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PubMed1998

Induced preference or conditioned aversion for sodium chloride in rats with chronic bile duct ligation.

We examined whether a learned aversion to saline could account for the reduction in saline intake produced by bile duct ligation (BDL) in rats and whether increased saline intake by BDL rats was associated with hypotension. In three experiments, rats were given continuous access to water in choice with saline after surgery. In Experiment 1, rats were deprived of food and fluid for 24 h and then given 2-h access to either 0.15 or 0.3 M saline. Rats received a BDL or sham-ligation immediately (paired) or 48 h after (nonpaired) the 2-h bout of saline ingestion. The results show that nonpaired BDL rats increased their daily saline intake relative to nonpaired sham-ligated or paired BDL rats approximately 1-4 weeks after surgery. In Experiment 2, when water and either cherry or grape Kool-Aid (0.05% w/v) dissolved in 0.15 M saline to distinguish the flavor of the solution was offered prior to surgery, BDL rats reduced their ingestion of grape-flavored saline after surgery regardless of whether they were exposed to grape- or cherry-flavored saline prior to surgery. In Experiment 3, when rats were offered water and 0.3 M saline 48 h after surgery, BDL rats ligated for 4 weeks increased their saline intake relative to sham-ligated controls and this elevation in saline intake by BDL rats was associated with hypotension. The results suggest that the symptoms associated with the BDL surgery can serve as effective unconditioned stimuli in the acquisition of learned flavor aversions, and that hypotension may play a role in the elevated intake of saline by BDL rats.

Animals↗
PubMed2003

Proton NMR study of chemically modified horse heart ferricytochrome c confirms the presence of histidine and lysine-ligated conformers in 30% acetonitrile solution.

Comparison of the 1H NMR spectra for guanidinated ferricyt c and chloro(terpyridine)platinum(II)-modified ferricyt c in 30% acetonitrile (ACN) solution with that for ferricyt c in 30% ACN is reported. The absence of the heme methyl proton resonances characteristic of the IV*-form (Lys-ligated) in the NMR spectrum of guanidinated ferricyt c in 30% ACN solution confirms that a lysine-ligated form of ferricyt c is produced in 30% ACN solution. The absence of the 8-methyl heme proton resonance of the V*-form in the NMR spectrum of chloro(terpyridine)platinum(II)-modified ferricyt c in 30% ACN solution demonstrates that a bis-His-ligated form of ferricyt c is produced in 30% ACN, not a hydroxide ligated form, as previously proposed. The revised assignment for the V* form of ferricyt c in mixed media explains differences between the exchange network we previously reported for ferricyt c in 30% ACN [Protein Sci. 10 (2001) 2291] as versus that reported by Dopner et al. at high pH [J. Am. Chem. Soc. 120 (1998) 11246]. Lys- and His-ligated forms are known to be produced in the presence of denaturants in protein folding studies of ferricyt c. Consequently, the exchange network between these non-native forms of ferricyt c in 30% ACN may have biological relevance for ferricyt c folding.

Acetonitriles↗
PubMed1997

Expression of the liver Na+-independent organic anion transporting polypeptide (oatp-1) in rats with bile duct ligation.

