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PubMed1997

Thoracoscopic loop ligation of parenchymal blebs and bullae: is it effective and safe?

Surgeons who have gained experience and confidence with video-assisted thoracic surgery are now routinely applying the minimally invasive approach to treat patients with spontaneous pneumothorax. Although the endoscopic stapling device may be a preferred method for resection of parenchymal blebs or bullae, the stapling device is not inexpensive. In an effort to contain costs since we started performing the video-assisted thoracoscopic procedure in chest surgical diseases, we have used a self-made endoscopic loop as an alternative method. It has assisted us in performing bulla ablation in a cost-effective manner. Over a 4-year period (1992 to 1996), we assessed the efficacy of ligating parenchymal blebs and bullae with a self-made endoscopic loop by video-assisted techniques. A total of 263 ligations were performed in 250 patients. Surgical indications included recurrence (n = 146), bilaterality of the disease (n = 13), hemopneumothorax (n = 7), radiologically demonstrated large bulla (n = 9), persistent air leak (n = 52), and nonexpansion of the lung (n = 23). There were no operative deaths. Early postoperative complications included a dislodged endoscopic loop necessitating reexploration in one patient and postoperative minor wound infections in 13. The average postoperative hospitalization was 4.5 days. Two hundred seventeen patients (86.8% of all patients) were followed up for a median of 28 months (1 to 46 months) after the operation. There have been no recurrences to date. Our results showed that thoracoscopic loop ligation is safe and effective in managing blebs and parenchymal bullae, with a lower cost, fewer complications, and a lower recurrence rate than provided by standard surgical techniques. On the basis of our results, we advocate the use of the self-made endoscopic loop for ligation of parenchymal blebs and bulla in patients with spontaneous pneumothorax to achieve a truly cost-effective and minimally invasive thoracoscopic procedure.

Adolescent↗
PubMed1982

The pathologic effect of ureteral ligation, with clinical implications.

Inadvertent, ureteral ligation is a frequent occurrence with pelvic surgery. We determined the effect of the ureteral ligation of varying lengths of time in an animal model in order to provide a rational basis for the clinical management of such injuries in humans. In the rat, simple deligation of a ureter that has been ligated for as long as 24 hours results in complete resolution of the injury with no gross or microscopic evidence of the trauma. After 48 and 72 hours of ligation, hydroureter was apparent.

Animals↗
PubMed2000

A new thioether-ligated iron porphyrin as a model of a protonated form of P450 active site.

Thioether-ligated iron porphyrin (complex 1) was synthesized as a model of the protonated form of P450 to explore the possible involvement of the protonated form in the catalytic cycle, and ether-ligated iron porphyrin (complex 2) was also synthesized for comparison. The thioether and ether ligands enhanced heterolytic O-O bond cleavage of peroxy acid-iron porphyrin complex even in highly hydrophobic media without the assistance of acid or base, using mCPPAA as an oxidant. Competitive oxidation of cyclooctane/cyclooctene catalyzed by iron porphyrins showed that complexes 1 and 2 are less effective than heme thiolate (P450 and a synthetic heme thiolate (SR complex)) in oxidizing alkane. The possibility that thiol-ligated heme, which is a protonated form of heme thiolate, is not involved in the active intermediate structure of P450 is indicated by this result. This is the first report concerning the oxidizing ability of a thioether-ligated iron porphyrin.

Binding Sites↗
PubMed2000

Effects of galanin on wide-dynamic range neuron activity in the spinal dorsal horn of rats with sciatic nerve ligation.

