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1.
Protein engineering of Bacillus thuringiensis
δ-endotoxin: Mutations at domain II of CryIAb enhance receptor
affinity and toxicity toward gypsy moth larvae
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Francis Rajamohan Oscar Alzate JeffreyA. Cotrill April Curtiss DonaldH. Dean 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(25):14338-14343
Substitutions or deletions of domain II loop residues of Bacillus thuringiensis δ-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) ≈4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering δ-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions. 相似文献
2.
Dorothy J. Fiete Mary C. Beranek Jacques U. Baenziger 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(5):2089-2093
A critical element of lutropin bioactivity in vivo is its rapid removal from the blood by a receptor, located in hepatic endothelial cells, that recognizes the terminal sulfated carbohydrate structure SO4-4-GalNAcβ1,4GlcNAcβ1,2Manα (S4GGnM). We have previously shown that the macrophage mannose (Man)-receptor cDNA directs the synthesis of a protein that binds oligosaccharides with either terminal S4GGnM or terminal Man, at independent sites. We now show that the cysteine-rich (Cys-Rich) domain at the N terminus of the Man/S4GGnM receptor accounts for binding of oligosaccharides with terminal GalNAc-4-SO4, whereas calcium-dependent carbohydrate recognition domains (CRDs) account for binding of ligands containing terminal Man. The Cys-Rich domain is thus a previously unrecognized carbohydrate binding motif. Cys-Rich domains have been described on the three other members of the endocytic C-type lectin family of receptors. The structural relationship of these receptors to the Man/S4GGnM receptor raises the possibility that their Cys-Rich domains also bind carbohydrate moieties and contribute to their function. 相似文献
3.
A synthetic cryIC gene, encoding a Bacillus
thuringiensis δ-endotoxin, confers Spodoptera
resistance in alfalfa and tobacco
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Nicolai Strizhov Menachem Keller Jaideep Mathur Zsuzsanna Koncz-Klmn Dirk Bosch Evgenia Prudovsky Jeff Schell Baruch Sneh Csaba Koncz Aviah Zilberstein 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(26):15012-15017
Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation–PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01–0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA. 相似文献
4.
5.
Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor
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Chris Dealwis Elias J. Fernandez Darren A. Thompson Reyna J. Simon Michael A. Siani Elias Lolis 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(12):6941-6946
Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response. 相似文献
6.
J R Gimbel 《Heart (British Cardiac Society)》2003,89(6):e18
A patient with palpitations and suspected arrhythmia underwent Holter and external loop recorder monitoring. No arrhythmias were detected by these traditional monitoring methods. An insertable loop recorder (ILR) was placed on the patient’s chest and used as an extended loop recorder. An arrhythmia was ultimately recorded by the externally placed ILR leading to appropriate treatment. 相似文献
7.
An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus
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Xiao-Ping Zhong Michael S. Krangel 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(10):5219-5224
T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development. 相似文献
8.
Protein Hal: Partial Deletion of a “γ” Immunoglobulin Gene(s) and Apparent Reinitiation at an Internal AUG Codon
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Blas Frangione Loretta Lee Edgar Haber Kurt J. Bloch 《Proceedings of the National Academy of Sciences of the United States of America》1973,70(4):1073-1077
Protein Hal is a human gamma4 heavy-chain disease protein whose molecular weight is reduced from 55,000 to 25,000 in 6 M guanidine due to the lack of disulfide bonds between heavy chains. Studies of aminoacid sequence indicate that it contains a gap of about 240 residues, starting 10 residues from the N-terminal end and including the rest of the Fd fragment, as well as the hinge region. Normal sequence apparently resumes at a methionine residue (position 252) in the second constant domain (C(H)2) and seems normal from there to the carboxyl end of the molecule. These results imply that reinitiation of translation at an internal AUG codon occurs in protein Hal. 相似文献
9.
10.
