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At least 829 records · Page 46Linked to original sources
PubMed2007

Salmeterol enhances pulmonary fibrinolysis in healthy volunteers.

OBJECTIVE: Various lung diseases are associated with local activation of coagulation and concurrent inhibition of fibrinolysis. Although salmeterol, a beta2-adrenoceptor agonist with profound bronchodilatory properties, has been studied extensively, the effects of this compound on the pulmonary hemostatic balance are not elucidated. DESIGN: A single-blinded, placebo-controlled study. SETTING: University hospital and laboratory. SUBJECTS: A total of 32 human volunteers. INTERVENTIONS: Subjects inhaled 100 microg of salmeterol or placebo (t = -30 mins) followed by 100 microg of lipopolysaccharide (LPS) or normal saline (t = 0 mins; n = 8 per group). MEASUREMENTS AND MAIN RESULTS: Measurements were performed in bronchoalveolar lavage fluid obtained 6 hrs postchallenge. Inhalation of LPS enhanced pulmonary coagulation as determined by an increase in the concentrations of thrombin-antithrombin complexes, factor VIIa, and soluble tissue factor in bronchoalveolar lavage fluid (all p < .05 vs. saline). LPS concurrently inhibited pulmonary fibrinolysis, as reflected by a decrease in bronchoalveolar lavage fluid plasminogen activator activity together with an increase in plasminogen activator inhibitor type 1 (both p < .05 vs. saline). Moreover, LPS inhalation was associated with a suppression of the anticoagulant protein C pathway, as indicated by an increase in soluble thrombomodulin and decreases in protein C and activated protein C levels in bronchoalveolar lavage fluid (all p < .05 vs. saline). Salmeterol, either with or without LPS inhalation, enhanced fibrinolysis (plasminogen activator activity and tissue-type and urokinase-type plasminogen activator levels) but did not influence LPS-induced changes in coagulation or the protein C pathway. CONCLUSIONS: Salmeterol has profibrinolytic properties in the normal lung and when applied in a model of sterile pulmonary inflammation.

Administration, Inhalation↗
PubMed2006

Assessment of risk and special observations in mental health practice: a comparison of forensic and non-forensic settings.

The use of special observations in psychiatric practice may be employed as an alternative to more restrictive methods such as the use of seclusion and restraint. From the literature, special observations are used for a complex array of signs and symptoms (and risk behaviours) which include suicidal intent, self-injurious behaviour, hallucinatory experiences, and absconding. This paper reports on research into the use of special observations in both forensic and non-forensic psychiatric settings. A comparative approach was adopted to establish if the perceived risk factors leading to the adoption of special observations were similar in both settings. Three groups of nursing staff were requested to assess 30 patients who were placed on special observations. Before this, nurses were requested to rate the risk factors in terms of their severity on a 7-point Likert scale. The rank-ordering analysis revealed a similarity of identified risk factors and anova (one-way, unrelated) and the Jonckheere Trend Test revealed that there were significant differences between the scores in the forensic and the non-forensic settings. The statistical differences existed for risk factors relating to harm to self and others but not for psychiatric symptomatology.

Analysis of Variance↗
PubMed2005

Direct binding of respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal adherence to human epithelial cells and pneumococcal invasiveness in a murine model.