Galanin is a 29-amino acid peptide with a suggested role in nociception. The effect of galanin on wide-dynamic range neuron discharge frequency in rats with nerve ligation, used as a model of neurogenic pain, was investigated by extracellular recording methods. Seven to 14 days after sciatic nerve ligation, 0.1, 0.5 or 1 nmol of galanin was administered directly on the dorsal surface of the L3-L5 spinal cord of rats with sciatic nerve ligation. It was found that galanin inhibited the activity of wide-dynamic range neurons dose-dependently, an effect was more pronounced in sciatic nerve ligated rats than intact rats. Furthermore, when 1 nmol of galantide, the galanin antagonist, was administered on the dorsal surface of the L3-L5 spinal cord, the wide-dynamic range neuron discharge frequency increased significantly. The results suggest that galanin plays an important role in the modulation of presumed nociception in mononeuropathy.

Animals↗
PubMed2002

PCR ligation mutagenesis in transformable streptococci: application and efficiency.

PCR ligation mutagenesis is a novel technique that can easily be adapted for many gene modification purposes. Successful application of this versatile technique involves sequence identification of the target gene region, creation of a mutagenic construct consisting of two gene-flanking proximal sequences specifically ligated to a selectable marker, and incorporation of this construct into the genome via genetic transformation and homologous recombination. In this study, we demonstrate the use of PCR, followed by restriction digestion and re-ligation to generate transforming constructs for the rapid deletion of open reading frames in transformable streptococci. Moreover, we characterized the dependence of transformation efficiency for mutant generation on the length of the homologous regions harbored by the mutagenic construct. Our results indicated that PCR ligation mutagenesis could be reliably employed for the systematic generation of gene deletion mutants in both highly transformable Streptococcus mutans and S. pneumoniae. Evaluation of the method showed a strong influence of the length of homologous flanking region on integration efficiency.

DNA, Bacterial↗
PubMed1997

Effects of simvastatin, pentoxifylline and spironolactone on hepatic fibrosis and portal hypertension in rats with bile duct ligation.

AIMS/METHODS: Our aim was to study the antifibrotic and hemodynamic effects of simvastatin (SMV), pentoxifylline (PTX) and spironolactone (SPN), three drugs which may have antifibrotic and/or portal hypotensive properties, in a model of hepatic fibrosis and portal hypertension induced in rats by bile duct ligation. A blind study was performed in five groups of 53 Sprague-Dawley rats: sham, placebo (PL), SMV (2.5 mg x kg(-1) x J(-1)), PTX (50 mg x kg(-1) x J(-1)) and SPN (100 mg x kg(-1) x J(-1)). Drugs were administered by daily gavage over a 4-week period as soon as bile duct ligation was performed. At day 28, the following parameters were evaluated: area of hepatic fibrosis by image analysis after staining collagen with picrosirius and plasma concentrations of hyaluronate, splanchnic and systemic hemodynamics (radiolabeled microspheres). RESULTS: Portal venous pressure (PL: 15.5+/-1.5, SMV: 15.8+/-2.5, PTX: 15.9+/-1.8, SPN: 13.5+/-2.1 mmHg, p<0.05) and porto-systemic shunts (PL: 30+/-31, SMV: 18+/-27, PTX: 25+/-24, SPN: 5+/-4%, p<0.05) were significantly reduced in the SPN group; other hemodynamic parameters were not significantly altered. There was a significant correlation between portosystemic shunts and portal pressure (r(s)=0.47, p<0.01). The area of fibrosis was not significantly different among the four groups of bile duct ligated rats (PL: 8.7+/-3.9, SMV: 7.1+/-3.6, PTX: 7.8+/-2.7, SPN: 6.6+/-3.3%) but was higher than in sham rats (1.5+/-0.5%, p<0.001). Hyaluronate was significantly higher in bile duct ligated rats (from 374+/-162 to 420+/-131 microg/l, among the four groups) than in sham rats (52+/-16 microg/l, p<0.0001). CONCLUSIONS: In this model, none of the drugs prevented hepatic fibrosis. On the other hand, spironolactone decreased portal pressure and prevented porto-systemic shunts. Therefore, this drug may have beneficial effects in patients with early portal hypertension.

