Search PubMed⌕ Search

Biomedical subjects

J Qin

Publications and source records attributed to J Qin.

At least 145 records · Page 8Linked to original sources
PubMed1995

Expression of human stem cell factor in the baculovirus expression system.

A partial cDNA of human Stem Cell Factor (SCF) was isolated and transferred into the genome of Bombyx Mori Nuclear Polyhedrosis Virus adjacent to the polyhedrin promoter. The soluble form of the SCF protein was expressed in Bombyx Mori cells and silkworm larvae and was secreted into the medium in an active state. The recombinant SCF was similar to natural SCF with respect to its interaction with monoclonal antibody and its synergistic biological activity in conjunction with colony-stimulating factor.

Baculoviridae↗
PubMed1994

The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin.

BACKGROUND: Thioredoxin is a ubiquitous protein and is involved in a variety of fundamental biological functions. Its active site is conserved and has two redox active cysteines in the sequence Trp-Cys-Gly-Pro-Cys. No structures of the oxidized and reduced states from the same species have been determined at high resolution under the same conditions and using the same methods. Hence, any detailed comparison of the two oxidation states has been previously precluded. RESULTS: The reduced and oxidized states of the (C62A, C69A, C73A) mutant of human thioredoxin have been investigated by multidimensional heteronuclear NMR. Structures for both states were determined on the basis of approximately 28 experimental restraints per residue, and the resulting precision of the two structures is very high. Consequently, subtle differences between the oxidized and reduced states can be reliably assessed and evaluated. Small differences, particularly within and around the active site can be discerned. CONCLUSIONS: Overall, the structures of the reduced and oxidized states of the (C62A, C69A, C73A) mutant of human thioredoxin are very similar (with a backbone atomic root mean square difference of about 0.9 A) and the packing of side chains within the protein core is nearly identical. The conformational change between oxidized and reduced human thioredoxin is very small and localized to areas in spatial proximity to the redox active cysteines. These subtle structural differences, in addition to the restriction of conformational freedom within the active site upon oxidation, may be important for the different activities of thioredoxin involving a variety of target proteins.

Amino Acid Sequence↗
PubMed1994

Correlation between the steric bulk of the distal E7 and E11 residues and the tilt of the FeCN unit in cyanometmyoglobin as determined by NMR from the orientation of the magnetic axes in single and double point mutants.

The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)-->Gly (H[E7]G), Val68(E11)-->Ile (V[E11]I), and Val68(E11)-->Ala (V[E11]A) and the double mutants His64-(E7)-->Gly:Val68(E11)-->Ile (H,V[E7,E11]G,I) and His64(E7)-->Gly:Val68(E11)-->Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (approximately 8 degrees) from that in the wild type (WT) (approximately 16 degrees), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C delta H3 directed away from the iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

1H NMR study of the solution molecular and electronic structure of engineered distal myoglobin His64(E7) Val/Val68(E11) His double mutant. Coordination of His64(E11) at the sixth position in both low-spin and high-spin states.

A genetically engineered human myoglobin (Mb) in which the distal His, His64(E7), and the distal Val, Val68(E11), are replaced by Val and His, respectively, has been expressed in Escherichia coli, for the purpose of assessing the potential role of a E11 residue in providing a hydrogen bond donor to the coordinated ligand. Molecular modeling indicates that such an interaction is possible. The 1H NMR spectrum of the ferric form of the double mutant Mb exhibits large hyperfine shifts and strong paramagnetic relaxation for which the temperature dependence of the hyperfine shifts reveals a thermal equilibrium between a low-spin and high-spin state (70, 30% at 25 degrees C, respectively). Standard sequence specific two-dimensional (2D) NMR assignments of the E and F helical backbones allow the identification of the peptide protons for the proximal His93(F8) and substituted distal His68(E11). Steady-state nuclear Overhauser effect from these peptide protons locate strongly hyperfine shifted His93(F8) and His68(E11) side chain protons which dictate that both the imidazole rings are coordinated to the iron. 2D bond correlation and one-dimensional and 2D dipolar correlation experiments locate and assign the resonances for the heme. The pattern of the heme contact shifts in both the low-spin and high-spin state, together with the nature of the temperature dependence of the His93(F8) and His68(E11) resonances, establish that the two His are ligated in the high-spin as well as low-spin forms. The pattern of heme methyl hyperfine shifts in the low-spin state, and the smaller hyperfine shifts for His68(E11) as compared to His93(F8) in the high-spin state, indicate that the axial bond to the distal His68(E11) is weakened or strained as compared with that for the proximal His93(F8) in both spin states. This weak ligation originates from a tilted iron-His68 bond, the only conformation in which His68 can place its imidazole group sufficiently close to bind to the heme iron in the conventional Mb folding. Not only do these results support the belief that distal His is indispensable for the control of the ligand binding in Mb and hemoglobin, but also reveal the significance of the evolution that the stereochemical disposition of both His64 and Val68 are unique and non-exchangeable for interacting with the bound ligand.