In a previous study we showed that pneumococcal adherence to epithelial cells was enhanced by a preceding respiratory syncytial virus (RSV) infection. RSV-glycoproteins, expressed on the infected cell surface, may play a role in this enhanced pneumococcal binding, by acting as bacterial receptors. In the current study, it was attempted to analyze the capacity of pneumococci to interact directly with RSV virions. By flow-cytometry, a direct interaction between RSV and pneumococci could be detected. Heparin, an inhibitor of RSV infectivity that interacts with RSV protein-G, blocked RSV-pneumococcal binding, indicating that the latter interaction is indeed mediated by protein-G. RSV-pneumococcal complexes showed enhanced adherence to uninfected human epithelial cells, compared with pneumococcal adherence without bound RSV, and this enhancement was also blocked by heparin. In addition, the significance of these findings in vitro was explored in vivo in a murine model. Both mice that were pretreated with RSV at day 4 before pneumococcal challenge and mice infected with both agents simultaneously showed significantly higher levels of bacteraemia than controls. Simultaneous infection with both agents enhanced the development of pneumococcal bacteraemia most strongly. It was hypothesized that direct viral binding is another mechanism by which RSV can induce enhanced pneumococcal binding to epithelial cells, a phenomenon that is translated in vivo by a higher invasiveness of pneumococci when administered simultaneously with RSV to mice. Apparently, RSV acts in this process as a direct coupling particle between bacteria and uninfected epithelial cells, thereby increasing colonization by and enhancing invasiveness of pneumococci.

Animals↗
PubMed2003

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets.

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T.AT triplets are unstable and melt below 30 degrees C. In contrast, triplets with repeats of TTC, TCC and CTCT melt at 67, 72 and 76 degrees C, respectively. The most stable complex is generated by the sequence containing alternating C+*GC and T*AT triplets. All four triplexes are stabilised by increasing the ionic strength or by the addition of magnesium, although triplexes with a higher proportion of C+*GC triplets are much less sensitive to changes in the ionic conditions. The enthalpies of formation of these triplexes were estimated by examining the concentration dependence of the melting profiles and show that, in the presence of 200 mM sodium at pH 5.0, each C+*GC triplet contributes about 30 kJ x mol(-1), while each T*AT contributes only 11 kJ x mol(-1). Kinetic experiments with these oligonucleotides show that in 200 mM sodium (pH 5.0) repeats of TCC and TTC have half-lives of approximately 20 min, while the triplex with alternating C+*GC and T.AT triplets has a half-life of approximately 3 days. In contrast, the dissociation kinetics of the triplex containing only T*AT are too fast to measure.

Base Sequence↗
PubMed2006

Nanoliter high throughput quantitative PCR.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

Cells, Cultured↗
PubMed2005

Quantitative trait locus (QTL) isogenic recombinant analysis: a method for high-resolution mapping of QTL within a single population.

In the quest for fine mapping quantitative trait loci (QTL) at a subcentimorgan scale, several methods that involve the construction of inbred lines and the generation of large progenies of such inbred lines have been developed (Complex Trait Consortium 2003). Here we present an alternative method that significantly speeds up QTL fine mapping by using one segregating population. As a first step, a rough mapping analysis is performed on a small part of the population. Once the QTL have been mapped to a chromosomal interval by standard procedures, a large population of 1000 plants or more is analyzed with markers flanking the defined QTL to select QTL isogenic recombinants (QIRs). QIRs bear a recombination event in the QTL interval of interest, while other QTL have the same homozygous genotype. Only these QIRs are subsequently phenotyped to fine map the QTL. By focusing at an early stage on the informative individuals in the population only, the efforts in population genotyping and phenotyping are significantly reduced as compared to prior methods. The principles of this approach are demonstrated by fine mapping an erucic acid QTL of rapeseed at a subcentimorgan scale.

Brassica rapa↗
PubMed2003

Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties.