BACKGROUND/AIMS: In rats with cholestasis due to bile duct ligation, the expression of the Na+-dependent taurocholate co-transporting polypeptide, the major uptake system for conjugated bile acids in hepatocytes, is down-regulated. Our purpose was to examine the expression of the organic anion transporting polypeptide, a Na+-independent uptake system for bile acids and organic anions, in rats with bile duct ligation, and to compare the expression of organic anion transporting polypeptide to that of Na+-dependent taurocholate co-transporting polypeptide. METHODS: Rats with bile duct ligation were studied after 1, 3 or 7 days. The expression of organic anion transporting polypeptide and Na+-dependent taurocholate co-transporting polypeptide proteins was examined by Western blot analysis and steady-state mRNA levels were determined by Northern blot analysis using cDNAs encoding organic anion transporting polypeptide and Na+-dependent taurocholate co-transporting polypeptide. Sham-operated animals were used as controls. RESULTS: The expression of organic anion transporting polypeptide protein was slightly, but not significantly, decreased 1 day after ligation (10.3%); it was markedly decreased after 3 days (56.9%; p<0.03) and 7 days (46.8%; p<0.05) compared to sham-operated animals. Steady-state mRNA levels of organic anion transporting polypeptide were decreased by 79.7% (p<0.04), 48.8% (p<0.02) and 57.4% (p<0.02) after 1, 3 and 7 days respectively. For comparison, Na+-dependent taurocholate co-transporting polypeptide protein and mRNA levels were decreased by 73.8% (p<0.03) and 70.0% (p<0.05) at 1 day and remained low after 3 and 7 days. CONCLUSIONS: In rats with bile duct ligation, the expression of organic anion transporting polypeptide protein and mRNA is down-regulated. Down-regulation of organic anion transporting polypeptide seems less pronounced than that of Na+-dependent taurocholate co-transporting polypeptide. Nevertheless, it could contribute to a decreased uptake of potentially toxic bile acids or organic anions in this situation.

Animals↗
PubMed2001

Changes in the rate of tubal ligation done after cesarean section.

OBJECTIVE: We studied tubal ligations done after cesarean section in a Spanish hospital during a 20-year period, in order to analyze changes in patient characteristics and indications for cesarean delivery. STUDY DESIGN: We reviewed the clinical records, for the period from 1978 to 1997, of 1996 cases of cesarean section followed by tubal ligation in 108776 births in which the fetus weighed 1000 g or more. RESULTS: During the 20-year period of study, the proportion of cesarean sections relative to vaginal deliveries increased, as did the frequency of cesarean section followed by tubal ligation relative to cesarean and vaginal deliveries. The proportion of women who underwent tubal ligation after a second cesarean section decreased from 60% during 1978-1982 to 5.6% during 1993-1997. The most frequent maternal pathology associated with gestation was previous cesarean section (60.5%), although 50% of the women had no underlying pathology. CONCLUSIONS: In our setting, the rate of cesarean section followed by tubal ligation has been increasing steadily since the early 1980s. The proportion of women who requested tubal sterilization and who had only one living child, or who had had a previous cesarean birth, also increased.

Adolescent↗
PubMed1996

Ligation of lateral carotid artery attenuates disturbance of brain function caused by subsequent cerebral ischemia in rabbits.

The effects of right carotid artery ligation on the subsequent cerebral ischemia, induced by iron particle injection, in rabbits, were evaluated by recording somatosensory evoked potentials (SEPs) and cerebral blood flow (CBF) using laser Doppler flowmetry. Iron particle injection decreased CBF over 120 min and delayed SEP onset latency in rabbits with no previous carotid artery ligation (control group). In rabbits with a carotid ligation 3 days before, iron particle injection induced the decrease of CBF, as in the control group, but did not prolong the latency of SEP. Injection of iron particles induced only a transient decrease of CBF (less than 10 min) followed by an abrupt recovery, and no prolongation of SEP latency was observed in rabbits with a carotid ligation 6 days before. These results suggest that carotid artery ligation induces a beneficial effect on cerebral function during the subsequent ischemia, which is independent on the CBF changes.

Animals↗
PubMed2003

Induction of left ventricular remodeling and dysfunction in the recipient heart after donor heart myocardial infarction: new insights into the pathologic role of tumor necrosis factor-alpha from a novel heterotopic transplant-coronary ligation rat model.