Animals↗
PubMed2000

DNA synthesis by CHO cell extracts on fork-like DNA templates containing the major cisplatin adduct requires a ligation step.

In this work we have examined the role of DNA ligation in the in vitro replication catalyzed by CHO crude extracts on fork-like oligonucleotide substrates containing a unique d(GpG) intrastrand cross-link produced by the antitumor drug cisplatin. We show here that this reaction involves a ligation step, which necessitates excision of the flap strand of the forked substrate. By constructing substrates in which the unannealed tail could not be degraded by a 5' exonuclease, we obtained evidence suggesting that this type of activity participates in the removal of the flap strand. Furthermore, we found that the ligation event played a predominant role in the synthesis of fully replicated products from both intact and platinated templates. Finally, we investigated whether translesion synthesis of the cisplatin lesion could occur concomitantly to ligation by monitoring the incorporation of labeled precursors downstream of the adduct. Our results are compatible with the possibility that some translesion syntheses of the Pt-d(GpG) adduct by the extracts also contributed to the generation of full length molecules.

Animals↗
PubMed1997

Tertiary and quaternary chloride effects of the partially ligated (CN-met) hemoglobin intermediates.

Heterotropic effects of NaCl were studied using CN-met hemoglobin, which has been found to follow the same rules of homotropic cooperativity as CO-Hb and O2-Hb [Huang and Ackers, Biochemistry, 35 (1996) 704; Huang et al., Biophys. J., 71 (1996) 2094]. Modulation of heme site cooperativity by NaCl was determined in this study for all partially ligated CN-met intermediates by measuring their dimer-to-tetramer assembly free energies as a function of NaCl concentration (0.08-1.4 M; pH 7.4, T = 21.5 degrees C). Thermodynamic linkage analysis yielded the contributions to heme site binding cooperativity for all 16 reactions of the binding cascade, and also their apparent changes in bound salt. The principal findings were as follows: (i) At each [NaCl] the ten tetrameric species exhibited three discrete cooperative free energy levels; (ii) positional isomers of the doubly ligated tetramers were distributed among two of these levels according to their specific configurations of ligated sites, in conformity with the symmetry rule mechanism of hemoglobin cooperativity [Ackers et al., Science 255 (1992) 54]; (iii) the apparent moles of NaCl release followed the same configuration-specific distribution as that of heme site cooperativity, i.e., this parameter was synchronized according to the same response clusters. The system thus manifests both a "tertiary chloride effect" and a "quaternary chloride effect", which parallel the tertiary and quaternary Bohr effects [Daugherty et al., Biochemistry, 33 (1994) 10345; Perrella et al., Biochemistry, 33 (1994) 10358] and the tertiary and quaternary enthalpy effects [Huang and Ackers, Biochemistry, 34 (1995) 6316]. Comparison with findings on the stoichiometric O2-binding linkages over an identical range of conditions [Doyle et al., Biophys. Chem., 64 (1997)] revealed that the overall NaCl release upon ligating all four hemes is identical for O2 and CN-met, whereas the detailed distributions of apparent chloride release showed variations between the two ligands, i.e., CN-met Hb showed only a negligible quaternary enhancement at all [NaCl] conditions and a larger tertiary chloride effect compared with O2-Hb. Possible origins of these variations are considered.

Allosteric Regulation↗
PubMed1998

Amplification of target-specific, ligation-dependent circular probe.

We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe). The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region. The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix. Subsequent ligation of the two termini results in a covalently linked C-probe that becomes 'locked on to' the target. The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to 'rolling circle' replication of bacteriophage in vivo. This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification. This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR. Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein-Barr virus early RNA. The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons. As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion. Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified. The combined advantages of the C-probe and thermocycling have a broad applicability for the detection of both DNA and RNA. Finally, we described a novel isothermal amplification method, ramification extension amplification, utilizing circular nature of C-probe and displacement activity of DNA polymerase.

