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Biomedical subjects

S M Wu

Publications and source records attributed to S M Wu.

At least 91 records · Page 5Linked to original sources
PubMed1996

Low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein.

Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

Animals↗
PubMed1996

Effect of positioning on pulmonary function of newborns: comparison of supine and prone position.

The effect of positioning on pulmonary function has been previously evaluated, and the prone position has been reported to be preferable for neonates with various respiratory diseases. Studies in healthy neonates have yielded conflicting results. Using a crying pulmonary function test, we examined the effect of positioning on pulmonary function in healthy full-term neonates. Thirty-nine infants with a mean birthweight (+/- SD) of 3,140 +/- 379 g and a mean gestational age (+/- SD) of 39.8 +/- 1.6 weeks were investigated during the first 6 hours of life. Measurements were obtained in both supine and prone positions using a computerized volume-flow system. There were statistically significant decreases in crying vital capacity (CVC) and peak expiratory flow rate (PEF) in the prone compared with the supine position. However, there were no significant differences in forced expiratory flow rate at 75% (V(75)), 50% (V(55)), and 25% (V(25)) of vital capacity between the two positions. These results suggest that prone positioning decreases lung volume and increases resistance of upper airways. We conclude that healthy neonates should be in the supine posture for optimal ventilation.

Humans↗
PubMed1996

Heterogeneity of activating mutations of the human luteinizing hormone receptor in male-limited precocious puberty.

Male-limited precocious puberty (MPP) is a gonadotropin-independent disorder that occurs sporadically or is inherited in an autosomal dominant, male-limited pattern. Recent studies have identified constitutively activating missense mutations in the human luteinizing hormone receptor (hLHR) gene leading to Leydig cell activation and precocious puberty. Patients with sporadic MPP (SMPP) or with different ethnic backgrounds appear to have a greater likelihood of having novel mutations. In the current study we examined genomic DNA from two unrelated cases of SMPP of African-American descent for novel mutations of the hLHR gene. A heterozygous A to C transversion at nucleotide 1723 resulting in substitution of Leu for lle575 in transmembrane helix 6 was identified. Human embryonic kidney cells transfected with cDNA for the mutant hLHR-I575L, created by polymerase chain reaction-based mutagenesis of the wild-type (hLHR-wt) cDNA, exhibited increased basal levels of cAMP production in the absence of agonist, indicating constitutive activation. Surface expression of hLHR-I575L, as reflected by human chorionic gonadotropin binding, was diminished compared to hLHR-wt, while agonist affinity was unaffected. With the exception of two polymorphic bases, no mutation was identified within the coding sequence of the hLHR in the second case of SMPP. We conclude that I575L is a unique constitutively activating mutation that impairs cell surface expression of the receptor but does not alter agonist affinity. Furthermore, mutations of the hLHR gene causing SMPP are highly heterogeneous and may be found in regions other than exon 11 of the hLHR. Last, patients with MPP from different ethnic backgrounds are likely to have novel mutations.

Amino Acid Sequence↗
PubMed1996

The G1691A mutation of the coagulation factor V gene (factor V Leiden) is rare in Chinese: an analysis of 618 individuals.

To understand the allele frequency of the G1691A mutation of the coagulation factor V gene (factor V Leiden) in Chinese, 618 Chinese individuals, including 54 cases with venous thrombosis, were analyzed. Only one case in the control group was heterozygous for the 1691G allele and the 1691A allele. Our data suggest that the factor V Leiden is rare in Chinese.

Alleles↗
PubMed1996

Synaptic inputs mediating bipolar cell responses in the tiger salamander retina.

Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringer's solution, but in normal Ringer's it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringer's, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.

Ambystoma↗
PubMed1996

Response surface method: a novel strategy to optimize iontophoretic transdermal delivery of thyrotropin-releasing hormone.