OBJECTIVES: The present study investigated the effects of tumor necrosis factor (TNF)-alpha and angiotensin II (ANG II) on cardiac remodeling and dysfunction at the early stage of acute myocardial infarction (MI) by using a novel heterotopic cardiac transplantation-coronary ligation model. BACKGROUND: A recent clinical study has demonstrated a possible role of monocytosis in the development of left ventricular (LV) remodeling in patients with acute MI reperfusion. METHODS: We performed isogenic heterotopic cardiac transplantation and simultaneous coronary ligation to produce MI in the donor heart and to evaluate the hearts of both donors and recipients in Lewis rats. RESULTS: A significant decrease in LV fractional shortening and positive rate of rise in LV pressure and a significant increase in LV end-diastolic dimension/body weight and LV end-diastolic pressure were observed in the recipient hearts in the ligation group on day 7. TNF-alpha was significantly elevated not only in the plasma but also in the recipient hearts in the ligation group. In contrast, ANG II was significantly increased only in the infarct region of the donor hearts, but not in the plasma. Furthermore, the recipients' transient LV remodeling and dysfunction were completely abolished by the intravenous administration of a TNF-alpha antagonist. CONCLUSIONS; We developed a novel cardiac transplantation-coronary ligation model capable of inducing MI in the absence of downstream hemodynamic effects and allowing differential quantification of indexes of cardiac remodeling in vivo, including the local and remote effects of ANG II and TNF-alpha on cardiac remodeling.

Angiotensin II↗
PubMed1996

Ultrastructural study on the epithelium of ligated fallopian tubes in women of reproductive age.

The epithelium of normal and ligated Fallopian tubes in nine women of reproductive age was studied by using scanning electron microscopy (SEM). The results were as follows: at a distance of 0.5 cm from the ligated scar, numerous cilia of the epithelium were twisted and adhered, the shape of the epithelial cells was irregular, and even many microvilli and cilia lost from the local epithelium. However, the microvilli and cilia of the epithelium were normal on the mucosa at 1.0 cm from the ligated scar. This may account for the continued infertility following re-anastomosis of the ligated Fallopian tubes, due to the ultrastructural changes of oviducts and inability to convey the ova. Thus, at the Fallopian tube re-anastomosis, it is recommended that the removal of the mucosa should be at least 0.5 cm away from the ligated scar so as to increase the success rate of the fertilization. On the epithelium of the hydrosalpinx, Fallopian tube stomata located among the secretory cells were found for the first time. These stomata are small openings of lymphatic capillaries with a diameter of 3.25 microns to 4.25 microns. They are likely to be connected between the lumen of the Fallopian tube and subserous lymphatic vessels, and also involved in the drainage of the hydrosalpinx. Most or all cilia were lost from the ciliated cell surface on the epithelium of the hydrosalpinx, whereas the microvilli were flourishing. Therefore, many ciliated cells turned into secretory cells. In addition, a new pathogenesis of the hydrosapinx is discussed.

Adult↗
PubMed1997

Surprising fidelity of template-directed chemical ligation of oligonucleotides.

BACKGROUND: Nucleic acid replication via oligonucleotide ligation has been shown to be extremely prone to errors. If this is the case, it is difficult to envision how the assembly and replication of short oligonucleotides could have contributed to the origin of life and to the evolution of a putative RNA world. In order to assess the fidelity of oligonucleotide replication more accurately, chemical ligation reactions were performed with constant-sequence DNA templates and random-sequence DNA pools as substrates. RESULTS: In keeping with earlier results, constant-sequence hairpin templates were not faithfully copied by random-sequence substrates. Linear templates, however, showed exceptional replication fidelity, particularly when random hexamers were ligated at 25 degrees C. Surprisingly, at low temperatures the formation of G.A base pairs was common and sometimes occurred even more readily than the formation of the corresponding Watson-Crick A-T and G-C base pairs. CONCLUSIONS: The fidelity of ligation reactions increases with temperature and decreases with the length of the random-sequence substrates. Oligonucleotides with a defined sequence can be copied faithfully in the absence of enzymes. Thus, to the extent that short oligonucleotides could readily have been generated by prebiotic mechanisms, it is possible that the earliest self-replicators arose via oligonucleotide ligation.

Base Composition↗
PubMed1993

2'-Hydroxyl groups important for exon polymerization and reverse exon ligation reactions catalyzed by a group I ribozyme.

