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Biomedical subjects

L S Huang

Publications and source records attributed to L S Huang.

At least 37 records · Page 2Linked to original sources
PubMed1998

Translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apolipoprotein B are determined by the presence of beta sheet domains.

Apolipoprotein (apo) B100 is an atypical secretory protein in that its translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal alpha globular domains but no beta sheet domains, and apoB13,16,beta, which has an amphipathic beta sheet domain of apoB100 inserted into apoB13,16. These constructs, along with carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induced proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,beta, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined by the presence of a lipid binding beta sheet domain. It is possible that beta sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel.

Apolipoprotein B-100↗
PubMed1998

The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus.

The movement of many transcription factors, kinases and replication factors between the nucleus and cytoplasm is important in regulating their activity. In some cases, phosphorylation of a protein regulates its entry into the nucleus; in others, it causes the protein to be exported to the cytoplasm. The mechanism by which phosphorylation promotes protein export from the nucleus is poorly understood. Here we investigate how the export of the yeast transcription factor Pho4 is regulated in response to changes in phosphate availability. We show that phosphorylation of Pho4 by a nuclear complex of a cyclin with a cyclin-dependent kinase, Pho80-Pho85, triggers its export from the nucleus. We also find that the shuttling receptor used by Pho4 for nuclear export is the importin-beta-family member Msn5, which is required for nuclear export of Pho4 in vivo and binds only to phosphorylated Pho4 in the presence of the GTP-bound form of yeast Ran in vitro. Our results reveal a simple mechanism by which phosphorylation can control the nuclear export of a protein.

Biological Transport↗
PubMed1998

The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms. In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p. Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton. Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner. In response to pheromones, Far1p moves from the nucleus to the cytoplasm. Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

Adaptor Proteins, Signal Transducing↗
PubMed1998

ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice.

To study the role of low levels of high density lipoprotein (HDL) and apolipoprotein (apo) A-I in atherosclerosis risk, human apoB transgenic mice (HuBTg) were crossed with apoA-I-deficient (apoA-I-/-) mice. After a high fat challenge, total cholesterol levels increased drastically due to an increase in the non-HDL cholesterol as confirmed by FPLC analysis. In addition, total cholesterol levels in A-I-/- HuBTg mice were lower than the control HuBTg mice, due mainly to decreased HDL-C in A-I-/- HuBTg mice. Analysis of atherosclerosis in the proximal aorta in mice fed a high-fat Western-type diet for 27 weeks revealed a 200% greater lesion area in female apoA-I-/- HuBTg mice (49740+/-9751 microm2) compared to control HuBTg mice (23320+/-4981 microm2, P = 0.03). Lesion size (12380+/-3281 microm2) in male A-I-/- HuBTg mice was also about 200% greater than that in the control HuBTg mice (5849+/-1543 microm2), although not statistically significant. Very few and small lesions were observed in both apoA-I-/- HuBTg and control HuBTg animals fed a chow diet. Therefore, the adverse effect of low HDL on atherosclerosis in mice was only evident when LDL-cholesterol was markedly elevated by high-fat challenge. Male apoA-I-/- HuBTg mice exhibited hypertriglyceridemia when challenged with a high-fat diet. This correlated with both a reduction in lipoprotein lipase activity and a decrease in lipoprotein lipase activation by HDL. In summary, low high density lipoprotein levels due to apolipoprotein A-I deficiency exacerbated the development of atherosclerotic lesions in mice with elevated atherogenic lipoproteins. This mouse model mimics human conditions associated with low HDL levels and provides additional evidence for the anti-atherogenic role of apoA-I.

Animals↗
PubMed1996

A novel functional role for apolipoprotein B in male infertility in heterozygous apolipoprotein B knockout mice.

Male infertility, affecting as many as 10% of the adult population, is an extremely prevalent disorder. In most cases, the cause of the condition is unknown, and genetic factors that might affect male fertility, other than some sequences on the Y chromosome, have not been identified. We report here that male mice heterozygous for a targeted mutation of the apolipoprotein B (apo B) gene exhibit severely compromised fertility. Sperm from these mice failed to fertilize eggs both in vivo and in vitro. However, these sperm were able to fertilize eggs once the zona pellucida was removed but displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization-related and other experiments revealed reduced sperm motility, survival time, and sperm count also contributed to the infertility phenotype. Recognition of the infertility phenotype led to the identification of apo B mRNA in the testes and epididymides of normal mice, and these transcripts were substantially reduced in the affected animals. Moreover, when the genomic sequence encoding human apo B was introduced into these animals, normal fertility was restored. These findings suggest that this genetic locus may have an important impact on male fertility and identify a previously unrecognized function for apo B.