Amino Acid Sequence↗
PubMed1994

Expression of biologically active monomeric form of human M-CSF in baculovirus infected silkworm, Bombyx mori.

The human macrophage colony stimulating factor (hM-CSF) in its monomeric form has been over-produced in BmN cells and in silkworm larvae infected with the recombinant baculovirus Bm284M-CSF. The recombinant monomeric M-CSF (rhM-CSF) exhibited the activity of 8-14 x 10(4) units/ml of cell culture medium. When the insect larvae were infected with the recombinant virus, the maximum rhM-CSF was expressed 4-5 days post infection with an activity of 3 x 10(6) units/ml hemolymph. The monomeric rhM-CSF was purified to homogeneity through three steps of purification. A pilot purification yielded 1 mg of homogeneous monomeric rhM-CSF from 10 larvae. The purified rhM-CSF monomers gradually dimerized in vitro. In contrast, the crude or the semi-purified monomers did not dimerize in vitro, indicating that the presence of an unknown moiety in the rhM-CSF preparations obtained from hemolymph interfered with dimerization.

Alkylation↗
PubMed1994

[24-hour electroencephalography monitoring in children: analysis of 403 cases].

24-hour EEG monitoring was performed in 403 children with repeated seizure of various types. The positive rate was 82.7% for defined epilepsy. Epileptic discharges mainly occurred in NREM stage and focal discharges accounted for 69%. More than 70% of the children suspected of epilepsy were excluded from epileptic seizures. 24-hour EEG monitoring is of significant value in determining seizure characteristics, epilepsy types, the relations between epilepsy and sleep and between clinical seizures and EEGs.

Adolescent↗
PubMed1993

1H NMR study of the dynamics of the pH modulation of axial coordination in Aplysia limacina (Val(E7)) and sperm whale double mutant His(E7)-->Val,Thr(E10)-->Arg metmyoglobin.

The molecular and electronic structure, thermodynamics, dynamics, and mechanism of interconversion of the pH-modulated reversible equilibria of Aplysia limacina metmyoglobin, (metMb), have been investigated by 1H NMR spectroscopy. The four identified species which interconvert slowly on the NMR time scale (lifetime > 1 ms) are metMbOH (B) at alkaline pH, five coordinate metMb (N) at acidic to neutral pH, an acidic form, A, near pH approximately 4 and an extremely low pH form, D, attributed to an equilibrium unfolded species. The presence of strong distal hydrogen bonding by Arg (E10) to bound hydroxide is detected via a significant solvent isotope effect on the metMbOH (B) hyperfine shifts. Integration of the peak intensities yields pK values of 7.7 and approximately 4 for the B<-->N and N<-->A equilibria, respectively. Saturation transfer via chemical exchange is observed for B<-->N and N<-->A, where the rates for forming metMbOH (B) and the acidic form A from N are base- and acid-catalyzed, respectively, while the reverse rates are first-order. The much slower interconversion rate for N<-->B in A. limacina metMb than His(E7) containing mammalian metMb is attributed to the fact that a ligand bond is broken rather than just proton transferred and that the equilibrium involves a major rearrangement of the orientation of Arg(E10). This conclusion is supported by 1H NMR data for the sperm whale double mutant His(E7)-->Val/Thr(E10)-->Arg metMb, which exhibits a pK approximately 8.7 for the equilibrium between five-coordinate metMb (N) and metMbOH (B) with an even slower interconversion rate than in A. limacina metMb. This double mutant metMbOH (B) exhibits hydrogen bonding by Arg (E10) with coordinated hydroxide similar to that in A. limacina metMbOH. The slow but acid-catalyzed rates of conversion of A. limacina metMb (N) to the acid species A with significantly weakened bonding of the heme iron to the axial His(F8) residue is consistent with protonation of an inaccessible residue and/or a structural change accompanying the protonation equilibrium. It is concluded that metMb will coordinate water strongly only when there is a distal hydrogen bond acceptor residue, while the hydroxide ion is coordinated strongly only if there is a distal hydrogen bond donor residue.