The functional importance of ribose moieties in both exons and in intron sequences proximal to the 3' splice site of a group I intron has been analyzed using a novel exon polymerization reaction. The ribozyme is a modified version of a self-splicing bacterial tRNA intron (I) that attacks a 20-nucleotide synthetic ligated exon substrate (E1.E2), yielding E1 and I.E2 by reverse exon ligation. A series of repetitive reactions then polymerize E2 on the 3' end of the intron; attack by E1 subsequently generates E1.(E2)n. Systematic deoxyribonucleotide substitution within E1.E2 was used to probe the function of 2'-hydroxyl groups in each exon and the 3'-terminal nucleotides of the intron. We find that ribose at the splice junction (U-1) and at the two adjacent positions with E1 (A-2, C-3) is important for reverse exon ligation. Within E2, deletion of 2'-hydroxyl groups of the nucleotides that form P10 does not affect reactivity. In contrast, ribose at the 3' end of the intron is essential for reverse exon ligation, and the presence of a 2'-OH group in each of the nucleotides comprising P9.0[3'] contributes to reaction efficiency. These results support a model in which specific 2'-hydroxyl groups at and adjacent to the reaction sites form tertiary contacts that serve to stabilize interactions with the catalytic core of the ribozyme. Furthermore, they suggest that the mechanism by which guanosine at the 3' end of the intron is activated for reverse exon ligation is the same as that by which guanosine mononucleotide is activated in the first step of splicing.

Bacteria↗
PubMed1977

Binding of CO to mutant alpha chains of hemoglobin M Iwate; evidence for distal imidazole ligation.

The optical contribution of the beta chains to the spectrum of hemoglobin M Iwate (alpha87his leads to tyr)2beta2a was subtracted with the aid of a computer so that the spectrum of ferric alpha chains was obtained. Tyrosinate binding to the heme is suggested from spectral resemblance to ferric heme phenolate in dimethyl sulfoxide. The slow reduction of the abnormal ferric alpha chains in hemoglobin M Iwate by dithionite was studied spectrophotometrically both in the presence and absence of CO. The rate of reduction was found to be dependent on the state of ligation of the normal beta chains. The CO-ligated form of the reduced alpha chains bears strong spectral resemblance to the CO-ligated form of the reduced beta chains suggesting similar structures for the heme-ligand complex. A model compound with similar optical properties to the CO-ligated protein can be prepared in dimethyl sulfoxide from hemin chloride, imidazole, and CO using chromous acetate as the heme reductant. Substitution of phenolate for imidazole produces a spectral entity so different from that observed in the protein as to rule out tyrosinate ligation to ferrous heme of the alpha chains when CO is bound.

Carbon Monoxide↗
PubMed2003

Does aspartate 170 of the D1 polypeptide ligate the manganese cluster in photosystem II? An EPR and ESEEM Study.

Aspartate 170 of the D1 polypeptide provides part of the high-affinity binding site for the first Mn(II) ion that is photooxidized during the light-driven assembly of the (Mn)(4) cluster in photosystem II [Campbell, K. A., Force, D. A., Nixon, P. J., Dole, F., Diner, B. A., and Britt, R. D. (2000) J. Am. Chem. Soc. 122, 3754-3761]. However, despite a wealth of data on D1-Asp170 mutants accumulated over the past decade, there is no consensus about whether this residue ligates the assembled (Mn)(4) cluster. To address this issue, we have conducted an EPR and ESEEM (electron spin-echo envelope modulation) study of D1-D170H PSII particles purified from the cyanobacterium Synechocystis sp. PCC 6803. The line shapes of the S(1) and S(2) state multiline EPR signals of D1-D170H PSII particles are unchanged from those of wild-type PSII particles, and the signal amplitudes correlate approximately with the lower O(2) evolving activity of the mutant PSII particles (40-60% compared to that of the wild type). These data provide further evidence that the assembled (Mn)(4) clusters in D1-D170H cells function normally, even though the assembly of the (Mn)(4) cluster is inefficient in this mutant. In the two-pulse frequency domain ESEEM spectrum of the 9.2 GHz S(2) state multiline EPR signal of D1-D170H PSII particles, the histidyl nitrogen modulation observed at 4-5 MHz is unchanged from that of wild-type PSII particles and no significant new modulation is observed. Three scenarios are presented to explain this result. (1) D1-Asp170 ligates the assembled (Mn)(4) cluster, but the hyperfine couplings to the ligating histidyl nitrogen of D1-His170 are too large or anisotropic to be detected by ESEEM analyses conducted at 9.2 GHz. (2) D1-Asp170 ligates the assembled (Mn)(4) cluster, but D1-His170 does not. (3) D1-Asp170 does not ligate the assembled (Mn)(4) cluster.

