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Query: UNIPROT:P06889 (Mol)
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The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.
Mol Microbiol 1990 Oct
PMID:HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide. 170 54

We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.
J Mol Biol 1991 Apr 20
PMID:Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin. 202 47

In the present study we have analyzed the genetic regulation of increased expression of transformation-associated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (E11-NMTneo) and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFcap). Parental E11-NMTneo cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFcap cells exhibited phenotypes opposite to those of E11-NMTneo cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFcap and E11-NMTneo cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which still exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with E11-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 E1A and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in Ad5-transformed cells and that this suppression is not associated with changes in the expression of Ad5 transforming genes.
Mol Cell Biol 1990 May
PMID:Suppression of the progression phenotype in somatic cell hybrids occurs in the absence of altered adenovirus type 5 gene expression. 213 70

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.
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PMID:The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin. 277 99

Assignments are reported for a substantial number of heme and amino acid proton resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of isolated hemoglobin alpha-chains. These resonances provide information on the solution conformation of the protein, particularly in the vicinity of the heme. The heme pocket structure is generally similar to that of carbonmonoxymyoglobin; several conserved residues adopt virtually identical positions relative to the heme in the two proteins. The largest conformational differences involve residues surrounding the ligand-binding site, notably Val62 (E11) and His58 (E7). The chemical shifts of the proximal His87 (F8) resonances are very similar in spectra of the two proteins, indicating a highly conserved coordination geometry and similar hydrogen bonding to the backbone carbonyl of Leu83 (F4).
J Mol Biol 1987 Mar 20
PMID:Assignment of resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of human hemoglobin alpha-chains. 303 52

We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.
Cell Mol Biol (Noisy-le-grand) 1995 Mar
PMID:Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear. 778 31

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.
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PMID:1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule. 796 51

The crystal structure of the aquo-met form of the sulfide-reactive hemoglobin (component I) from the gill of the symbiont-harboring mollusc, Lucina pectinata, has been solved and refined at 1.5 A resolution, based on synchrotron radiation X-ray diffraction data, and employing molecular replacement techniques. The crystallographic R-factor, calculated for the data in the 15.0 to 1.5 A resolution range, is 0.170, with highly regular stereochemical parameters for the protein model, and including 131 water molecules. The monomeric hemoglobin I chain consists of 142 amino acid residues, which have been partly identified on the basis of the crystallographic analysis. The molecule is characterized by an unusual distribution of aromatic residues, particularly in the region surrounding the distal site in the heme pocket. The heme distal residue is Gln(64)E7, while other notable amino acid substitutions include Trp(21)B2, Phe(29)B10, Leu(46)CD3, Phe(68)E11 and Trp(75)E18. An amino acid insertion (Ser44) is observed between sites CD1 and CD2. In the aquo-met protein, a water molecule is present at the sixth coordination position of the heme iron, and hydrogen bonded to Gln(64)E7. Simple model building shows that a dioxygen molecule, bound to ferrous protein, would contact with its free atom the ring edge of Phe(29)B10, being thus stabilized at the coordination site by an aromatic-electrostatic interaction. Similarly, the unique packing and organization of aromatic residues in the surroundings of the heme distal site is proposed as the molecular basis of the very high affinity of Lucina pectinata hemoglobin I for hydrogen sulfide, considered as one of the two physiological ligands of the protein.
J Mol Biol 1994 Nov 18
PMID:Structure of the sulfide-reactive hemoglobin from the clam Lucina pectinata. Crystallographic analysis at 1.5 A resolution. 796 24

As an initial step toward understanding its role in steroidogenesis, we studied the developmental profile of steroidogenic factor-1 (SF-1), a nuclear receptor that regulates the steroid hydroxylases. SF-1 transcripts first appear on embryonic day 9 (E9) in the urogenital ridge, the probable source of steroidogenic cells of both adrenals and gonads. By E11, after the adrenals and gonads are clearly separate, SF-1 transcripts are detected throughout the adrenal primordium. Thereafter, adrenal expression of SF-1 localizes to the cortex. Consistent with its proposed role in regulating cholesterol side-chain cleavage enzyme (SCC), SF-1 is expressed before SCC. During the sexually undifferentiated stage of gonadal development (E9-E12), all embryos express SF-1 in the genital ridge. As testicular cords form in males, SF-1 transcripts are diffusely expressed throughout the testis, whereas SCC mRNA is limited to the interstitium. These differences between SF-1 and SCC reflect SF-1 expression by Sertoli cells, as shown by Northern blotting and in situ hybridization. In contrast to its persistent expression in the embryonic testis, SF-1 transcripts disappear from the ovary between E13.5-E16.5, reappearing only during late gestation (E18.5). Thus, expression of SF-1 in the embryonic gonad is sexually dimorphic. Coupled with the demonstration of SF-1 mRNA in Sertoli cells, these data suggest that SF-1 plays a role in gonadal development distinct from regulating the steroidogenic enzymes. Additionally, SF-1 is expressed in the embryonic forebrain, implying a role in neural development.
Mol Endocrinol 1994 May
PMID:Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. 805 73

The structure of carbonmonoxide Glycera hemoglobin has been determined to 1.5 A resolution by X-ray diffraction. The model, including ordered solvent, has been refined by the method of restrained least-squares to an R-value of 0.146. The positions of 1104 protein atoms and the oxygens of 155 water molecules have been determined with an estimated r.m.s. error of 0.10 to 0.13 A. The r.m.s. errors in protein geometry are 0.027 A for bond distances, 0.038 A for angle distances and 0.012 A for deviations of planar groups from their least-squares planes. The iron lies exactly in the plane of the heme nitrogens and the heme is very slightly domed toward the proximal side. The carbon-oxygen bond in the carbon monoxide ligand is bent 7.9 degrees away from the normal to the plane of the heme nitrogens. The ligand is in close contact with, and slightly removed from the heme normal by distal residues Leu 58(E7) and Val62(E11). Comparison of the CO structure with the 1.5 A deoxy structure shows that the majority of the rather small structural changes occurring upon ligation are mediated by movement of the heme due to shortening of the five iron to nitrogen bonds. There is very little empty space inside the molecule, and no direct channel from the solvent into the heme pocket; however, rotation of the side-chain of the distal leucine residue Leu 58(E6) could provide a ligand pathway.
J Mol Biol 1994 Apr 22
PMID:Glycera dibranchiata hemoglobin. X-ray structure of carbonmonoxide hemoglobin at 1.5 A resolution. 814 55


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