly (through inhibition of P-glycoprotein phosphorylation via inhibition of the phosphatidylserine-dependent protein kinase C or changes in the conformation and functioning of the membrane-integrated proteins via changes in the structure organization of the surrounding membrane bilayer) to the reversal of MDR. Using differential scanning calorimetry and NMR techniques and artificial membranes composed of phosphatidylcholine or phosphatidylserines we found a significant correlation between the MDR-reversing activity of the drugs in doxorubicin-resistant human breast carcinoma MCF-7/DOX and murine leukaemia P388/DOX tumour cells (data taken from the literature) and their ability to interact with phosphatidylserines. Trans- and cis-flupentixol were found to interact most strongly with both the phospholipids, followed by trifluoperazine, chlorpromazine, triflupromazine, flunarizine, imipramine, quinacrine and lidocaine. Differences in the interaction of trans- and cis-flupentixol with the phospholipids studied are suggested to be responsible for their different MDR-reversing ability. Verapamil showed moderate membrane activity, assuming that the membrane interactions are not the only reason for its high MDR-reversing ability. Amiodarone showed very strong interactions with phosphatidylserines and is recommended for further MDR-reversal studies.

Amiodarone↗
PubMed1985

The reversion phenomenon in irradiated fern prothalli: effects of acute or chronic anoxia and LET.

In several systems a paradoxical reduction of radiation damage with increasing dose, termed reversion, has been observed. In the fern Osmunda regalis the percentage of cells which does not die but stays alive, although reproductively sterile, increases with dose. The assumed mechanism of this effect is a continuation of cytoplasmic growth during radiation-induced mitotic delay which induces terminal differentiation (early differentiation) thus preventing mitosis and the expression of chromosomal injury. Suppression of cytoplasmic growth after irradiation should abrogate reversion. This was tested using anoxia. Reversion was suppressed by storage of the sporelings in nitrogen for 8 h or more after X-rays, but was not suppressed by storage in 0.27 microM oxygen nor by a 60-min exposure to air after irradiation and before storage in nitrogen. Anoxia before irradiation in air had no effect. Anoxia only during irradiation showed an OER of about 2 for the reversion peak. The partial abrogation of reversion is consistent with the assumed mechanism. Marked reversion also was observed after 14.7 MeV neutrons.

Cell Survival↗
PubMed1984

Increase of cellular murine leukemia virus reverse transcriptase in interferon-treated cells.

The addition of mouse interferon (IFN) to AKR murine leukemia virus (MuLV)-producing NIH3T3 cells inhibited the extracellular appearance of AKR MuLV when assayed for reverse transcriptase activity or infectious virus production. IFN treatment had no detectable effect on proviral DNA formation during infection nor on the level of viral RNA in virus-producing cells. However, addition of IFN did alter the level of cellular viral reverse transcriptase activity. Chromatography of extracts from virus-producing cells on poly(G)-Sepharose columns revealed two peaks of reverse transcriptase activity. Peaks I and II eluted at 0.45 M and 0.65 M NaCl, respectively, while the cellular DNA polymerase beta eluted earlier at 0.3 M NaCl. IFN treatment of these chronic virus producer cells resulted in a 5-fold increase in peak I whereas peak II and polymerase beta remained essentially unchanged. When reverse transcriptase from purified virions was similarly chromatographed on poly(G)-Sepharose, all of the enzymatic activity eluted as peak I. Thus, the reverse transcriptase in peak I from cell extracts appears to be the form which is present in mature virions. Contrary to the results with chronic virus-producing cells, IFN treatment prior to exogenous infection with MuLV did not alter levels of reverse transcriptase peaks I and II or polymerase beta. These results provide further evidence that the major effect of IFN occurs at the level of MuLV maturation and assembly.

AKR murine leukemia virus↗
PubMed1995

Structure, function, and evolution of bacterial reverse transcriptase.

The discovery of retroelements in the prokaryotes raises intriguing questions concerning their roles in bacteria and the origin and evolution of reverse transcriptases. We first discuss a possible structure of bacterial reverse transcriptases on the basis of the known three-dimensional structure of HIV-1 reverse transcriptase, and how such a putative three-dimensional structure is able to recognize a single primer-template RNA molecule to initiate DNA chain elongation from the 2'-OH group of an internal G residue. This reaction leads to the production of a unique RNA-DNA complex called msDNA (multicopy single-stranded DNA) in which a single-stranded DNA branches out from an RNA molecule via a 2',5'-phosphodiester linkage. Second, the mobility of the bacterial retroelements called retrons, responsible for the production of msDNA, are discussed and compared with the mobility of group I and group II introns. Third, the original and evolution of bacterial reverse transcriptases are discussed in light of the question of whether the bacterial reverse transcriptases are older than eukaryotic reverse transcriptases.