Binding Sites↗
PubMed2003

Evaluation of friction of stainless steel and esthetic self-ligating brackets in various bracket-archwire combinations.

This study measured and compared the level of frictional resistance generated between stainless steel self-ligating brackets (Damon SL II, SDS Ormco, Glendora, Calif), polycarbonate self-ligating brackets (Oyster, Gestenco International, Göthenburg, Sweden), and conventional stainless steel brackets (Victory Series, 3M Unitek, Monrovia, Calif), and 3 different orthodontic wire alloys: stainless steel (Stainless Steel, SDS Ormco), nickel-titanium (Ni-Ti, SDS Ormco), and beta-titanium (TMA, SDS Ormco). All brackets had a.022-in slot, whereas the orthodontic wire alloys were tested in 3 different sections:.016,.017 x.025, and.019 x 0.025 in. Each of the 27 bracket and archwire combinations was tested 10 times, and each test was performed with a new bracket-wire sample. Both static and kinetic friction were measured on a custom-designed apparatus. All data were statistically analyzed (Kruskal-Wallis and Mann Whitney U tests). Stainless steel self-ligating brackets generated significantly lower static and kinetic frictional forces than both conventional stainless steel and polycarbonate self-ligating brackets, which showed no significant differences between them. Beta-titanium archwires had higher frictional resistances than stainless steel and nickel-titanium archwires. No significant differences were found between stainless steel and nickel-titanium archwires. All brackets showed higher static and kinetic frictional forces as the wire size increased.

Dental Alloys↗
PubMed2003

Ligation of IFN-gamma-induced HLA-DR molecules on fibroblasts induces RANTES expression via c-Jun N-terminal kinase (JNK) pathway.

The role of human leukocyte antigen (HLA) class II molecules on non-antigen presenting cells has been a matter of controversy. We recently reported that ligation of HLA-DR molecule with anti-HLA-DR antibodies (L243) and/or antigenic peptide/T cell receptor complex resulted in a secretion of several chemokines such as RANTES. In the present study, we aimed to detect putative signal transduction pathway leading to RANTES production from fibroblasts when the DR molecules were ligated with L243. Protein tyrosine kinase inhibitor (GF109203X) suppressed RANTES expression in a dose dependent manner for up to 50% from gingival fibroblasts (GF), while protein kinase C inhibitor (genistein) had no inhibitory effect. Ligation of DR molecules with L243 resulted in tyrosine phosphorylation of 54 kDa cellular protein. Thus, we suspected that either Jun N-terminal kinase-2 (JNK-2) or Src family proteins were involved in HLA-DR-mediated signaling. JNK inhibitor (SP600125), but not Src inhibitor (PP2), suppressed both L243 stimulated RANTES mRNA expression and protein secretion. The maximum inhibition for RANTES production by SP600125 was more than 80%. Additionally, JNK inhibitor nearly completely blocked tumor necrosis factor-alpha (TNF-alpha)-induced RANTES production in GF. Furthermore, ligation of GF HLA-DR with L243 induced selective phosphorylation of JNK-2. We concluded that JNK-2 was one of the HLA-DR-mediated signal transduction pathways.

Blotting, Western↗
PubMed1996

Template-directed ligation of peptides to oligonucleotides.

BACKGROUND: Oligonucleotide-peptide conjugates have several applications, including their potential use as therapeutic agents. We developed a strategy for the chemical ligation of unprotected peptides to oligonucleotides in aqueous solution. The two compounds are joined via a stable amide bond in a template-directed reaction. RESULTS: Peptides, ending in a carboxy-terminal thioester, were converted to thioester-linked oligonucleotide-peptide intermediates. The oligonucleotide portion of the intermediate binds to a complementary oligonucleotide template, placing the peptide in close proximity to an adjacent template-bound oligonucleotide that terminates in a 3' amine. The ensuing reaction results in the efficient formation of an amide-linked oligonucleotide-peptide conjugate. CONCLUSIONS: An oligonucleotide template can be used to direct the ligation of peptides to oligonucleotides via a highly stable amide linkage. The ligation reaction is sequence-specific, allowing the simultaneous ligation of multiple oligonucleotide-peptide pairs.

