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Biomedical subjects

J L Song

Publications and source records attributed to J L Song.

16 recordsLinked to original sources
PubMed2002

Transcription initiation of genes associated with azole resistance in Candida albicans.

Oral infections with the opportunistic pathogenic yeast Candida albicans are one of the earliest and most frequent infections in immunosuppressed individuals. In these populations, drug-resistant isolates have emerged with the widespread use of antifungal azole drugs. Many molecular mechanisms of resistance have been identified, including overexpression of two types of efflux pumps, the major facilitator MDR1 and the ABC-transporters (CDR1 and CDR2), and the overexpression or mutation of the target enzyme, ERG11. With overexpression of these four genes implicated in multidrug resistance, identification of regulatory regions of the promoters is important. 5' rapid amplification of cDNA ends (RACE) was used to identify transcription initiation sites for genes associated with multidrug resistance (CDR1, CDR2, MDR1 and ERG11). These results were confirmed by cloning and sequencing of 5' RACE products and by primer extension. This research will allow further analysis of the regulation of transcription for these genes.

ATP-Binding Cassette Transporters↗
PubMed2002

Effect of dietary supplemental levels of vitamin A on the egg production and immune responses of heat-stressed laying hens.

Two experiments were conducted to evaluate the effect of vitamin A supplementation of a commercial layer diet on the laying performance and immune function of heat-stressed hens. In Experiment 1, two different levels of vitamin A supplementation (3,000 and 9,000 IU/kg) were used to investigate the laying performance and antibody titer against Newcastle disease virus (NDV) of heat-stressed hens. Results showed that the high level of vitamin A supplementation (9,000 IU/kg) had a beneficial effect on the feed intake and laying rate of heat-stressed hens (P < 0.05), compared with the control group (3,000 IU/kg). The antibody titers were not influenced by the level of vitamin A (P > 0.05). In Experiment 2, the effect of four levels of vitamin A (3,000, 6,000, 9,000, and 12,000 IU/kg) on the antibody titer to NDV and T lymphocyte proportion was studied. The experimental birds were exposed to a high temperature (31.5 C) 15 d after NDV vaccination (Treatment 1) or immediately (Treatment 2). The results showed that the egg weight was increased (P < 0.01) by the high levels of vitamin A supplementation (6,000 and 9,000 IU/kg), but feed intake, laying rate, and body weight loss were not (P > 0.05). In Treatment 1, vitamin A had no significant effect on antibody titers against NDV in normal or hot environments but increased (P < 0.01) the proportion of alpha-naphthyl acetate esterase (ANAE)-positive cells. Vitamin A supplementation had a significant effect on NDV antibody titer and ANAE-positive cell proportion in Treatment 2 (P < 0.01). The results of the present study suggested that vitamin A supplementation in commercial layer diets to layer chickens under heat stress was beneficial to laying performance and immune function.

Animal Feed↗
PubMed2001

A 11.5 A single particle reconstruction of GroEL using EMAN.

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to approximately 11.5 A performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.

Chaperonin 60↗
PubMed2001

Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain alpha-ketoacid decarboxylase in maple syrup urine disease.

Maple syrup urine disease is caused by deficiency in the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. The clinical phenotype includes often fatal ketoacidosis, neurological derangement, and mental retardation. The type IA mutations Y393N-alpha, Y368C-alpha, and F364C-alpha, which occur in the E1alpha subunit of the decarboxylase (E1) component of the BCKD complex, impede the conversion of an alphabeta heterodimeric intermediate to a native alpha(2)beta(2) heterotetramer in the E1 assembly pathway. In the present study, we show that a natural osmolyte trimethylamine N-oxide (TMAO) at the optimal 1 m concentration restores E1 activity, up to 50% of the wild type, in the mutant E1 carrying the above missense mutations. TMAO promotes the conversion of otherwise trapped mutant heterodimers to active heterotetramers. This slow step does not involve dissociation/reassociation of the mutant heterodimers, which are preformed in the presence of chaperonins GroEL/GroES and Mg-ATP. The TMAO-stimulated mutant E1 activity is remarkably stable upon removal of the osmolyte, when cofactor thiamine pyrophosphate and the transacylase component of the BCKD complex are present. The above in vitro results offer the use of chemical chaperones such as TMAO as an approach to mitigate assembly defects caused by maple syrup urine disease mutations.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

Interactions of GroEL/GroES with a heterodimeric intermediate during alpha 2beta 2 assembly of mitochondrial branched-chain alpha-ketoacid dehydrogenase. cis capping of the native-like 86-kDa intermediate by GroES.