Clemens L. Schpf Cornelia Ablinger Stefanie M. Geisler Ruslan I. Stanika Marta Campiglio Walter A. Kaufmann Benedikt Nimmervoll Bettina Schlick Johannes Brockhaus Markus Missler Ryuichi Shigemoto Gerald J. Obermair 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(14)
In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.In synapses neurotransmitter release is triggered by the entry of calcium through voltage-gated calcium channels (VGCCs). Neuronal VGCCs consist of an ion-conducting α1 subunit and the auxiliary β and α2δ subunits. α2δ subunits, the targets of the widely prescribed antiepileptic and antiallodynic drugs gabapentin and pregabalin, are membrane-anchored extracellular glycoproteins, which modulate VGCC trafficking and calcium currents (1–5). In nerve cells α2δ subunits have been linked to neuropathic pain and epilepsy (4) and they interact with mutant prion proteins (6) and regulate synaptic release probability (7). Importantly, all α2δ isoforms are implicated in synaptic functions. Presynaptic effects of α2δ-1, for example, may be mediated by an interaction with α-neurexins (8) or N-methyl-D-aspartate receptors (e.g., refs. 9 and 10). In contrast, postsynaptic α2δ-1 acts as a receptor for thrombospondins (11) and promotes spinogenesis via postsynaptic Rac1 (12). α2δ-2 is necessary for normal structure and function of auditory hair cell synapses (13); it has been identified as a regulator of axon growth and hence a suppressor of axonal regeneration (14) and was recently shown to control structure and function of cerebellar climbing fiber synapses (15). A splice variant of α2δ-2 regulates postsynaptic GABAA receptor (GABAAR) abundance and axonal wiring (16). In invertebrates, α2δ loss of function was associated with abnormal presynaptic development in motoneurons (17, 18) and in mice the loss of α2δ-3 results in aberrant synapse formation of auditory nerve fibers (19). Finally, α2δ-4 is required for the organization of rod and cone photoreceptor synapses (20, 21).Despite these important functions, knockout mice for α2δ-1 and α2δ-3 show only mild neurological phenotypes (5, 10, 22–25). In contrast, mutant mice for α2δ-2 (ducky) display impaired gait, ataxia, and epileptic seizures (26), all phenotypes consistent with a cerebellar dysfunction due to the predominant expression of α2δ-2 in the cerebellum (e.g., ref. 15). Hence, in contrast to the specific functions of α2δ isoforms (discussed above) the phenotypes of the available knockout or mutant mouse models suggest a partial functional redundancy in central neurons. Moreover, detailed mechanistic insights into the putative synaptic functions of α2δ subunits are complicated by the simultaneous and strong expression of three isoforms (α2δ-1 to -3) in neurons of the central nervous system (27).In this study, by transfecting cultured hippocampal neurons from α2δ-2/-3 double-knockout mice with short hairpin RNA (shRNA) against α2δ-1, we developed a cellular α2δ subunit triple-knockout/knockdown model. Excitatory synapses from these cultures show a severe failure of synaptic vesicle recycling associated with severely reduced presynaptic calcium transients, loss of presynaptic calcium channels and presynaptic vesicle-associated proteins, and a reduced size of the presynaptic active zone (AZ). Lack of presynaptic α2δ subunits also induces a failure of postsynaptic PSD-95 and AMPA receptor (AMPAR) localization and a thinning of the postsynaptic density (PSD). Each individual α2δ isoform (α2δ-1 to -3) could rescue the severe phenotype, revealing the highly redundant role of presynaptic α2δ isoforms in glutamatergic synapse formation and differentiation. Together our results show that α2δ subunits regulate presynaptic differentiation as well as the transsynaptic alignment of postsynaptic receptors and are thus critical for the function of glutamatergic synapses. 相似文献
11.
Michael D. Stern 《Proceedings of the National Academy of Sciences of the United States of America》1999,96(19):10746-10751
Homeostasis, the creation of a stabilized internal milieu, is ubiquitous in biological evolution, despite the entropic cost of excluding noise information from a region. The advantages of stability seem self evident, but the alternatives are not so clear. This issue was studied by means of numerical experiments on a simple evolution model: a population of Boolean network "organisms" selected for performance of a curve-fitting task while subjected to noise. During evolution, noise sensitivity increased with fitness. Noise exclusion evolved spontaneously, but only if the noise was sufficiently unpredictable. Noise that was limited to one or a few stereotyped patterns caused symmetry breaking that prevented noise exclusion. Instead, the organisms incorporated the noise into their function at little cost in ultimate fitness and became totally noise dependent. This "noise imprinting" suggests caution when interpreting apparent adaptations seen in nature. If the noise was totally random from generation to generation, noise exclusion evolved reliably and irreversibly, but if the noise was correlated over several generations, maladaptive selection of noise-dependent traits could reverse noise exclusion, with catastrophic effect on population fitness. Noise entering the selection process rather than the organism had a different effect: adaptive evolution was totally abolished above a critical noise amplitude, in a manner resembling a thermodynamic phase transition. Evolutionary adaptation to noise involves the creation of a subsystem screened from noise information but increasingly vulnerable to its effects. Similar considerations may apply to information channeling in human cultural evolution. 相似文献
12.
