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Biomedical subjects

W Hu

Publications and source records attributed to W Hu.

At least 235 records · Page 13Linked to original sources
PubMed1996

Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Functional regulation of proteins is central to living organisms. Here it is shown that a nonfunctional conformational state of a polypeptide can be kinetically trapped in a lipid bilayer environment. This state is a metastable structure that is stable for weeks just above the phase transition temperature of the lipid. When the samples are incubated for several days at 68 degrees C, 50% of the trapped conformation converts to the minimum-energy functional state. This result suggests the possibility that another mechanism for functional regulation of protein activity may be available for membrane proteins: that cells may insert proteins into membranes in inactive states pending the biological demand for protein function.

Magnetic Resonance Spectroscopy↗
PubMed1996

Phenotypical and functional characterization of intrahepatic bile duct cells from common duct ligated mice.

Bile duct cells (BDCs), especially intrahepatic cholangiocytes, are primary targets of immune-related injury in such pathologic processes as liver graft rejection, liver graft-versus-host disease, and primary sclerosing cholangitis. Cholestasis and progressive loss of intrahepatic BDCs indicate chronicity in these diseases. The present investigation characterizes the acquisition of immune markers of intrahepatic BDCs isolated from mice after bile duct ligation. Purified BDCs from cholestatic C57BL/6 (H-2b) mice express not only the major histocompatibility complex (MHC) class I and class II antigens but also the costimulatory molecules B7-1 (CD80) and B7-2 (CD86). The expression of the MHC class II molecules on BDCs before and after interferon (IFN)-gamma exposure is also demonstrated by immunohistochemistry on the cultured cells. Cytotoxicity assays indicate the vulnerability of these cells as targets for immunologic injuries. Effector cells generated from B10.BR splenocytes (H-2k) against C57BL/6 splenocytes (H-2b) show comparable killing of BDCs and EL4 cells, the latter being a lymphoma line that was established from the C57BL/6N (H-2b) mouse. An immortalized mouse BDC line (IBDC) is included in this study to validate some of the findings from BDCs isolated from bile duct-ligated mice. We suggest that cholestatic BDCs express surface antigens different from those of normal epithelial cells that result in increased susceptibility to damage by immune mechanisms.

Animals↗
PubMed1996

[Transplantation of pedicled pisiform bone to replace lunate bone with aseptic necrosis].

OBJECTIVE: To find out a new and effective method for the treatment of aseptic necrosis of lunate bone. METHODS: Blood supply and morphology of pisiform bone were investigated in 25 upper limbs and 57 sets of carpal bone from adult cadavers on the basis of anatomical study. The lunate bone was replaced with pisiform bone of pedicled blood vessel and tendon of musculus flexor earpiulnaris in 17 patients with aseptic necrosis of lunate bone (stage III). RESULTS: The patients were followed up for 18-46 months (average of 28 months). Complete relief of pain was obtained in all of the patients, and the range of motion of their wrists was improved. Grip strength was increased by 48.2%. Radiograph showed normal location of transferred pisiform bone without osteosclerosis and atrophy in 16 patients. Atrophy of pisiform bone was found 2 years after operation in one patient. In the 17 patients, 15 resumed their original jobs and 2 changed their jobs for other reasons. CONCLUSION: We consider that the carpal bone chain can be completely preserved by transplantation of pedicled pisiform bone, which it is an effective method to treat aseptic necrosis stage III of lunate bone.

Adult↗
PubMed1996

Expression and distribution of amyloid precursor protein-like protein-2 in Alzheimer's disease and in normal brain.

Amyloid precursor-like protein-2 (APLP-2) belongs to a family of homologous amyloid precursor-like proteins. In the present study we report on the expression and distribution of APLP-2 in fetal and adult human brain and in brains of patients with Alzheimer's disease. We demonstrate that APLP-2 mRNAs encoding isoforms predicted to undergo post-translational modification by chondroitin sulfate glycosaminoglycans are elevated in fetal and aging brains relative to the brains of young adults. Immunocytochemical labeling with APLP-2-specific antibodies demonstrates APLP-2 immunoreactivity in cytoplasmic compartments in neurons and astrocytes, in large part overlapping the distribution of the amyloid precursor protein. In Alzheimer's disease brain, APLP-2 antibodies also label a subset of neuritic plaques. APLP-2 immunoreactivity is particularly conspicuous in large dystrophic neurites that also label with antibodies specific for APP and chromogranin A. In view of the age-dependent increase in levels of chondroitin sulfate glycosaminoglycan-modified forms of APLP-2 in aging brain and the accumulation of APLP-2 in dystrophic presynaptic elements, we suggest that APLP-2 may play roles in neuronal sprouting or in the aggregation, deposition, and/or persistence of beta-amyloid deposits.

