A new mode for heme-heme interactions in hemoglobin associated with distal perturbations. (57/6391)

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.  (+info)

Different molecular consequences of the 1;19 chromosomal translocation in childhood B-cell precursor acute lymphoblastic leukemia. (58/6391)

The prognostically important 1;19 chromosomal translocation can alter the E2A gene on chromosome 19p13 in childhood B-cell precursor acute lymphoblastic leukemia (ALL), leading to formation of a fusion gene (E2A-PBX1) that encodes a hybrid transcription factor with oncogenic potential. It is not known whether this molecular alteration is a uniform consequence of the t(1;19) or is restricted to translocation events within specific immunologic subtypes of the disease. Therefore, we studied leukemic cells from 25 cases of B-cell precursor ALL, with or without evidence of cytoplasmic Ig mu heavy chains (cIg); 17 cases had the t(1;19) by cytogenetic analysis. Leukemic cell DNA samples were analyzed by Southern blotting to detect alterations within the E2A genomic locus; a polymerase chain reaction assay was used to identify expression of chimeric E2A-pbx1 transcripts in leukemic cell RNA; and immunoblotting with anti-Pbx1 antibodies was used to detect hybrid E2A-Pbx1 proteins. Of 11 cases of cIg+ ALL with the t(1;19), 10 had E2A-pbx1 chimeric transcripts with identical junctions and a characteristic set of E2A-Pbx1 hybrid proteins. Each of these cases had E2A gene rearrangements, including the one in which fusion transcripts were not detected. By contrast, none of the six cases of t(1;19)-positive, cIg- ALL had evidence of rearranged E2A genomic restriction fragments, detectable E2A-pbx1 chimeric transcripts, or hybrid E2A-Pbx1 proteins. Typical chimeric E2A-pbx1 transcripts and proteins were detected in one of eight cIg+ leukemias in which the t(1;19) was not identified by cytogenetic analysis, emphasizing the increased sensitivity of molecular analysis for detection of this abnormality. We conclude that the molecular breakpoints in cases of cIg- B-cell precursor ALL with the t(1;19) differ from those in cIg+ cases with this translocation. Leukemias that express hybrid oncoproteins such as E2A-Pbx1 or Bcr-Abl have had a poor prognosis in most studies. Thus, molecular techniques to detect fusion genes and their aberrant products should allow more timely and appropriate treatment of these aggressive subtypes of the disease.  (+info)

Functional dissection of AP-1 transcription factors using the Gal4 adaptor assay. (59/6391)

In the Gal4 adaptor assay, leucine zipper-containing proteins are tethered indirectly to the promoters of Gal4-responsive reporter genes via synthetic protein chimeras consisting of a Gal4 DNA-binding domain with an attached leucine zipper. Ternary complexes composed of the DNA binding site, the adaptor protein, and a leucine zipper factor can stimulate reporter gene activity, provided that the latter component possesses a transcriptional activation domain. This system is used to assay the transcriptional function and the interactions between various AP-1 factors.  (+info)

How M.MspI and M.HpaII decide which base to methylate. (60/6391)

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.  (+info)

Subunit folding and alpha delta heterodimer formation in the assembly of the nicotinic acetylcholine receptor. Comparison of the mouse and human alpha subunits. (61/6391)

We have used the mouse alpha (alpha M) and human alpha (alpha H) subunits to investigate the molecular mechanisms of assembly of the mammalian acetylcholine receptor (AChR) transiently expressed in COS cells. COS cells expressing hybrid receptors incorporating alpha H along with other mouse subunits exhibited a 2-fold higher level of surface alpha-bungarotoxin (BuTx) binding than cells expressing the wild-type mouse AChR. When expressed either alone or with the delta subunit in COS cells, alpha H acquired the BuTx binding conformation (alpha Tx) more efficiently than did alpha M. By oligonucleotide-directed mutagenesis we showed that 2 residues in the amino-terminal domain were responsible for the differences between alpha M and alpha H. Alpha MST, the modified mouse alpha subunit, both folded more efficiently to form alpha Tx and was more effective in forming a stable alpha delta heterodimer than was alpha M. The kinetics of alpha Tx and alpha delta heterodimer formation revealed that the delta subunit increased the conversion of immature forms of the alpha subunit into the BuTx binding form and therefore provides evidence for interaction between the delta subunit and the immature form of the alpha subunit. These results provide evidence of the importance of the amino-terminal domains of the AChR subunits in the assembly process.  (+info)