“What” and “how”: Evidence for the dissociation of object knowledge and mechanical problem-solving skills in the human brain
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John R. Hodges Josef Spatt Karalyn Patterson 《Proceedings of the National Academy of Sciences of the United States of America》1999,96(16):9444-9448
Patients with profound semantic deterioration resulting from temporal lobe atrophy have been reported to use many real objects appropriately. Does this preserved ability reflect (i) a separate component of the conceptual knowledge system ("action semantics") or (ii) the operation of a system that is independent of conceptual knowledge of specific objects, and rather is responsible for general mechanical problem-solving skills, triggered by object affordances? We contrast the performance of three patients-two with semantic dementia and focal temporal lobe atrophy and the third with corticobasal degeneration and biparietal atrophy-on tests of real object identification and usage, picture-based tests of functional semantic knowledge, and a task requiring selection and use of novel tools. The patient with corticobasal degeneration showed poor novel tool selection and impaired use of real objects, despite near normal semantic knowledge of the same objects' functions. The patients with semantic dementia had the expected deficit in object identification and functional semantics, but achieved flawless and effortless performance on the novel tool task. Their attempts to use this same mechanical problem-solving ability to deduce (sometimes successfully but often incorrectly) the use of the real objects provide no support for the hypothesis of a separate action-semantic system. Although the temporal lobe system clearly is necessary to identify "what" an object is, we suggest that sensory inputs to a parietal "how" system can trigger the use of objects without reference to object-specific conceptual knowledge. 相似文献
13.
Andrey Rzhetsky Francisco Jos Ayala Lily C. Hsu Cheng Chang Akira Yoshida 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(13):6820-6825
Whether or not nuclear introns predate the divergence of bacteria and eukaryotes is the central argument between the proponents of the “introns-early” and “introns-late” theories. In this study we compared the goodness-of-fit of each theory with a probabilistic model of exon/intron evolution and multiple nonallelic genes encoding human aldehyde dehydrogenases (ALDHs). Using a reconstructed phylogenetic tree of ALDH genes, we computed the likelihood of obtaining the present-day ALDH sequences under the assumptions of each competing theory. Although on the grounds of its own assumptions each theory accounted for the ALDH data significantly better than its rival, the introns-early model required frequent intron slippage, and the estimated slippage rates were too high to be consistent with reported correlations between the boundaries of ancient protein modules and the ends of ancient exons. Because the molecular mechanisms proposed to explain intron slippage are incapable of providing such high rates and are incompatible with the observed distribution of introns in higher eukaryotes, the ALDH data support the introns-late theory. 相似文献
14.
Dan Liu Yunyun Mao Xue Gu Yang Zhou Dong Long 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(11)
The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.Situated in a central position of the complex intracellular signaling network, Ras proteins play critical roles in regulating cell growth, differentiation, migration and apoptosis through cycling between the guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms (1, 2). Aberrant signaling caused by oncogenic mutations in Ras that break this physiological balance can result in uncontrolled cell proliferation and ultimately the development of human malignancies (3, 4). Despite its well-established role in tumorigenesis and the extensive efforts to target this oncoprotein in past decades, clinically approved therapies remain unavailable. One obstacle to the development of anti-Ras drugs lies in the native structures of active and inactive Ras that lack apparently druggable pockets for high-affinity interactions with inhibitory compounds (5–7).Both the active and inactive forms of Ras, however, are inherently flexible, populating rare conformers distinct from the native structures and presenting alternative opportunities for drug discovery (8–11). For example, in GTP-bound active Ras, a major and minor state (termed states 2 and 1, respectively) coexist in solution and exchange on a millisecond timescale, with state 1 showing surface roughness unobserved in the major state (12–18). The direct visibility of state 1 in the one-dimensional 31P NMR spectra of active Ras largely facilitated its early discovery and characterization (12, 19). And the available mutants of H-Ras (e.g., T35A), or the homolog M-Ras, which predominantly assume the state 1 conformation, further promoted the atomic-resolution studies of its structure and internal dynamics, as well as the concomitant drug discovery efforts targeting this low-populated conformer (17, 18, 20).In comparison to the intensive studies on active Ras, research on the dynamics of GDP-bound inactive Ras has lagged