Adult↗
PubMed1996

[Osteocartilaginous reconstruction, research and clinical application].

Facing the problem set by losses of osteo-cartilagenous substances around the little bones, the finger- and toe-joints for example, the authors scanned a way different from that of amputation or arthrodesis: the functional rebuilding with the help of substitution grafts. An exploratory research conducted on 21 rabbits in Bordeaux in 1990 allowed to test the coupling of two bio-materials used in surgery here and now, a coupling which has not shown any side-effect and whose benefit is to obtain a non-deformable mass, colonizable by osteoblastic cells. The loss of articular substance suffered by a patient in his fingers in 1992 profited by this bone-rebuilding technique. The use of an external articulated stabilizer was an important provisional support during the colonization of the grafts by the osteoblasts. The difference of time in the osseus rebuilding between the rabbits on the one hand, and the human being on the other, is recorded.

Animals↗
PubMed1996

[A randomized clinical study of sulperazone versus tienam in the treatment of LRTIS].

The purpose of the clinical study is to evaluate the efficacy and safety of Sulperazone (SPZ) (sulbactam/cefoperazone) comparited with tienam in the treatment of lower resperiatory tract infections. A total of 73 patients enrolled in the study. 61 patients were evaluated for efficacy (SPZ 31, TIM 30) and 63 patients were evaluated for safty (SPZ 31, TIM 32). Drugs were administered twice a day for 7-14 days, at a daily dose of either 2.0-4.0 g of sulperazone or 2.0-4.0 g tienam. The overall clinical efficacy rates of sulperazone and tienam were 93.5% and 93.3%, respectively. Total pathogens were isolated from 49 of 61 patients prior to treatment. Over two-thirds of the isolates (37/48, 77.1%) were beta-lactammase producing strains. The bacterialogical clearance rate was 92.0% and 91.7% for both groups. The incidence of adverse drug reactions for SPZ and TIM were 3.2% and 10.0%, respectively. The susceptibility rates of the bacteria isolated to five drugs: sulperazone, tienam, ceftazidime, ceftriaxone and cefotaxime were 95.9%, 93.9%, 96.0%, 70.0%, and 80.0% respectively. The susceptibility rate of sulperazoene was significant.

Adolescent↗
PubMed1995

Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel.

Tryptophans in the gramicidin channel are important for defining the conformation and the orientation with respect to the bilayer normal and for facilitating cation conductance. Here, high-resolution structure and dynamics of these rings are characterized by solid state NMR. Both oriented and unoriented lipid bilayer preparations are used. Fast frozen lipid bilayer preparations of unoriented samples have been used to obtain static characterizations of nuclear spin interaction tensors. The temperature dependence of these unoriented samples and the spectral features of fast frozen oriented samples were used to experimentally define the local motions of the individual indole rings. Local motions were shown to have amplitudes as high as +/- 29 degrees, and the motions were dominated by libration about the chi 2 axis. The high-resolution structure has been achieved by interpreting seven precise (+/- 0.3 degree) orientational constraints from 2H and 15N NMR for each indole ring in light of the motionally averaged interaction tensors. Each of the four indoles is restricted to a unique orientation of the ring with respect to the bilayer normal and one of two possible rotameric states. The side chain torsion angles for each residue are very similar, generating similar electric dipole moment orientations with respect to the channel.

Amino Acid Sequence↗
PubMed1995

Tryptophan hydrogen bonding and electric dipole moments: functional roles in the gramicidin channel and implications for membrane proteins.