Amino Acid Substitution↗
PubMed2004

Evidence that the C-terminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S1 to S2 transition: an isotope-edited FTIR study.

Isotope-edited FTIR difference spectroscopy was employed to determine if the C-terminal alpha-COO(-) group of the D1 polypeptide ligates the (Mn)(4) cluster in photosystem II (PSII) and, if so, if it ligates the Mn ion that undergoes an oxidation during the S(1) --> S(2) transition. Wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated photoautotrophically in the presence of L-[1-(13)C]alanine or unlabeled ((12)C) L-alanine. In wild-type cells, both the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 and all alanine-derived peptide carbonyl groups will be labeled. In D1-A344G and D1-A344S mutant cells, the C-terminal alpha-COO(-) group of the D1 polypeptide will not be labeled because this group is no longer provided by alanine. The resultant S(2)-minus-S(1) FTIR difference spectra of purified wild-type and mutant PSII particles showed that one symmetric carboxylate stretching mode that is altered during the S(1) --> S(2) transition is sensitive to L-[1-(13)C]alanine-labeling in wild-type PSII particles but not in D1-A344G and D1-A344S PSII particles. Because the only carboxylate group that can be labeled in the wild-type PSII particles but not in the mutant PSII particles is the C-terminal alpha-COO(-) group of the D1 polypeptide, we assign the L-[1-(13)C]alanine-sensitive symmetric carboxylate stretching mode to the alpha-COO(-) group of D1-Ala344. In unlabeled wild-type PSII particles, this mode appears at approximately 1356 cm(-1) in the S(1) state and at approximately 1339 or approximately 1320 cm(-1) in the S(2) state. These frequencies are consistent with unidentate ligation of the (Mn)(4) cluster by the alpha-COO(-) group of D1-Ala344 in both the S(1) and S(2) states. The apparent 17-36 cm(-1) downshift in frequency in response to the S(1) --> S(2) transition is consistent with the alpha-COO(-) group of D1-Ala344 ligating a Mn ion whose charge increases during the S(1) --> S(2) transition. Accordingly, we propose that the alpha-COO(-) group of D1-Ala344 ligates the Mn ion that undergoes an oxidation during the S(1) --> S(2) transition. Control experiments were conducted with Mn-depleted wild-type PSII particles. These experiments showed that tyrosine Y(D) may be structurally coupled to the carbonyl oxygen of an alanine-derived peptide carbonyl group.

Alanine↗
PubMed2005

No evidence from FTIR difference spectroscopy that aspartate-170 of the D1 polypeptide ligates a manganese ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II.

On the basis of mutagenesis and X-ray crystallographic studies, Asp170 of the D1 polypeptide is widely believed to ligate the (Mn)4 cluster that is located at the catalytic site of water oxidation in photosystem II. Recent proposals for the mechanism of water oxidation postulate that D1-Asp170 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions. To test these hypotheses, we have compared the FTIR difference spectra of the individual S state transitions in wild-type* PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 with those in D1-D170H mutant PSII particles. Remarkably, our data show that the D1-D170H mutation does not significantly alter the mid-frequency regions (1800-1000 cm(-1)) of any of the FTIR difference spectra. Therefore, we conclude that the oxidation of the (Mn)4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp170 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. The simplest explanation for these data is that the Mn ion that is ligated by D1-Asp170 does not increase its charge or oxidation state during any of these S state transitions. These data have profound implications for the mechanism of water oxidation. Either (1) the oxidation of the Mn ion that is ligated by D1-Asp170 occurs only during the transitory S3 --> S4 transition and serves as the critical step in the ultimate formation of the O-O bond or (2) the oxidation increments and O2 formation chemistry that occur during the catalytic cycle involve only the remaining Mn3Ca portion of the Mn4Ca cluster. Our data also show that, if the increased positive charge on the (Mn)4 cluster that is produced during the S1 --> S2 transition is delocalized over the (Mn)4 cluster, it is not delocalized onto the Mn ion that is ligated by D1-Asp170.