far behind, presumably due to its high degree of spectral homogeneity with little sign of resonance splitting or exchange broadening at room temperature (21). The previously reported cryptic pockets for covalent and noncovalent inhibitors of Ras•GDP (22–24), which are unseen in the compound-free structure, nevertheless indicate that the inactive form is also structurally plastic. The recent relaxation-based NMR experiments carried out at low temperature successfully captured the intrinsic microsecond timescale motions in Ras•GDP, which map to regions that overlap with those rearranged on the binding of inhibitors (11). However, the structural information of the transiently formed excited state, in the form of chemical shifts, is not available from the relaxation measurements, owing to the fast exchange rate on the chemical shift timescale. Moreover, unlike the case of active Ras, there are no known mutations that can stabilize the excited state of Ras•GDP for investigations using conventional biophysical techniques. Thus far, the sparsely populated conformations of inactive Ras derived from its microsecond dynamics remain poorly understood, precluding structure-based rational drug discovery.To address these challenges, in this work we constructed a solution ensemble of Ras•GDP by integrating atomistic computer simulation with diverse NMR experimental parameters containing complementary information about the intrinsic protein motions on timescales from picoseconds to microseconds. This NMR-based ensemble well covers the slow dynamics as probed by spin relaxation and provides an atomic-resolution delineation of thermally accessible conformations, including those bearing surface or buried pockets similar to the cryptic pockets previously observed in the inhibitor-bound forms. The utility of the Ras•GDP ensemble in the development of inhibitors is demonstrated by ensemble-based virtual screening, which achieves an impressive level of enrichment of known binders. 相似文献
15.
Arlin Stoltzfus John M. Logsdon Jr. Jeffrey D. Palmer W. Ford Doolittle 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(20):10739-10744
Alignments of homologous genes typically reveal a great diversity of intron locations, far more than could fit comfortably in a single gene. Thus, a minority of these intron positions could be inherited from a single ancestral gene, but the larger share must be attributed to subsequent events of intron gain or intron “sliding” (movement from one position to another within a gene). Intron sliding has been argued from cases of discordant introns and from putative spatial clustering of intron positions. A list of 32 cases of discordant introns is presented here. Most of these cases are found to be artefactual. The spatial and phylogenetic distributions of intron positions from five published compilations of gene data, comprising 205 intron positions, have been examined systematically for evidence of intron sliding. The results suggest that sliding, if it occurs at all, has contributed little to the diversity of intron positions. 相似文献
16.
Mihaela Pertea Petru Ciobanu Natalia Velenciuc Vladimir Poroch Alexandru Filip Dan Cristian Moraru Sorinel Lunca Bogdan Veliceasa 《Medicine》2021,100(38)
Injuries that result in thumb amputation cause a loss of 50% of hand function. Microsurgical replantation remains the gold standard of thumb reconstruction techniques. The non-microsurgical technical variants of thumb reconstruction described so far aim to create a neo-thumb of adequate length, stable, opposable, sensitive, and last but not least esthetically pleasing appearance. Avulsion of the distal phalanx and the absence of the nail will determine a functional deficit but also an unesthetic appearance. When replantation is not possible or the patient refuses to “sacrifice” another anatomical region for thumb reconstruction, the “reposition-flap” technique can be used. Although often controversial, this surgical technique deserves proper attention and should be used in some cases. We studied a group of 32 patients with distal thumb amputations. In patients with amputations in zone II according to Tamai, with interphalangeal joint preservation, the thumb was reconstructed using “reposition-flap” with an O’Brien flap in 15 cases. In the remaining 17 cases where the amputation was at the level of the interphalangeal joint, we used the same technique, but the thumb neopulp was reconstructed with the Littler heterodigital neurovascular flap harvested from the ulnar border of the middle finger in 11 cases or radial border of the ring finger in 6 cases. The results were evaluated from a functional (Kapandji score), sensitive (2-point discrimination, Semmes-Weinstein test) but also esthetically (patient satisfaction) point of view. Donor site morbidity, cold intolerance, the presence of nail dystrophy, and bone resorbtion were also assessed. The disabilities of the arm, shoulder and hand score was evaluated for each patient. Although various surgical and microsurgical techniques for thumb reconstruction are described, when choosing the technique to use we must first consider patient''s wishes. A well-informed patient will be able to make, with the surgeon, the best decision for him concerning the reconstruction option. 相似文献
17.