The known high-resolution structure and dynamics characterization of the lipid bilayer-bound polypeptide gramicidin A provides a unique opportunity to study structure-function and dynamics-function correlations in a model membrane protein. In particular, the indoles have a variety of very important functional roles in this cation channel that will undoubtedly be recognized in membrane proteins. That indoles and phenols are oriented at the hydrophobic-hydrophilic interface of lipid bilayers is already well-recognized in membrane proteins. The most buried indole of the gramicidin channel, Trp9, is shown by 15N solid state NMR to be exposed to the hydrophilic surface through hydrogen exchange. Here the importance of the indole dipole moments is described for cation conductance. Preparation of samples with high concentrations of Na+ is shown by high-resolution orientational constraints derived from 2H NMR to have no structural effect on the indole side chain conformations. These dipoles stabilize cations in the binding sites near the channel entrance and substantially reduce the potential energy barrier at the bilayer center. This latter finding conclusively documents that the rate-limiting step in cation conductance by this channel involves the barrier at the bilayer center. Furthermore, dynamics of the indole rings cause significant fluctuations in the energy of stabilization at the binding site that may result in a rapid mechanism for gating the channel.

Electrochemistry↗
PubMed1995

Zeon-1, a member of a new maize retrotransposon family.

We have previously shown that the tandemly duplicated 27 kDa maize storage protein locus underwent mitotic rearrangement to yield a single-copy allele in isolates of the inbred line A188. This rearrangement contains a new LTR retrotransposon, designated Zeon-1. This middle repetitive element of 7313 bp had two long terminal repeats, a primer binding site, a polypurine tract and a gag-related open reading frame of 375 amino acids. Transcripts of the gag-related region were detected by the polymerase chain reaction (PCR) in certain maize tissues, and Western blots detected the gag-related protein in the same tissues. Moreover, the product of this mitotic rearrangement was shown to contain the same insertion site and 3' LTR as Zeon-1, suggesting that this rearrangement occurs with unusual precision. Zeon elements were found to be present in teosinte and not present in the Gramineae wheat, barley, sorghum and rye.

Amino Acid Sequence↗
PubMed1995

Expression of Aequorea green fluorescent protein in plant cells.

The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

Arabidopsis↗
PubMed1995

A chimeric bacterial phosphofructokinase exhibits cooperativity in the absence of heterotropic regulation.

The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP. Whereas E. coli PFK (EcPFK) is profoundly inhibited by ATP, B. stearothermophilus PFK (BsPFK) is only slightly inhibited. The structural basis for this difference could be closure of the active site via a conformational transition that occurs in the ATP-binding domain of EcPFK, but is absent in BsPFK. To investigate the role of this transition in ATP inhibition of EcPFK, we have constructed a chimeric enzyme that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit. The chimeric PFK has the following characteristics: (i) tetrameric structure and kinetic parameters similar to those of the native enzymes, (ii) insensitivity to regulation by the effector phosphoenolpyruvate despite its ability to bind to the enzyme, and (iii) a sigmoidal (nH around 2) fructose 6-phosphate saturation curve. From the results, it is concluded that the active site regions of the two native enzymes are remarkably similar, but their effector sites and their mechanisms of heterotropic regulation are different. The chimeric subunit is locked in a structure resembling that of activated E. coli PFK, yet the enzyme can exist in two different conformational states. Mechanisms for its sigmoidal kinetics are discussed.

Adenylyl Imidodiphosphate↗
PubMed1995

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.

Amino Acid Sequence↗
PubMed1995

Low-temperature solid-state 15N NMR characterization of polypeptide backbone librations.

The local molecular dynamics of gramicidin A in dimyristoylphosphatidylcholine bilayers were probed by a combination of low-temperature solid-state 15N NMR and rapid freezing of samples using liquid propane. It has previously been shown that this approach leads to sharp discontinuities in powder-pattern spectra of the gramicidin channel in lipid bilayers, and hence, an accurate determination of tensor element magnitudes is achieved. The static-tensor-element magnitudes determined here using hydrated bilayer samples at low temperatures are different from the values obtained from dry-powder samples at 308 K. However, for the one site studied, the orientation of the 15N chemical-shift tensor in the molecular frame is the same for both types of samples. Averaging of the chemical-shift tensor between 200 and 263 K is shown to be anisotropic and consistent with a motional axis parallel with the C alpha-C alpha axis for adjacent residues. The librational amplitudes determined here range from +/- 14 degrees to +/- 22 degrees at 263 K and are significantly larger than the nanosecond librations characterized by relaxation studies. The nanosecond motions are thought to represent correlated motions, while the librations described here are the sum of correlated (nanosecond) and uncorrelated (picosecond) motions. The picosecond librational amplitudes become considerably larger and more isotropic above 0 degree for the residues near the bilayer surface.

Amino Acid Sequence↗