Coagulant properties of hybrid human/porcine factor VIII molecules. (62/6391)

Human and porcine factor VIII (fVIII) are activated by thrombin to form a heterotrimer composed of subunits designated A1 and A2 derived from the fVIII heavy chain (HC) and a subunit designated A3-C1-C2 derived from the fVIII light chain (LC). Human and porcine fVIII were activated at the same rate to the same peak levels but dissociation of the A2 subunit and concomitant loss of fVIIIa activity at pH 7.4 and 22 degrees C was 3-fold faster with human fVIIIa compared to porcine fVIIIa (0.35 min-1 versus 0.12 min-1, respectively). To determine structural requirements for the increased activity of porcine fVIII, plasma-derived hybrid human/porcine fVIII molecules were isolated. Porcine HC/human LC (pHC/hLC) fVIII had 44-fold higher coagulant activity than reconstituted human fVIII (hHC/hLC), 40-fold higher activity than hHC/pLC, and slightly (1.4-fold) higher activity than reconstituted porcine fVIII (pHC/pLC). Additionally, human and porcine A2 subunits and inactive A1/A3-C1-C2 human and porcine dimers were isolated and reconstitution experiments were done. Addition of the porcine A2 subunit to the human A1/A3-C1-C2 dimer produced coagulant activity similar to that found with porcine fVIIIa and superior to human fVIIIa. These results suggest that human fVIII has weaker coagulant activity than porcine fVIII due to faster dissociation of the A2 subunit and that the A2 subunit itself is responsible for the difference.  (+info)

Protein LG: a hybrid molecule with unique immunoglobulin binding properties. (63/6391)

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.  (+info)

Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits. (64/6391)

Background potassium currents carried by the KCNK family of two-pore-domain K+ channels are important determinants of resting membrane potential and cellular excitability. TWIK-related acid-sensitive K+ 1 (TASK-1, KCNK3) and TASK-3 (KCNK9) are pH-sensitive subunits of the KCNK family that are closely related and coexpressed in many brain regions. There is accumulating evidence that these two subunits can form heterodimeric channels, but this evidence remains controversial. In addition, a substantial contribution of heterodimeric TASK channels to native currents has not been unequivocally established. In a heterologous expression system, we verified formation of heterodimeric TASK channels and characterized their properties; TASK-1 and TASK-3 were coimmunoprecipitated from membranes of mammalian cells transfected with the channel subunits, and a dominant negative TASK-1(Y191F) construct strongly diminished TASK-3 currents. Tandem-linked heterodimeric TASK channel constructs displayed a pH sensitivity (pK approximately 7.3) in the physiological range closer to that of TASK-1 (pK approximately 7.5) than TASK-3 (pK approximately 6.8). On the other hand, heteromeric TASK channels were like TASK-3 insofar as they were activated by high concentrations of isoflurane (0.8 mm), whereas TASK-1 channels were inhibited. The pH and isoflurane sensitivities of native TASK-like currents in hypoglossal motoneurons, which strongly express TASK-1 and TASK-3 mRNA, were best represented by TASK heterodimeric channels. Moreover, after blocking homomeric TASK-3 channels with ruthenium red, we found a major component of motoneuronal isoflurane-sensitive TASK-like current that could be attributed to heteromeric TASK channels. Together, these data indicate that TASK-1 and TASK-3 subunits coassociate in functional channels, and heteromeric TASK channels provide a substantial component of background K(+) current in motoneurons with distinct modulatory properties.  (+info)