Amino Acids↗
PubMed1993

Solution nuclear magnetic resonance determination of active site structure for a paramagnetic protein: cyanomet Aplysia myoglobin.

The active site structure of a paramagnetic protein, cyanomet myoglobin (Mb) from Aplysia limacina, has been determined in solution by NMR in order to investigate the mechanism of ligand stabilization in this protein, which lacks the usual distal His residue. The structure determination relies on using the experimental dipolar shifts from two-dimensional NMR assignments, NOEs and paramagnetic-induced relaxations as the constraints with a combination of the magnetic susceptibility tensor. The magnetic susceptibility tensor was obtained by using the high resolution crystal co-ordinates of other derivatives of Aplysia Mb (notably metMb, metMbF and metMbN3) for the part of the molecule which is unperturbed by the different ligands, together with the observed dipolar shifts of Aplysia metMbCN, to search for the Euler rotation that correctly converts the crystal co-ordinates to the magnetic axes. The magnetic anisotropies, as well as the orientation of the magnetic axes, are well defined by using the various input data sets based on the conserved structural portions of three sets of crystal co-ordinates. An excellent fit between the calculated versus the observed shifts was obtained for both backbone portions (E, F, G helical segments and FG corner) and the proximal side-chains, which demonstrates that the solution structure of the backbone portions and the proximal side for Aplysia metMbCN is identical to those for three other derivatives investigated by crystallography. On the other hand, the distal side was shown to present minor but important structural changes relative to Aplysia metMbF, which reflect the binding of a diatomic ligand (CN-) rather than a monoatomic one. In particular, the guanidinium group of Arg E10(66) is repositioned and further away from the iron at a distance consistent with H-bonding to the bound cyanide. The bound cyanide is tilted approximately 8 degrees (as detected by the major magnetic axis) away from the heme normal approximately towards the gamma-meso-H. This tilt appears to be due to the attractive hydrogen-bonding with Arg E10(66) and the steric repulsion from Ile E11(67). The results strongly support the role of Arg E10 in stabilizing the anionic ligands by hydrogen bonding as proposed before and demonstrate the validity of the dipolar shifts as a unique and sensitive constraint in determining the active site structure of paramagnetic proteins in the low-spin ferric state.

Animals↗
PubMed1993

Structural and functional characterization of sperm whale myoglobin mutants: role of arginine (E10) in ligand stabilization.

1H NMR and ligand-binding data were used to assess the role of residue Arg(E10) in ligand stabilization of several site-directed mutants, all carrying the His(E7) to Val substitution, obtained using a synthetic sperm whale myoglobin gene. Arg(E10) was previously found to form a hydrogen bond with the ligand in fluoro-, azido- and cyanomet derivatives of Aplysia limacina myoglobin, which lacks the distal His(E7) [Qin, J., La Mar, G. N., Ascoli, F., Bolognesi, M., & Brunori, M. (1992) J. Mol. Biol. 224, 891-897]. NMR analysis of the paramagnetically induced relaxation, hyperfine shift patterns, and dipolar connectivities shows that Arg(E10) also falls into the distal pocket in the engineered sperm whale myoglobin mutants and resides at an H-bonding distance from the Fe(3+)-bound cyanide. The rate constant for cyanide dissociation from the ferrous derivative was determined by stopped-flow experiments; the ligand stabilization achieved by Arg(E10) is similar to that exerted by His(E7) in wild-type sperm whale myoglobin, and both are very different from the His(E7)Val single mutant. Contrary to that for the wild-type, the cyanide dissociation rate constant for the mutant containing Arg(E10) is essentially independent of pH (from 6 to 9), as expected on the basis of the guanidinium group of Arg having a pK > 10. This finding is consistent with the NMR data in which the chemical shift of the Arg(E10) N epsilon H is insensitive to pH (6-9), as is also observed in Aphysia limacina cyanometmyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Solution structure determination of the heme cavity in the E7 His-->Val cyano-met myoglobin point mutant based on the 1H NMR detected dipolar field of the iron: evidence for contraction of the heme pocket.