Amides↗
PubMed1997

Pyranosyl-RNA: chiroselective self-assembly of base sequences by ligative oligomerization of tetranucleotide-2',3'-cyclophosphates (with a commentary concerning the origin of biomolecular homochirality).

BACKGROUND: Why did Nature choose furanosyl-RNA and not pyranosyl-RNA as her molecular genetic system? An experimental approach to this problem is the systematic comparison of the two isomeric oligonucleotide systems with respect to the chemical properties that are fundamental to the biological role of RNA, such as base pairing and nonenzymic replication. Pyranosyl-RNA has been found to be not only a stronger, but also a more selective pairing system than natural RNA; both form hairpin structures with comparable ease. Base sequences of pyranosyl-RNA can be copied by template-controlled replicative ligation of short activated oligomers (e.g. tetramer-2',3'-cyclophosphates) under mild and potentially natural conditions. The copying proceeds with high regioselectivity as well as chiroselectivity: homochiral template sequences mediate the formation of the correct (4'-->2')-phosphodiester junction between homochiral tetramer units provided they have the same sense of chirality as the template. How could homochiral template sequences assemble themselves in the first place? RESULTS: Higher oligomers of pyranosyl-RNA can self-assemble in dilute solutions under mild conditions by ligative oligomerization of tetramer-2',3'-cyclophosphates containing hemi self-complementary base sequences. The only side reaction that effectively competes with ligation is hydrolytic deactivation of 2',3'-cyclophosphate end groups. The ligation reaction is highly chiroselective; it is slower by at least two orders of magnitude when one of the (D)-ribopyranosyl units of a homochiral (D)-tetramer-2',3'-cyclophosphate is replaced by a corresponding (L)-unit, except when the (L)-unit is at the 4' end of the tetramer and carries a purine, when the oligomerization rate can be approximately 10% of that shown for a homochiral isomer. The oligomerization of homochiral tetramers is not, or only weakly, inhibited by the presence of the non-oligomerizing diastereomers. CONCLUSIONS: Available data on the chiroselective self-directed oligomerization of tetramer-2',3'-cyclophosphates allow us to extrapolate that sets of tetramers with different but mutually fitting base sequences can be expected to co-oligomerize stochastically and generate sequence libraries consisting of predominantly homochiral (D)- and (L)-oligomers, starting from the racemic mixture of tetramers containing all possible diastereomers. Such a capability of an oligonucleotide system deserves special attention in the context of the problem of the origin of biomolecular homochirality: breaking molecular mirror symmetry by de-racemization is an intrinsic property of such a system whenever the constitutional complexity of the products of co-oligomerization exceeds a critical level.

Biopolymers↗
PubMed2000

Bimolecular exon ligation by the human spliceosome bypasses early 3' splice site AG recognition and requires NTP hydrolysis.

Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3' splice site AG is supplied by a different RNA molecule than that containing the 5' splice and branch sites. By varying the time during splicing reactions when the 3' splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3' splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.

Adenosine Triphosphate↗
PubMedDate not supplied

Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation.

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.

Aerobiosis↗
PubMed1995

1H NMR investigation of the paramagnetic cluster environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific assignment of ligated cysteines independent of tertiary structure.