Aspartic Acid↗
PubMed2005

Evidence from biosynthetically incorporated strontium and FTIR difference spectroscopy that the C-terminus of the D1 polypeptide of photosystem II does not ligate calcium.

Recent FTIR studies have provided evidence that the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 is a unidentate ligand of a Mn ion in photosystem II [Chu, H.-A., Hiller, W., and Debus, R. J. (2004) Biochemistry 43, 3152-3166; Kimura, Y., Mizusawa, N., Yamanari, T., Ishii, A., and Ono, T.-A. (2005) J. Biol. Chem. 280, 2078-2083]. However, the FTIR data could not exclude Ca ligation. Furthermore, the recent approximately 3.5 A X-ray crystallographic structural model positions the alpha-COO(-) group of D1-Ala344 near a Ca ion [Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Science 303, 1831-1838]. Therefore, to conclusively establish whether the alpha-COO(-) group of D1-Ala344 ligates Mn or Ca, the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344 was identified in the S(2)-minus-S(1) FTIR difference spectrum of PSII particles having Sr substituted for Ca. Cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated in media having Sr substituted for Ca and containing either l-[1-(13)C]alanine or unlabeled ((12)C) alanine. The S(2)-minus-S(1) FTIR difference spectra of the purified PSII particles show that substituting Sr for Ca alters several carboxylate stretching modes, including some that may correspond to one or more metal ligands, but importantly does not alter the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344. In unlabeled PSII particles, this mode appears at approximately 1356 cm(-)(1) in the S(1) state and at either approximately 1337 or approximately 1320 cm(-)(1) in the S(2) state, irrespective of whether the PSII particles contain Ca or Sr. These data are inconsistent with Ca ligation and show, therefore, that the C-terminal alpha-COO(-) group of the D1 polypeptide ligates a Mn ion. These data also show that substituting Ca with the larger Sr ion perturbs other unidentified carboxylate groups, at least one of which may ligate the Mn(4) cluster.

Calcium↗
PubMed1999

Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

Bacterial Proteins↗
PubMed2006

Reaction mechanism and kinetics of the traceless Staudinger ligation.

The traceless Staudinger ligation enables the formation of an amide bond between a phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here, the coupling of peptides by this reaction was characterized in detail. Experiments with [(18)O]H(2)O indicated that the reaction mediated by (diphenylphosphino)methanethiol proceeded by S-->N acyl transfer of the iminophosphorane intermediate to form an amidophosphonium salt, rather than by an aza-Wittig reaction and subsequent hydrolysis of the resulting thioimidate. A continuous (13)C NMR-based assay revealed that the rate-determining step in the Staudinger ligation of glycyl residues mediated by (diphenylphosphino)methanethiol was the formation of the initial phosphazide intermediate. Less efficacious coupling reagents and reaction conditions led to the accumulation of an amine byproduct (which resulted from a Staudinger reduction) or phosphonamide byproduct (which resulted from an aza-Wittig reaction). The Staudinger ligation mediated by (diphenylphosphino)methanethiol proceeded with a second-order rate constant (7.7 x 10(-3) M(-1) s(-1)) and yield (95%) that was unchanged by the addition of exogenous nucleophiles. Ligations mediated by phosphinoalcohols had lower rate constants or less chemoselectivity. Accordingly, (diphenylphosphino)methanethiol was judged to be the most efficacious known reagent for effecting the traceless Staudinger ligation.

Azides↗
PubMed2004

Observation of internal cleavage and ligation reactions of a ribozyme.

We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.

Binding Sites↗
PubMed1997

Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28.

The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.

B7-1 Antigen↗