The 1H NMR spectrum of the cyanomet complex of the sperm whale His[E7]Val myoglobin (Mb) point mutant has been analyzed by 2D methods to yield the assignments for the active site residues, including the substituted Val E7. The dipolar shifted proximal residues are used to quantitatively locate the magnetic axes for the paramagnetic susceptibility tensor in the molecular framework. The orientation of the major axis, which correlates with the ligand tilt, is approximately 15 degrees from the heme normal, as found in wild-type (WT) Mb, but is tilted in a direction rotated approximately 40 degrees toward the heme gamma-meso position with respect to WT and similar to that in the His[E7]Gly mutant [Rajarathnam, K., La Mar, G. N., Chiu, M., & Sligar, S. G. (1992) J. Am. Chem. Soc. 114, 9048-9058]. The altered direction of an unchanged tilt angle for the Fe+3-CN unit is shown to be qualitatively consistent with earlier computations of the potential energy surface for MbCO [Kuriyan, J., Wilz, S., Karplus, M., & Petsko, G. A. (1986) J. Mol. Biol. 192, 133-154]. It is concluded that His E7 does not significantly contribute to the ligand tilt but strongly influences the direction of tilt. Deviations between observed and predicted dipolar shifts for the E-helix backbone protons and perturbed patterns of their respective nuclear Overhauser effect between the E-helix and the heme 1,8-methyls are separately analyzed for movement of the E-helix and agree on a translation of the E-helix of the order of 0.8 A in a direction toward the iron. The discrepancy between observed and predicted dipolar shifts for Phe CD1 indicates a approximately 0.5-A movement by the ring parallel to the heme and towards the E-helix. The E-helix and Phe CD1 movements are consistent with a contraction of the pocket to fill the space created by the His-->Val substitution. The correlation between the observed dipolar shifts of the substituted Val E7 side chain and those calculated as a function of rotation of the residue with and without movement of the E-helix confirm the movement of the E-helix and allow a quantitative description of the Val orientation. It is concluded that the dipolar field of the paramagnetic susceptibility tensor provides an important quantitative constraint for defining the heme cavity structure in cyanomet complexes of distal point mutants of myoglobin and hemoglobin.

Amino Acid Sequence↗
PubMed1993

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.

Amino Acid Sequence↗
PubMed1992

Solution 1H nuclear magnetic resonance determination of hydrogen bonding of the E10 (66) Arg side-chain to the bound ligand in Aplysia cyano-met myoglobin.

A combined one-dimensional nuclear Overhauser effect, paramagnetic-induced relaxation and two-dimensional sequence-specific 1H n.m.r. assignment of the spectrum of portions of the distal pocket of Aplysia cyano metMyoglobin (metMbCN) has been carried out in order to establish the presence and identity of distal residues in the heme pocket. In the absence of the usual distal E7 His in Aplysia Mb (E7 Val), the sequence-specific assignment of the E7 and E10 residues, together with their hyperfine shift patterns, relaxivities and dipolar connectivities to each other and the remainder of the E helix, reveal that the E10 Arg is turned into the pocket and hydrogen bonds to the bound cyanide group. We have previously found a similar rearrangement of the E10 Arg in Aplysia fluoro metMyoglobin, and the stabilizing effect of this residue was proposed to be responsible for the slow rate of cyanide dissociation from rapidly reduced ferrous Aplysia myoglobin. Based on the similar distal E7 His hydrogen-bonding interaction to the bound ligand in the crystal of sperm whale MbO2 and in solution of its cyano met complex, we propose that the E10 Arg similarly hydrogen bonds to the bound O2 in Aplysia MbO2 and accounts for its strong ligand binding and slow dissociation rate.

Animals↗
PubMed1992

Complete sequence-specific 1H NMR resonance assignment of hyperfine-shifted residues in the active site of a paramagnetic protein: application to Aplysia cyano-metmyoglobin.