One- and two-dimensional 1H NMR data tailored to detect paramagnetically relaxed protons near the S = 1/2, three-iron-sulfur cluster of the ferredoxin from the hyperthermophile Pyrococcus furiosus are analyzed to sequence specifically assign the hyperfine shifted ligated cysteine signals, to determine the nature of the secondary structural elements on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterized secondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse, S.C., Howard, J.B., Adams, M. W. W., & La Mar, G. N. (1994) Biochemistry 33, 6316-6326]. Inspection of the geometry of the cluster ligating cysteines in the six structurally characterized cubane ferredoxin (Fd) clusters reveals a pattern of distances from the cluster iron(s) that indicate that each Cys will exhibit one backbone proton that will allow the detection of dipolar connectivities to the backbone of adjacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibit weakly relaxed peptide NH and strongly relaxed C alpha H signals, while the two central Cys in that sequence will exhibit strongly relaxed peptide NH but weakly relaxed C alpha H peaks. These dipolar contacts are clearly observed for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explicitly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferredoxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys17 is on a short helix through Leu20 and that Cys56 likely initiates a type I turn, as observed in the crystal structure of the 3Fe Fd for Desulfovibrio gigas [Kissinger, C. R., Sieker, L. C., Adman, E. T., & Jensen, L. H. (1991) J. Mol. Biol. 219, 693-715]. The observed relaxation rates of resolved or partially resolved signals are shown to correlate with their proximity to the various iron in the cluster, as determined for the homologous residues in D. gigas Fd, providing additional qualitative information on tertiary contacts of the cluster.

Amino Acid Sequence↗
PubMed1995

Enthalpic and entropic components of cooperativity for the partially ligated intermediates of hemoglobin support a "symmetry rule" mechanism.

Subunit assembly reactions of hemoglobin's 10 ligation microstates have been studied as a function of temperature to evaluate the enthalpic and entropic components of cooperativity. It is found that the cooperative enthalpies and cooperative entropies distribute in close agreement with predictions of the symmetry rule mechanism (Ackers et al., 1992) previously deduced from the free energy distribution, in combination with structure-sensitive probes (Doyle & Ackers, 1992; LiCata et al., 1993; Daugherty et al., 1994). Principal findings of the present study are as follows: (1) In unligated hemoglobin (quaternary T), dimer-tetramer assembly is driven by a large negative enthalpy, whereas in the fully ligated (quaternary R) species, the driving force for quaternary assembly is entropic. For the eight intermediate ligation species, the switchover from enthalpic to entropic control follows precisely the symmetry rule predictions; i.e., switching from enthalpic to entropic control occurs at each of the six steps that create ligated heme sites on both sides of the dimer-dimer interface. The combinatorial distribution found previously with free energies does not therefore arise from coincidental enthalpy-entropy compensation that masks a more fundamental distribution. (2) The free energy of tertiary constraint delta Gtc, which pays for intradimer cooperativity prior to quaternary switching, contains large enthalpic and entropic components delta Htc and delta Stc. Like delta Gtc, these terms vanish at the second binding step within the T tetramer. It is found that delta Gtc arises from a net enthalpic dominance over an almost equally large T delta Stc. (3) The stepwise enthalpies correlate with stepwise values of Bohr protons and Bohr free energies (Daugherty et al., 1994) throughout the cascade of 16 stepwise reactions; the correlated clusters of these values follow predictions of the symmetry rule mechanism. (4) These results obtained with cyanomethemoglobin are consistent with the corresponding data on oxygenated hemoglobin which has been resolved at each stage of oxygenation.

Allosteric Regulation↗
PubMed1994

Nonenzymatic plasmid ligation mediated by minor groove-binding molecules.

DNA ligation is the weak link in the chain of gene cloning. We have developed a straightforward nonenzymatic alternative to this reaction that employs easily available commercial reagents. The method uses the affinity of distamycin for the minor groove to join DNA ends together. Phosphodiester bonds are formed after cyanoimidazole-promoted phosphate activation in the presence of manganese(II) cations. When transfected into eukaryotic cells, the chemically ligated plasmid is transcribed even more efficiently than after enzymatic ligation. At present this technique compares favorably with T4 ligation for AT-rich cohesive termini, but in principle it could be extended to any restriction site.

Cloning, Molecular↗