We showed previously that the interaction of an alphabeta heterodimeric intermediate with GroEL/GroES is essential for efficient alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase. In the present study, we further characterized the mode of interaction between the chaperonins and the native-like alphabeta heterodimer. The alphabeta heterodimer, as an intact entity, was found to bind to GroEL at a 1:1 stoichiometry with a K(D) of 1.1 x 10(-)(7) m. The 1:1 molar ratio of the GroEL-alphabeta complex was confirmed by the ability of the complex to bind a stoichiometric amount of denatured lysozyme in the trans cavity. Surprisingly, in the presence of Mg-ADP, GroES was able to cap the GroEL-alphabeta complex in cis, despite the size of 86 kDa of the heterodimer (with a His(6) tag and a linker). Incubation of the GroEL-alphabeta complex with Mg-ATP, but not AMP-PNP, resulted in the release of alpha monomers. In the presence of Mg-ATP, the beta subunit was also released but was unable to assemble with the alpha subunit, and rebound to GroEL. The apparent differential subunit release from GroEL is explained, in part, by the significantly higher binding affinity of the beta subunit (K(D) < 4.15 x 10(-9)m) than the alpha (K(D) = 1.6 x 10(-8)m) for GroEL. Incubation of the GroEL-alphabeta complex with Mg-ATP and GroES resulted in dissociation and discharge of both the alpha and beta subunits from GroEL. The beta subunit upon binding to GroEL underwent further folding in the cis cavity sequestered by GroES. This step rendered the beta subunit competent for reassociation with the soluble alpha subunit to produce a new heterodimer. We propose that this mechanism is responsible for the iterative annealing of the kinetically trapped heterodimeric intermediate, leading to an efficient alpha(2)beta(2) assembly of human branched-chain alpha-ketoacid dehydrogenase.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

GroEL/GroES promote dissociation/reassociation cycles of a heterodimeric intermediate during alpha(2)beta(2) protein assembly. Iterative annealing at the quaternary structure level.

Whereas the mechanism of GroEL/GroES-mediated protein folding has been extensively studied, the role of these chaperonins in oligomeric protein assembly remains poorly understood. In the present study, we investigated the interaction of the chaperonins with an alphabeta heterodimeric intermediate during the alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase/decarboxylase (BCKD). Incubation of the recombinant His(6)-tagged BCKD in 400 mM KSCN for 45 min at 23 degrees C caused a complete dissociation of the alpha(2)beta(2) heterotetramers into inactive alphabeta heterodimers. Dilution of the denaturant resulted in a rapid recovery of BCKD independent of the chaperonins GroEL/GroES. Prolonged incubation of BCKD in 400 mM KSCN resulted in the generation of nonproductive or "bad" heterodimers, which were unable to undergo spontaneous reactivation but capable of binding to GroEL to form a stable GroEL-alphabeta complex. Incubation of this complex with GroES and Mg-ATP led to the slow reactivation of BCKD with a second-order rate constant k = 480 M(-1) s(-1). Mixing experiments with radiolabeled and unlabeled protein substrates provided direct evidence that GroEL/GroES promote dissociation and subunit exchange between bad heterodimers. This was accompanied by the transformation of bad heterodimers to their "good" or productive counterparts. The good heterodimers were capable of spontaneous dimerization to initially form an inactive heterotetrameric species, followed by conversion to active heterotetramers. However, a large fraction of bad heterodimers were regenerated and rebound to GroEL. The cycle was perpetuated until the reconstitution of active BCKD was complete. Our data support the thesis that chaperonins GroEL/GroES mediate iterative annealing of nonproductive assembly intermediates at the quaternary structure level. This step is essential for an efficient subsequent higher order oligomerization.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1999

Chaperone activity of DsbC.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.