Amino Acid Sequence↗
PubMed1996

Olanzapine, a novel atypical antipsychotic, reverses d-amphetamine-induced inhibition of midbrain dopamine cells.

This study compared the ability of the novel atypical antipsychotic olanzapine with that of clozapine to reverse the d-amphetamine-induced inhibition of substantia nigra (A9) and ventral tegmental area (A10) dopamine (DA) cells. Extracellular single-unit recordings were made from A9 and A10 DA cells in anesthetized rats. When administered alone, neither olanzapine nor clozapine altered the firing rate of A9 or A10 DA cells. Administration of d-amphetamine (0.5, 1.0 and 2.0 mg/kg, IV, decreased the firing rate of A9 and A10 DA cells. Olanzapine completely reversed the inhibitory effects of d-amphetamine on A10 DA cells (ED100 = 0.18 mg/kg, IV) and on A9 DA cells (ED100 = 1.0 mg/mg, IV). Clozapine completely reversed the inhibitory effects of d-amphetamine on A10 DA cells (ED100 = 3.8 mg/kg, IV), but only partially reversed the effects of d-amphetamine on A9 DA cells at the highest dose tested (8.0 mg/kg, IV). Thus, olanzapine, like clozapine, was more potent in reversing the effects of d-amphetamine on A10 than A9 DA cells. In addition, olanzapine was more potent than clozapine in the reversal of d-amphetamine effects on A9 and A10 DA cells. These results indicate that olanzapine and clozapine have similar effects on DA unit activity and predict that olanzapine should have an atypical antipsychotic profile in man.

Amphetamine↗
PubMedDate not supplied

Effects of experimental scotomata on sequential pattern-onset, pattern-reversal and pattern-offset visual evoked potentials.

The effect of experimental scotomata on visual evoked potentials to half-field stimulation using sequential checkerboard onset, reversal and offset was investigated in 10 normal subjects to assess the relative sensitivity of the three stimulus modes, and the contributions of pathways subserving macular and paramacular parts of the visual field. Four scotoma sizes (0-1.5 degrees , 0 degrees - 2 degrees, 0 degrees -3 degrees and 0 degrees -4.5 degrees ) were used to mask the central part of the stimulus field (0 degrees -12 degrees ). Five check sizes (6', 12', 20', 50' and 80') were presented for each scotoma size. Peak-to-peak amplitudes and peak latencies of components on the ipsilateral and contralateral sides of the scalp to the stimulated half-field were measured. Scotoma size was highly significant in influencing component amplitude (p < 0.0001) and latency (p < 0.03) of all ipsilateral and contralateral onset components and all ipsilateral reversal and offset components. Components that were attenuated to the greatest extent with the smallest 0 degrees -1.5 degrees scotoma were the contralateral onset P105 and ipsilateral reversal P100 and N145. Onset CIII, reversal N80, and offset N85 and P110 only showed a significant attenuation after the use of scotomata of 0 degrees -3 degrees and larger. Our results show that scotoma size is a significant factor in influencing all the major visual evoked potential components, with the exception of reversal and offset contralateral potentials (N105 and N115), probably reflecting their paramacular origins. Reversal, ipsilateral P100 and N145, and onset, contralateral P105, appear to be predominantly of macular origin and the most sensitive potentials for detecting effects of small central scotomata.

Adult↗
PubMedDate not supplied

Pattern-onset visual evoked potentials: more useful than reversal for patients with nystagmus.

PURPOSE: The visual evoked potential is often used to assess visual function in neurologically impaired patients, a group in whom nystagmus is a common feature. Pattern-reversal stimuli are commonly used to produce visual evoked potentials in clinical practice. Previous reports have shown that this stimulus is not optimal when subjects have nystagmus. The present study aimed to compare the efficacy of pattern-onset and reversal stimuli when used to measure visual evoked potentials from subjects with idiopathic nystagmus. METHODS: In five adults with congenital nystagmus and 10 visually normal adults, VEPs were recorded and reproduced for checkerboard stimuli of two sizes (120' and 60'). Each size was presented as both pattern-onset and reversal check. RESULTS: Visually normal adults demonstrated similar visual evoked potential amplitudes and quality in response to pattern-reversal and pattern-onset. However, in the presence of nystagmus, visual evoked potentials recorded to pattern-reversal stimuli were significantly smaller and of poorer quality than those obtained to pattern-onset stimuli (analysis of variance p<0.05; Kendall's tau, p<0.05). CONCLUSIONS: Pattern-onset stimuli produce larger and clearer visual evoked potentials in patients with nystagmus compared with those produced to pattern-reversal stimuli.

Adult↗