Animals↗
PubMed1996

Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of ApoB-containing lipoproteins.

Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.

Apolipoproteins B↗
PubMed1995

Apoprotein B100, an inefficiently translocated secretory protein, is bound to the cytosolic chaperone, heat shock protein 70.

Apoprotein B100 (apoB) is a secretory protein that appears to be constitutively translated but inefficiently translocated into the lumen of the endoplasmic reticulum. Using several experimental approaches, we found that apoB is bound to the cytosolic chaperone protein, heat shock protein 72/73 (commonly referred to as Hsp70). Similar to other chaperone-protein interactions, this binding was transient and ATP-sensitive. The binding of apoB to Hsp70 in HepG2 cells was decreased by treatment with oleic acid, which increases both translocation and secretion of apoB, and was increased by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor which efficiently protects apoB from cellular degradation without affecting translocation. The N-terminal 16% of apoB, which is efficiently translocated into the endoplasmic reticulum lumen in stably transfected Chinese hamster ovary (CHO) cells, showed minimal, if any, binding to Hsp70. The N-terminal 50% of apoB, which is very poorly translocated in CHO cells, was found to bind significantly to Hsp70. These results suggest that domains of nascent apoB localized on the C-terminal regions of the molecule are transiently exposed to the cytosol during translation and/or translocation, and that Hsp70 functions as a molecular chaperone to maintain apoB in a translocational competent conformation until translocation is completed.

Adenosine Triphosphate↗
PubMed1995

A new crystal form of bovine heart ubiquinol: cytochrome c oxidoreductase: determination of space group and unit-cell parameters.

Ubiquinol:cytochrome c oxidoreductase, the middle segment of the mitochondrial respiratory chain, is a multi-subunit transmembrane redox enzyme. The purified protein from beef heart mitochondria has been crystallized by three groups in three different forms, but progress toward a structure has been hampered by the limited order (resolution) of the crystals. It has been found that under certain conditions the enzyme crystallizes in a new form suitable for X-ray diffraction studies. These crystals belong to space group C222(1) in the orthorhombic system. The cell dimensions are a = 384, b = 118 and c = 177 A. These new crystals at present diffract to 3.8 A at best. This is not significantly better than hexagonal [P6(1(5))22] crystals grown, but the new crystals have the advantage of less spot overlap because of face-centered packing which results in systematic extinctions. More importantly, the availability of the same enzyme in multiple crystal forms may allow phase refinement and extension by the method of molecular replacement.

Journal Article↗
PubMed1995

LET-23-mediated signal transduction during Caenorhabditis elegans development.

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.

Animals↗
PubMed1995

apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes.

apo B is a structural constituent of several classes of lipoprotein particles, including chylomicrons, VLDL, and LDL. To better understand the role of apo B in the body, we have used gene targeting in embryonic stem cells to create a null apo B allele in the mouse. Homozygous apo B deficiency led to embryonic lethality, with resorption of all embryos by gestational day 9. Heterozygotes showed an increased tendency to intrauterine death with some fetuses having incomplete neural tube closure and some live-born heterozygotes developing hydrocephalus. The majority of male heterozygotes were sterile, although the genitourinary system and sperm were grossly normal. Viable heterozygotes had normal triglycerides, but total, LDL, and HDL cholesterol levels were decreased by 37, 37, and 39%, respectively. Hepatic and intestinal apo B mRNA levels were decreased in heterozygotes, presumably contributing to the decreased LDL levels through decreased synthesis of apo B-containing lipoproteins. Kinetic studies indicated that heterozygotes had decreased transport rates of HDL cholesterol ester and apo A-I. As liver and intestinal apo A-I mRNA levels were unchanged, the mechanism for decreased apo A-I transport must be posttranscriptional. Heterozygotes also had normal cholesterol absorption and a normal response of the plasma lipoprotein pattern to chronic consumption of a high fat, high cholesterol, Western-type diet. In summary, we report a mouse model for apo B deficiency with several phenotypic features that were unexpected based on clinical studies of apo B-deficient humans, such as embryonic lethality in homozygotes and neural tube closure defects, male infertility, and a major defect in HDL production in heterozygotes. This model presents an opportunity to study the mechanisms underlying these phenotypic changes.