PURPOSE: Epratuzumab is a novel humanized antihuman CD22 IgG1 antibody that has recently shown promising clinical activity, both as a single agent and in combination with rituximab, in patients with non-Hodgkin's lymphomas (NHL). In an attempt to better understand the mode of action of epratuzumab, the antibody was tested in vitro in a variety of cell-based assays similar to those used to evaluate the biological activity of other therapeutic monoclonal antibodies, including rituximab. In this report, we present epratuzumab activities as they relate to binding, signaling, and internalization of the receptor CD22. METHODS: Chinese hamster ovary-expressed CD22 extracellular domain was used to measure epratuzumab affinity on Biacore. CD22 receptor density and internalization rate were measured indirectly using a monovalently labeled, noncompeting (with epratuzumab) anti-CD22 antibody on Burkitt lymphoma cell lines, primary B cells derived from fresh tonsils, and B cells separated from peripheral blood samples obtained from patients with chronic lymphocytic leukemia or healthy volunteers. Epratuzumab-induced CD22 phosphorylation was measured by immunoprecipitation/Western blot and compared with that induced by anti-IgM stimulation. RESULTS: Epratuzumab binds to CD22-extracellular domain, with an affinity of K(D) = 0.7 nM. Binding of epratuzumab to B cell lines, or primary B cells from healthy individuals and patients with NHL, results in rapid internalization of the CD22/antibody complex. Internalization appears to be faster at early time points in cell lines than in primary B cells and NHL patient-derived B cells, but the maximum internalization reached is comparable for all B cell populations after several hours of treatment and appears to reach saturation at antibody concentrations of 1-5 micro g/ml. Finally, epratuzumab binding results in modest but significant CD22 phosphorylation. CONCLUSIONS: Epratuzumab represents an excellent anti-CD22 ligating agent, highly efficacious in inducing CD22 internalization, and can induce phosphorylation. Although we cannot unequivocally demonstrate here that epratuzumab-induced internalization and signaling of CD22 directly contribute to its therapeutic efficacy, these properties are the fundamental characteristics of the target CD22 and its interaction with epratuzumab. Similar results were observed when epratuzumab was tested in vitro on Burkitt B cell lines as well as on primary normal B cells and neoplastic B cells separated from fresh peripheral blood samples from patients with chronic lymphocytic leukemia.

Animals↗
PubMed2003

Enhanced tumor immunogenicity through coupling cytokine expression with antigen presentation.

The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function in an active antitumor immune response. Thus, methods to enhance antigen expression/presentation could augment the effect of cancer immune therapy. In the present study, we investigated the feasibility of modifying a cytokine signal peptide with a tumor antigenic epitope. We inserted the genes encoding the MHC class I-restricted antigenic epitope of chicken ovalbumin and tyrosinase-related protein 2 into the signal sequence of the interleukin-2 gene, replacing part of the signal sequence at different positions. Our results showed that these modified signal peptides still functioned, as indicated by cytokine secretion. The antigenic epitope within the modified signal peptide could be processed properly and presented on tumor cell surface. Tumor cells demonstrated enhanced immunogenicity as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor grow in vivo after gene modification. These data provide potential perspectives in designing therapeutic or vaccine strategies in immuno-gene therapy of cancer.

Amino Acid Sequence↗
PubMed2005

Identification of the larval aggregation pheromone of codling moth, Cydia pomonella.

Mature larvae of the codling moth, Cydia pomonella L. (Lepidoptera: Olethreutidae), exit the fruit and seek sites suitable for pupation. Spinning cocoons in such sites, larvae produce a complex, cocoon-derived blend of volatiles recently shown to attract and/or arrest both conspecific larvae and the prepupal parasitoid Mastrus ridibundus Gravenhorst (Hymenoptera: Ichneumonidae). Here we report components of this blend that constitute the pheromone of fifth-instar C. pomonella larvae. Thirty-one two-choice olfactometer experiments showed that a blend of synthetic (E)-2-octenal, (E)-2-nonenal, sulcatone, and geranylacetone, in combination with either 3-carene and/or three saturated aldehydes (octanal, nonanal, decanal), elicited behavioral responses from C. pomonella larvae. In on-tree experiments with corrugated cardboard bands as pupation sites for larvae affixed to tree trunks, and with laboratory-reared larvae released onto such trees, more larvae cocooned in those halves of cardboard bands baited with cocoon-spinning conspecific larvae, or with synthetic pheromone components, than in unbaited control halves of the bands. With the larval aggregation pheromone identified in this study, there might be an opportunity to manipulate C. pomonella larvae in commercial fruit or nut orchards.

Animals↗
PubMed2001

DNA ligase I and proliferating cell nuclear antigen form a functional complex.

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.

Casein Kinase II↗
PubMed2005

Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs.

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.