Five TGA “stop” codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II 总被引:17,自引:0,他引:17
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Thomas D. Fox 《Proceedings of the National Academy of Sciences of the United States of America》1979,76(12):6534-6538
A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan. 相似文献
18.
Osamu Shimomura Frank H. Johnson Hiroshi Morise 《Proceedings of the National Academy of Sciences of the United States of America》1974,71(12):4666-4669
Fatty aldehydes, present in the luminescent cells of Photobacterium phosphoreum and Achromobacter fischeri, and to a very slight extent in the cells of a visually dark, "aldehydeless" mutant of the latter species, were extracted, purified, and oxidized to the corresponding acids. The acids were analyzed by mass spectrometry. The results, in conjunction with various other lines of evidence, indicate that saturated fatty aldehydes, comprising mostly dodecanal, tetradecanal, and hexadecanal, function in the bioluminescent reaction in living cells of these luminous bacteria. The amount of these aldehydes in the cells was computed to be sufficient to sustain steady-state luminescence for a period of about 1 sec, and under such conditions the rate of oxidation of the aldehydes in the process of luminescence must be balanced by their rate of production. 相似文献
19.
Cytolytic and IFN-γ-producing activities of γδ T cells in the mouse intestinal epithelium are T cell receptor-β-chain dependent
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Masako Kohyama Masanobu Nanno Mariko Kawaguchi-Miyashita Shinichiro Shimada Mamoru Watanabe Toshifumi Hibi Schuichi Kaminogawa Hiromichi Ishikawa 《Proceedings of the National Academy of Sciences of the United States of America》1999,96(13):7451-7455
We analyzed the cytolytic activity of intraepithelial T cells (IEL) isolated from the small intestines of 2- to 3-month-old mutant mice rendered deficient in different gene(s) in which the number of IEL expressing either T cell receptor (TCR)-alpha beta (alpha beta-IEL) or TCR-gamma delta (gamma delta-IEL) were absent or markedly diminished. When compared with wild-type littermates, cytolytic activity of gamma delta-IEL was sharply attenuated in TCR-beta mutant mice but remained unaltered in TCR-alpha mutant mice in which a minor population of dull TCR-beta+ (betadim)-IEL was also present. Cytolytic activity of gamma delta-IEL was maintained in mice doubly homozygous for beta2-microglobulin and transporter associated with antigen processing 1 gene mutations in which a conspicuous decrease was noted in absolute numbers of alpha beta-IEL. In contrast, both TCR-delta and IL-7 receptor-alpha gene mutations that lead to lack of gamma delta-IEL generation did not affect the development or cytolytic activity of the remaining alpha beta-IEL. The anti-CD3 and anti-TCR-gamma delta mAb-induced IFN-gamma production of gamma delta-IEL showed the same TCR-alpha and TCR-beta mutation-dependent variability. These results indicate that cytolytic and IFN-gamma-producing activities of gamma delta T cells in mouse intestinal epithelium are TCR-beta-chain-dependent. 相似文献
20.
Folding of the β-propeller domain of the integrin αL subunit is independent of the I domain and dependent on the β2 subunit
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Chichi Huang Timothy A. Springer 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(7):3162-3167
We have studied the folding during biosynthesis of the lymphocyte function-associated antigen 1 (LFA-1) αL subunit using mAb to epitopes that map to seven different regions within the amino acid sequence. The N-terminal portion of αL is predicted to contain a β-propeller domain, consisting of seven β-sheets, and an I domain that is predicted to be inserted between β-sheet 2 and β-sheet 3 of the β-propeller. The I domain of αL folds before association with the β2 subunit, as shown by immunoprecipitation of the unassociated αL subunit by mAbs specific for four different sequence elements within the I domain. By contrast, the β-propeller domain is not folded in unassociated αL after a chase of as long as 12 h after synthesis, but does fold upon association with β2. This is shown with mAbs to regions of αL, that precede and follow the I domain in the primary structure. A mAb that maps near the junction of the C terminus of the I domain with the β-propeller domain suggests that this region is partially folded before subunit association. The results show that the I domain and β-propeller domains fold independently of one another, and suggest that the β-propeller domain bears an interface for association with the β subunit. 相似文献