Enhanced photon generation in a Nb/n-InGaAs/p-InP superconductor/semiconductor-diode light emitting device.

We experimentally demonstrate Cooper pairs' drastic enhancement of the band-to-band radiative recombination rate in a semiconductor. Electron Cooper pairs injected from a superconducting electrode into an active layer by the proximity effect recombine with holes injected from a p-type electrode. The recombination of a Cooper pair with p-type carriers dramatically increases the photon generation probability of a light-emitting diode in the optical-fiber communication band. The measured radiative decay time rapidly decreases with decreasing temperature below the superconducting transition temperature of the niobium electrodes. Our results indicate the possibility to open up new interdisciplinary fields between superconductivity and optoelectronics.

Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain.

A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60 degrees C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5'-->3' exonuclease activity, which was enhanced by increasing the temperature to 50 degrees C. Mg(2+), Mn(2+) or Co(2+) ions were required to activate both proteins, whereas Ca(2+) and Zn(2+) had no effects.

Cloning and overexpression of the oah1 gene encoding O-acetyl-L-homoserine sulfhydrylase of Thermus thermophilus HB8 and characterization of the gene product.

The oah1 gene of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned, sequenced, and overexpressed in Escherichia coli cells. The gene product having a high O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity was purified to homogeneity, with a recovery of approximately 40% and a purification ratio of 81-fold, both calculated from the cell-homogenate. The protein showed molecular masses of approximately 163000 (for the native form) and 47000 (for the subunit). The isoelectric point was pH 6.0. The optimum temperature and pH for the activity were approximately 70 degrees C and pH 7.8, respectively. The enzyme was also shown to be very stable at high temperature (90% activity remaining at 90 degrees C for 60 min at pH 7.8) and in a wide range of pH (pH 4-12 at room temperature). The absorption spectrum showed a peak at 425 nm, and hydroxylamine hydrochloride (0.1 mM) inhibited approximately 90% of the activity, suggesting formation of a Schiff base with pyridoxal 5'-phosphate. The enzyme showed an apparent K(m) value of 6.8 mM for O-acetyl-L-homoserine, a V(max) value of 165 micromol/min per mg of protein at a fixed sulfide concentration of 5 mM, and also an apparent K(m) value of approximately 1.3 mM for sulfide (with 25 mM acetylhomoserine). L-Methionine (1 mM) inhibited the enzyme activity by 67%. Based on these findings, it was discussed that this enzyme might be inactive under ordinary conditions but might become active as an alternative homocysteine synthase in T. thermophilus HB8, only under such conditions as deficiency in transsulfuration, bringing about a sufficient amount of sulfide available in the cell.

Direct observation of three conformations of MutS protein regulated by adenine nucleotides.

Mismatched base-pairs, which are caused by either DNA replication errors, DNA damage or genetic recombination, are repaired by the mismatch-repair system. The MutS protein, a component of the mismatch-repair system, recognizes mismatched base-pairs in DNA, and its DNA-binding activity is affected by ATP and ADP. Here, we show that the MutS protein from Thermus thermophilus HB8 can have three different conformations in solution, based on direct observations made by small-angle X-ray scattering. The conformation of MutS in solution is drastically influenced by the presence of ADP and ATP; the ATP-bound form has the most compact conformation, the ADP-bound form the most stretched, and the nucleotide-free form has a conformation intermediate between the two. Based on these findings, we conclude that the DNA-binding activity of MutS may depend on conformational changes triggered by both the binding and hydrolysis of ATP.

N-terminal 33 amino acid residues of Escherichia coli RecA protein contribute to its self-assembly.