Bacteriophage T7↗
PubMed2007

Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B5). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP+ and pantoate bound, solved to 2.3 A of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn98 as well as Lys176 and Glu256 in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser244 for substrate binding and of Arg31 and Lys72 for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.

Alcohol Oxidoreductases↗
PubMed2002

CD19 signaling pathways play a major role for murine AIDS induction and progression.

Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus (BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions between B cells and CD4(+) T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19 integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine leukemia viruses. Infected CR2(-/-) mice developed MAIDS with a time course and severity indistinguishable from that of wild-type mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal induction and progression of MAIDS.

Animals↗
PubMed2003

Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells.

In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.

Animals↗
PubMed2006

Temporary esophageal stenting allows healing of esophageal perforations following atrial fibrillation ablation procedures.

BACKGROUND: Left atrial catheter ablation (LACA) has emerged as a successful method to eliminate atrial fibrillation (AF). Recent reports have described atrio-esophageal fistulas, often resulting in death, from this procedure. Temporary esophageal stenting is an established therapy for malignant esophageal disease. We describe the first case of successful temporary esophageal stenting for an esophageal perforation following LACA. CASE: A 48-year-old man with symptomatic drug refractory lone AF underwent an uneventful LACA. Fifty-nine ablations with an 8-mm tip ablation catheter (30 seconds, 70 Watts, 55 degrees C), as guided by 3-D NavX mapping, were performed in the left atrium to isolate the pulmonary veins as well as a left atrial flutter and roof ablation line. In addition, complex atrial electrograms in AF and sites of vagal innervation were ablated. Two weeks later, he presented with sub-sternal chest pain, fever, and dysphagia. A chest CT showed a 3-mm esophageal perforation at the level of the left atrium with mediastinal soiling and no pericardial effusion. An urgent upper endoscopy with placement of a PolyFlex removable esophageal stent to seal off the esophago-mediastinal fistula was performed. After 3 weeks of i.v. antibiotics, naso-jejunal tube feedings, and esophageal stenting, the perforation resolved and the stent was removed. Over 18 months of follow-up, there have been no other complications, and he has returned to a physically active life and remains free from AF on previously ineffective anti-arrhythmic drugs. CONCLUSION: Early diagnosis of esophageal perforations following LACA may allow temporary esophageal stenting with successful esophageal healing. Prompt chest CT scans with oral and i.v. contrast should be considered in any patient with sub-sternal chest pain or dysphagia following LACA.

Atrial Fibrillation↗
PubMed2000

Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP).

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.

Arabinofuranosylcytosine Triphosphate↗
PubMed2002

Targeted disruption of Dkc1, the gene mutated in X-linked dyskeratosis congenita, causes embryonic lethality in mice.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome associated with increased cancer susceptibility. The X-linked form is due to mutations in the DKC1 gene encoding dyskerin, a nucleolar protein predicted to be involved in rRNA processing and associated with the telomerase complex. Available evidence suggests the pathology of DC is due to telomerase defects. We have used the inducible Cre/loxP system to produce deletions in the murine Dkc1 gene in early embryogenesis. A large deletion lacking exons 12-15 and a small deletion lacking only the last exon, were produced. We found both deletions showed a parent-of-origin effect with 100% embryonic lethality when the mutation occurred on the maternal Dkc1. Embryonic analysis at day E7.5 and E9.5 showed no male embryos carrying either deletion whereas females with maternally derived deletions died around day E9.5, with degeneration of the extra embryonic tissue, in which the paternal X-chromosome is inactivated. Female mice carrying the deletion in the paternally derived Dkc1 show extreme skewing of X-inactivation with the wild type X-chromosome active in all cells. Since mice with no telomerase are viable in the first generations the lethality we observe is unlikely to be due to the effects of mutated dyskerin on telomerase activity.

Animals↗
PubMed2004

Two structural features of lambda integrase that are critical for DNA cleavage by multimers but not by monomers.

Despite many years of genetic and biochemical studies on the lambda integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.

Amino Acid Substitution↗