Bacterial Proteins↗
PubMed1999

GroEL/GroES-dependent reconstitution of alpha2 beta2 tetramers of humanmitochondrial branched chain alpha-ketoacid decarboxylase. Obligatory interaction of chaperonins with an alpha beta dimeric intermediate.

The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits. In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M-1 s-1. Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-alpha and GroEL-beta complexes as combined substrates. However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL. The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP. In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent. Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter. The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M-1 s-1. Dimers were regenerated from the GroEL-alpha beta complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 alpha2 beta2 tetramers.

Centrifugation, Density Gradient↗
PubMed1997

Does DsbA have chaperone-like activity?

DsbA showed chaperone-like activity similar to but weaker than that of protein disulfide isomerase in increasing reactivation and decreasing aggregation during the refolding of guanidine hydrochloride-denatured D-glyceraldehyde-3-phosphate dehydrogenase and rhodanese. The fact that both enzymes are devoid of disulfide bonds indicates the independence of the chaperone-like activity of DsbA from its thiol-protein oxidoreductase activity. The increased reactivation of D-glyceraldehyde-3-phosphate dehydrogenase by DsbA can be suppressed with increasing concentrations of a peptide of 21 amino acid residues, suggesting that the peptide binding ability of DsbA is responsible for its chaperone-like activity.

Glyceraldehyde-3-Phosphate Dehydrogenases↗
PubMed1997

Maternal HIV-1 viral load and vertical transmission of infection: the Ariel Project for the prevention of HIV transmission from mother to infant.

Most HIV-1 infections of children result from mother-to-infant transmission, which may occur perinatally or postnatally, as a consequence of breast feeding. In this study, the influence of maternal viral load on transmission of infection to infants from non-breast-feeding mothers was examined using samples of plasma and peripheral blood mononuclear cells (PBMCs) collected at several time points during pregnancy and the 6-month period after delivery. These samples were analyzed by several quantitative methods, including virus cultures of PBMCs and polymerase chain reaction (PCR) assays for HIV-1 RNA in plasma and DNA in PBMCs. The risk of transmission increased slightly with a higher viral load, but transmission and nontransmission occurred over the entire range of values for each assay. No threshold value of virus load was identified which discriminated between transmitters and nontransmitters. We also noted a significant rise in viral load and a decline in CD4+ lymphocytes in the six months after delivery. These findings suggest that a high maternal viral load is insufficient to fully explain vertical transmission of HIV-1. Additional studies are needed to examine the post-partum increase in viremia.

Anti-HIV Agents↗
PubMed1996

The second web bilobed island flap for thumb reconstruction.

The second web bilobed island flap is described. The flap includes skin from the second web of the hand and the dorsum of the index and middle fingers. Usually the flap is based on the first and second dorsal metacarpal arteries, but it can be used with just the first dorsal metacarpal vessels in its pedicle. Sensation of the flap is provided by branches of the radial nerve. The flap has been successfully used to reconstruct 11 injured thumbs.

Adult↗
PubMed1995

Chaperone-like activity of protein disulfide-isomerase in the refolding of rhodanese.

Protein disulfide-isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine-hydrochloride-denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above-mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515-1517] that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperone-like activity of PDI acting on rhodanese is independent of its disulfide-isomerase activity.

Animals↗
PubMed1993

[The third dorsal metacarpal artery neuromuscular island flap].

This paper describes the usefulness of the third dorsal metacarpal artery neurovascular island flap in the repair of soft tissue defect of the hand. The flap is based on the third dorsal metacarpal artery as its vascular pedicle, and it has been successfully used in 12 cases since 1990. We found the third dorsal metacarpal artery was larger and more constant than the first, and so the blood supply was highly guaranteed. There were no failures or donor site complications. However, the flap has its limitation. It is only suitable for covering relatively small defects. The anatomy and clinical dissection of the flap are described.

Adolescent↗