Two-dimensional sequence-specific 1H NMR resonance assignment methodology (Wüthrich, 1986) has been applied for the first time to a 18-kDa paramagnetic hemoprotein (cyano-met Aplysia Mb) to identify all the hyperfine-shifted residues. The assignment was greatly facilitated by the fact that hyperfine shifts of residues impart a strong temperature dependence to the cross peaks, which aids location and identification, and provides improved spectral dispersion, particularly in the fingerprint region. 2D COSY and TOCSY were found to be surprisingly effective in locating the complete spin connectivities of all of the hyperfine-shifted residues, with the exception of the axially coordinated His95 imidazole ring, whose proton resonances were found to exhibit severe line broadening (> 400 Hz). Conventional 1D NOE and NOESY with short mixing times, combined with paramagnetic-induced relaxation effects, led to the successful assignment of even extremely broad proton signals. Three helical stretches and two loop regions were identified as the source of all hyperfine-shifted residues: the F helical residues 3-9, the E-helix residues 6-14, the G-helix residues 5-9, the FG-loop residues 1-4 and the CD-loop residues 1-4. These segments comprise all the residues that make contact with the heme and modulate the reactivity of the prosthetic group. The sequence-specific identifications of the active-site residues revealed that the solution structure of Aplysia metMbCN is fully consistent with that observed by X-ray diffraction in single crystals for a variety of other derivatives, except for the distal Arg66 (E10), which is turned into the heme pocket, as found only in the metMbF crystal structure (Bolognesi et al., 1990). The ready identification, by their temperature sensitivity, and the complete assignments of all hyperfine-shifted residues of Aplysia metMbCN demonstrate that sequence-specific assignment can be profitably applied to paramagnetic proteins, and that it should be possible to determine the solution structures of paramagnetic proteins, at least for low-spin complexes, by using NMR techniques used for diamagnetic proteins.

Amino Acids↗
PubMed1992

Determination of carcinogenic potency of alkytoxynol-741 (AP-741) by rat peritoneal cell cultures.

The carcinogenic potency of AP-741 was tested in rats using the Nashed method of rat peritoneal cell short-term carcinogenic test. N-methyl-N-nitro-N'-nitrosoguanidian (MNNG) was used as positive control; saline and nonoxynol-9 (NP-9) were used as negative control. Two doses of AP-741 (4 mg/kg and 40 mg/kg equivalent to 4 and 40 times human doses) were tested. The result showed that no colonies of more than 9 cells were seen in the saline and NP-9 groups, and the two dose groups of AP-741. However, 12 +/- 8.3 colonies (9-29 cell/colony) and 4.5 +/- 4.2 colonies (30-300 cell/colony) were seen in the MNNG group. According to the Nashed criterion, we can say MNNG has potential carcinogenesis, while AP-741 does not have any potential carcinogenesis and its use as a vaginal contraceptive drug is safe.

Animals↗
PubMed1992

[The segmental distribution of the afferent neurons of the "zusanli" point and the caecum in rabbits--a study with the horseradish peroxidase method].

Ten adult rabbits were used in this experiment. A solution of 10-20% HRP (sigma IX, RZ = 3.2) was injected into the "Zusanli" point and the subserosa of the caecum. The uptake and retrograde transmission of HRP in the afferent neurons of both the somatic and visceral nerves were traced to the spinal ganglia. The results showed that: 1. Labelled afferent neurons from the region of "Zusanli" point were found in the spinal ganglia T12-S2 with a higher concentration in L4-S2. 2. Labelled afferent neurons from the region of the caecum were found in the spinal ganglia T2-S2 with a higher concentration in T12-L2. 3. The ranges of distribution of labelled afferent neurons from the regions overlapped in the segments T12-S2.

Acupuncture Points↗
PubMed1992

[Related factors for the development of delayed encephalopathy following acute carbon monoxide poisoning].

Two hundred and twenty three cases of acute carbon monoxide poisoning were observed during acute stage and followed-up subsequently for three months. Through single factor and multiple factors analyses, six out of 97 observed factors were demonstrated as risk factors for the development of delayed encephalopathy, namely, elderliness, mental work, previously with hypertension, coma lasting for 2-3 days, long standing dizziness and fatigue after regaining consciousness and mental stimulation during recovery. Based on these factors, a regression equation for predicting the probability of developing delayed encephalopathy in individual patient with acute carbon monoxide poisoning has been established In order to minimize the development of delayed encephalopathy, it is advisable to keep the patients of acute carbon monoxide poisoning with above mentioned related factors under constant monitoring and surveillance.

Adolescent↗