To identify the functional domains in the RecA protein, we prepared the truncated RecA protein lacking its N-terminal 33 amino acid residues by limited tryptic digestion and found that this truncated protein was inefficient at self-assembly. To investigate the function of the N-terminal region further, we constructed the N-terminal truncated recA gene lacking the portion corresponding to the N-terminal 33 residues and prepared a large amount of its gene product. This truncated protein could bind to ATP, but it was defective in self-assembly, binding to single-stranded (ss)DNA and hydrolysis of ATP under normal conditions, although no significant alteration in its stability in comparison with the wild-type protein was observed. In the presence of MgCl2, however, this truncated protein could self-assemble, although a higher protein concentration and longer time than for the wild-type protein were required to complete the process. This truncated protein inhibited the ssDNA-dependent ATPase and ssDNA-binding activities of the wild-type protein. Furthermore, gel filtration chromatography showed that this truncated protein interacted with the wild-type protein and reduced the apparent size of its aggregates. These results suggest that this truncated protein interfered with polymerization of the wild-type protein via a direct protein-protein interaction, which resulted in inhibition of ssDNA-binding and ssDNA-dependent ATP hydrolysis. On the basis of these observations, we concluded that the N-terminal 33 amino acid residues of the RecA protein play an important role not only in protein-protein interaction but also in regulation of the self-assembly process.

Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

Cloning, sequencing and expression of the uvrA gene from an extremely thermophilic bacterium, Thermus thermophilus HB8.

One of the most important DNA repair systems is the nucleotide (nt) excision repair system. The uvr A gene, which plays an essential role in the prokaryotic excision repair system, was cloned from an extremely thermophilic eubacterium, Thermus thermophilus (Tt) HB8, and its nt sequence was determined. In the amino acid (aa) sequence of Tt UvrA, a characteristic duplicated structure, two nt-binding consensus sequences (Walker's A-type motif) and two zinc finger DNA-binding motifs were found. The aa sequence showed 73% homology with that of Escherichia coli (Ec). These features suggest that Tt has the same excision repair system as Ec. Upon comparison of the Tt and Ec UvrA, some characteristic aa substitutions were found. The numbers of Arg and Pro residues were increased (from 66 to 81 and from 47 to 55, respectively), and the numbers of Asn and Met residues were decreased (from 33 to 18 and from 18 to 11, respectively) in Tt. The Tt uvr A gene was expressed in Ec under control of the lac promoter. Purified UvrA was stable up to 80 degrees C (at neutral pH) and at pH 2-11 (at 25 degrees C).

Cloning and characterization of the uvrD gene from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The uvrD gene encodes a DNA helicase which plays an important role in prokaryotic nucleotide (nt) excision repair, mismatch repair and DNA replication. A cosmid-based genomic DNA library for Thermus thermophilus (Tt) HB8 was constructed, and this was screened by Southern hybridization using a uvrD fragment amplified by PCR as the probe. The nt sequence of cloned Tt uvrD was then determined. Characteristic helicase motifs, made up of seven elements, were all conserved in the amino acid (aa) sequence of Tt UvrD. The aa sequence showed 41% homology with that of Escherichia coli (Ec). In the aa composition of Tt UvrD, the number of Asn, Gln, Met and Cys residues was decreased, and the number of Pro residues was increased. The distribution of Pro residues and recent data on X-ray crystallographic structure suggested the importance of the structural dynamics of the protein. These changes are thought to stabilize the native protein conformation against heat denaturation. Tt uvrD complemented the UV sensitivity of a Ec uvrD mutant. Thus, the thermophilic bacterium has a UvrD helicase, whose function is common to Ec UvrD.

Disruption of Thermus thermophilus genes by homologous recombination using a thermostable kanamycin-resistant marker.

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were disrupted by homologous recombination using a recently developed, thermostable kanamycin-resistant marker. First, the trpE gene was disrupted with various constructions of DNA. The transformation efficiency was exponentially increased as the length of the homologous regions flanking the marker gene increased above the minimum length (200-300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. The convenience and high reliability of this method should prompt its application to the high-throughput systematic disruption of the genes of this thermophilic bacterium.

Observation of RecA protein monomer by small angle X-ray scattering with synchrotron radiation.

RecA protein is capable of forming homo-oligomers in solution. The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X-ray scattering, a direct method used to measure the overall dimensions of a macromolecule. In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R(g)) of 52 A. The value of R(g) decreased to 36 A at a higher lithium perchlorate concentration (1.0 M). The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.

RecA protein from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T.th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T.th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein.

Difference absorption spectra, circular dichroism, and disulfide cleavage of hen and turkey lysozymes in the alkaline pH region.

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points. The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried tyrosyl residue on alkali denaturation. The disulfide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of Tyr 23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 degrees C from the CD titration data. Comparison of the CD titration of turkey lysozyme with that of hen lysozyme suggested that Tyr 3 and Tyr 23 in turkey lysozyme have apparent pK values of 11.9 and 9.8, respectively.