Alleles↗
PubMed1994

The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development.

During Caenorhabditis elegans vulval development, an inductive signal from the anchor cell stimulates three of the six vulval precursor cells (VPCs) to adopt vulval rather than nonvulval epidermal fates. Genes necessary for this induction include the lin-3 growth factor, the let-23 receptor tyrosine kinase, and let-60 ras. lin-15 is a negative regulator of this inductive pathway. In lin-15 mutant animals, all six VPCs adopt vulval fates, even in the absence of inductive signal. Previous genetic studies suggested that lin-15 is a complex locus with two independently mutable activities, A and B. We have cloned the lin-15 locus by germline transformation and find that it encodes two nonoverlapping transcripts that are transcribed in the same direction. The downstream transcript encodes the lin-15A function; the upstream transcript encodes the lin-15B function. The predicted lin-15A and lin-15B proteins are novel and hydrophilic. We have identified a molecular null allele of lin-15 and have used it to analyze the role of lin-15 in the signaling pathway. We find that lin-15 acts upstream of let-23 and in parallel to the inductive signal.

Amino Acid Sequence↗
PubMed1994

[Memory-improving effect of aqueous extract of Astragalus membranaceus (Fisch.) Bge].

The study shows that the aqueous extract of Astragulus membranaceus (AMWE) can improve the anisodine-induced impairment on memory acquisition as well as the alcohol-elicited deficit of memory retrieval (number of errors were reduced and latent period was prolonged) in step down, and can also prolong the gasping duration of mice after decapitation at doses of 50g/kg (ig) for 7 days.

Animals↗
PubMed1994

Alternative polyadenylation of apolipoprotein B RNA is a major cause of B-48 protein formation in rat hepatoma cell lines transfected with human apoB-100 minigenes.

The human apoB gene encodes an mRNA of 14121 nucleotides. In liver the apoB gene products a full-length mature protein of 4,536 amino acids (B-100), whereas in the intestine this gene produces a truncated protein of 2,152 amino acids (B-48). B-48 results from RNA editing of nucleotide 6666 from C to U, thereby producing a stop codon at position 2153. Rat liver has been shown to contain apoB RNA editing capability resulting in production of both B-100 and B-48. To create an in vitro expression system for human B-100, a minigene with a wild type coding sequence for the entire B-100 protein (B-100/Gln) was stably transfected into rat hepatoma cells (McA-RH7777). Similarly, a minigene with mutation at nucleotide 6667 that allowed translation even after editing of nucleotide 6666 (B-100/Leu, nonstop mutant), a minigene with an additional nonsense mutation at nucleotide 7053 to produce B-50 (B-50/Leu), and a truncated wild type minigene with a stop signal at codon 3261 to produce B-74 and an mRNA of 10 kb (B-74/Gln) were also transfected. Very little full-length B-100 and B-74 was produced by any of the respective constructions, including the B-100/Leu with the nonstop mutation. Transfection with B-100/Gln, B-100/Leu and B-74/Gln constructions produced greater than 90% of apoB as B-48, whereas the B-50/Leu construction produced 76% B-50 and 24% B-48. The inability of the B-100/Leu construction to produce B-100 suggested an explanation for B-48 production other than RNA editing. Northern blot analysis showed that the RNA produced by all four transfectants was shortened to a size of about 7 kb. A 10-kb but no 7-kb RNA was observed in the B-74/Leu construction when transfected to Chinese hamster ovary cells suggesting cell type specificity in generation of a shortened RNA. The 3' end of apoB RNA from McA-RH7777 B-100/Leu transfectants was reverse transcribed, cloned, and sequenced. This revealed two species of RNA: one polyadenylated at or near nucleotide 6775 capable of coding for B-48, the other polyadenylated at nucleotide 7080 capable of coding for B-50. In 18% of the cDNA clones, nucleotide 6666 was edited from C to T. In 6 of 34 clones, addition of the poly(A) tail after nucleotide 6774 created a TAA stop codon, whereas no stop signals could be detected in the remaining clones.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

X-ray diffraction by crystals of beef heart ubiquinol: cytochrome c oxidoreductase.

Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.

Animals↗
PubMed1991

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.

Carotenoids↗