Analysis of the acid-base titration curve of hen lysozyme.

The acid-base titration curves of hen egg-white lysozyme [EC 3.2.1.17] obtained by three groups (Sakakibara & Hamaguchi (1968) J. Biochem. 64, 613--619; Tanford & Roxby (1972) Biochemistry 11, 2192--2198; Pfeil & Privalov (1976) Biophys. Chem. 4, 23--32) were analyzed using the empirical formula of Linderstrøm-Lang. Hen lysozyme has 32 ionizable groups including the alpha-amino and alpha-carboxyl groups. Of the 21 groups other than the 11 arginyl residues, the pK values of 17 ionizable groups have been determined by various methods. Using these pK values, the pK values of the other four ionizable groups were estimated. The apparent pK values obtained were 2.0 (pKmit = 3.4), 2.1 (pKint = 3.5), 2.5 (pKint = 3.8), and 7.9 (pKint = 8.5) at 0.1 ionic strength and 25 degrees C. The titration curves obtained by the above three groups were consistently explained in terms of the same set of pKint values. The results obtained also showed that no buried and untitratable groups are present in the native lysozyme molecule.

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme.

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.

Binding of N-acetyl-chitotriose to Asp 52-esterified hen lysozyme.

The pH dependence of the binding constant of (GlcNAc)3 to Asp 52-esterified lysozyme was determined by the fluorescence technique. The pK values of Asp 101 in the modified lysozyme and its complex with (GlcNAc)3 were determined to be 4.5 and 3.6, respectively, at 25 degrees C and 0.1 ionic strength. This result is different from that obtained by Parsons and Raftery ((1972) Biochemistry 11, 1633--1638), who observed no pK shift of Asp 101. The macroscopic pK value of Asp 52 in intact lysozyme determined by them using the pH difference titration data of Asp 52-esterified lysozyme relative to intact lysozyme ((1972) Biochemistry 11, 1623--1629) was 4.5, which is higher by about one pH unit than the pK value determined by our group (Kuramitsu et al. (1974) J. Biochem. 76, 671--683; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170. We found that their pH difference titration data in the absence and presence of saccharides can be consistently interpreted in terms of our pK values of Asp 52, Glu 35, and Asp 101, if we assume that the pK value of another ionizable group (probably Asp 48) is perturbed on esterification of Asp 52.

Participation of the catalytic carboxyls, Asp 52 and Glu 35, and Asp 101 in the binding of substrate analogues to hen lysozyme.

The interactions of the substrate analogues, GlcNAc, beta-methyl GlcNAc, (GlcNAc)2, and (GlcNAc)3, with turkey egg-white lysozyme [ED 3.2.1.17], in which the Asp 101 of hen lysozyme is replaced by Gly, were studied at various pH values by measuring changes in the circular dichroic (CD) band at 295 nm. Results were compared with those for hen egg-white lysozyme. The modes of binding of these substrate analogues to turkey lysozyme were very similar to those hen lysozyme except for the participation of Asp 101 in hen lysozyme. The ionization constants of the catalytic carboxyls, Glu 35 and Asp 52, in the turkey lysozyme-(GlcNAc)3 complex were determined by measuring the pH dependence of the CD band at 304 nm, which originates from Trp 108 near the catalytic carboxyls. The ionization behavior of the catalytic carboxyls of turkey lysozyme in the presence and absence of (GlcNAc)3 was essentially the same as that for hen lysozyme. The pH dependence of the binding constant of (GlcNAc)3 to hen lysozyme was compared with that to turkey lysozyme between pH 2 and 8. The pH dependence of the binding constant for (GlcNAc)3 to turkey lysozyme could be interpreted entirely in terms of perturbation of catalytic carboxyls. In the case of hen lysozyme, it was interpreted in terms of perturbation of the catalytic carboxyls and Asp 101 in the substrate-binding site. The pK values of Asp 101 in hen lysozyme and the hen lysozyme-(GLcNAc)3 complex were 4.5 and 3.4, respectively. The binding constants of (GlcNAc)3 to lysozyme molecules with different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated. The binding constant of lysozyme, in which Asp 52 and Glu 35 are deprotonated, to (GlcNAc)3 was the smallest. The other three species had similar binding constant to (GlcNAc)3.

Hydrolysis of 4-methylumbelliferyl N-acetyl-chitooligosaccharides catalyzed by human lysozyme.

Binding of N-acetyl-chitotetraose, 4-methylumbelliferyl chitotrioside ((GlcNAc)3-MeU), and 4-methylumbelliferyl chitotetraoside ((GlcNAc)4-MeU) to human lysozyme [EC 3.2.1.17] was studied by fluorescence measurement. Hydrolysis of (GlcNAc)3-MeU and (GlcNAc)4-MeU catalyzed by human lysozyme was studied by measuring the release of 4-methylumbelliferone fluorimetrically and the kinetic constants were determined in the pH range of 2 to 8 at 0.1 ionic strength and 42 degrees C. On the basis of binding and kinetic data, it was shown that (GlcNAc)3-MeU binds mainly to subsites A to D with the terminal MeU group bound to subsite D (nonproductive binding) and that (GlcNAc)4-MeU binds to subsites A to E (productive binding) and subsites A to D with the nonreducing sugar residue extending beyond subsite A (nonproductive binding). The pH dependences of the kinetic constants for hydrolysis of (GlcNAc)3-MeU and (GlcNAc)4-MeU were analyzed assuming that nonproductive binding occurs competitively, that an ionizable group in addition to the catalytic groups (Asp 52 and Glu 35) participates in the catalysis, and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Analyses of the kinetic constants for (GlcNAc)3-MeU and (GlcNAc)4-MeU both gave the same pK values of the catalytic groups (pK52 = 3.6(3) and pK35 = 6.6(8) at 0.1 ionic strength and 42 degrees C). These pK values were very close to the values determined previously by spectroscopic methods in our laboratory (Kuramitsu et al. (1974) J. Biochem. 76, 671-683; (1978) ibid. 83, 159-170; (1980) ibid. 87, 771-778).

RecA protein has extremely high cooperativity for substrate in its ATPase activity.

The single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated. To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined. Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly(dT)-dependent ATPase activity was measured. At 25 degrees C, increasing the concentration of RecA protein from 0.3 to 1.0 microM increased the turnover number (kcat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6. At 0.5 microM RecA protein, increasing the temperature from 25 to 37 degrees C increased kcat from 0.18 to 0.35 s-1 but decreased nH from 9.8 to 6.6. Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date. Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far. These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules. This implies that ATP binding induces structural change of the whole nucleoprotein helical filament. Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.

Ionization of the catalytic groups and tyrosyl residues in human lysozyme.

The pH difference absorption spectra of human lysozyme [EC 3.2.1.17] were measured. The difference spectra in the acidic region had a peak at 300 nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300 nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25 degrees C) determined from the pH dependence of the circular dichroism at 303.5 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671--683) and the fluorescence excited at 305 nm (Kuramitsu et al. (1978) J. Biochem. 83, 159--170). The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (Kuramitsu & Hamaguchi (1979) J. Biochem. 85, 443--456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.

Interactions on 3-deoxy and 6-deoxy derivatives of N-acetyl-D-glucosamine with hen lysozyme.

The interactions of deoxy derivatives of GlcNAc, 6-deoxy-GlcNAc, and 3-deoxy-GlcNAc with hen egg-white lysozyme [EC 3.2.1.17] were studied at various pH's by measuring the changes in the circular dichroic (CD) band at 295 nm. It was shown that 6-deoxy-GlcNAc and 3-deoxy-GlcNAc bind at subsite C of lysozyme and compete with GlcNAc. The pH dependence of the binding constant of 6-deoxy-GlcNAc was the same as that of GlcNAc. On the other hand, the binding constants of 3-deoxy-GlcNAc were 3--10 times smaller than those of GlcNAc in the pH range from 3 to 9. X-ray crystallographic studies show that O(6) and O(3) of GlcNAc at subsite C are hydrogen-bonded to the indole NH's of Trp 62 and Trp 63, respectively, but the above results indicate that Trp 63, not Trp 62, is important for the interaction of GlcNAc with lysozyme.