PURPOSE: To maximize the iontophoretic transdermal delivery rate of thyrotropin-releasing hormone (TRH) facilitated by periodically monophase-pulsed current across excised skin. METHODS: The pH of the buffer, the ionic strength in the solution, the frequency of the periodically monophase-pulsed current and the current on/off ratio were chosen as the key variables. A response surface method was applied to optimize the transdermal delivery rate of TRH under different operational conditions. RESULTS: The optimum operating conditions were achieved via experimentation based on the response surface method by systematically adjusting the pH of the buffer, the ionic strength in the solution, the current amplitude, frequency and the active temporal ratio of the pulsed current. The rate of permeation of TRH crossing the skin during iontophoresis varied from two to ten-fold, depending on operating conditions. CONCLUSIONS: Only a few steps, two in this work, were needed to reach the optimal. The response surface near the region of the maximal point was thoroughly described with a quadratic function. A maximal transdermal rate of permeation of TRH, 103.2 micrograms h-1 cm-2, was obtained when the donor solution was at pH = 7.0, ionic strength = 0.037, and with a periodically monophase-pulsed current iontophoresis with duty cycle = 75%. The effect of pulse frequency was not statistically significant.

Administration, Cutaneous↗
PubMed1996

Response sensitivity and voltage gain of the rod- and cone-horizontal cell synapses in dark- and light-adapted tiger salamander retina.

1. Rods, cones, and horizontal cells (HCs) were recorded in superfused, flat-mounted isolated retinas of the larval tiger salamander, Ambystoma tigrinum, under dark- and light-adapted conditions. 2. Under dark-adapted conditions, HC responses to dim 500-nm light stimuli were mediated only by rods. In the linear voltage range (near the dark potentials), the average response to a light step of 0.5 s (500 nm, 0.438 photons per micron2 per s) was 0.41 +/- 0.06 (SD) mV for rods and 1.86 +/- 0.52 mV for HCs. The step sensitivity of rods was approximately 0.94 mV per photon micron2 s, or 0.032 mV per activated rhodopsin molecule (Rh*) rod second, and the step sensitivity of HCs was approximately 4.25 +/- 1.19 mV per photon micron2 s or 0.14 +/- 0.04 mV per Rh* rod second. The chord voltage gain of the rod-HC synapse had an average value of 4.54 and a range from 2.68 to 7.32. 3. By the use of the spectral subtraction method, we found that the average cone-mediated HC response to a 750-nm light step that elicited an average cone response of 0.73 +/- 0.20 mV was 1.15 +/- 0.31 mV. The step sensitivity of cones under dark-adapted conditions was 0.0012 mV per photon micron2 s, and that of the cone-mediated-HC response was 0.0019 mV per photon micron2 s. The chord voltage gains of the cone-HC synapses under dark-adapted conditions had an average value of 1.58 and a range from 0.82 to 2.05. 4. Under light-adapted conditions (with a 500-nm/-2.40 background light, which desensitized rod responses but did not substantially reduce the cone responses), the cones had an average response to a light step of 0.5 s (500 nm/-3.3) of 0.78 +/- 0.09 mV, and this response did not vary with time. The HC response to the same light step had an average value of 3.95 +/- 3.41 mV 3 min after the background light onset, and it increased with time until reaching a steady-state value of 5.95 +/- 3.63 mV approximately 15 min after the background light onset. The average chord voltage gain of the cone-HC synapse under such light-adapted conditions was 5.06 at 3 min after background light onset and 7.63 at 15 min after background light onset. These values are approximately 3-5 times higher than the chord voltage gain of the cone-HC synapse under dark-adapted conditions. 5. The background-induced increase of the chord voltage gain of the cone-HC synapse suggests that similarly to the rod-HC synapse, the voltage gain of the cone-HC synapse in the tiger salamander retina can also be modulated by light. Additionally, our results suggest that certain time-dependent process(es) in the synaptic cleft or postsynaptic membrane may be responsible for such modulation. 6. In addition to determining the average values of response sensitivity and chord voltage gains of the rod- and cone-HC synapses, we studied the variation of these parameters among different HCs. HCs with higher rod-HC synaptic gain had lower cone-HC synaptic gain, and HCs with lower rod-HC synaptic gain exhibited higher cone-HC synaptic gain under both dark- and light-adapted conditions. This suggests that the rod-HC and cone-HC synaptic gains in HCs are complementary to each other, and voltage responses of all HCs under dark- or light-adapted conditions are of comparable amplitudes.

Adaptation, Physiological↗
PubMed1996

Compound heterozygous mutations of the luteinizing hormone receptor gene in Leydig cell hypoplasia.

The human LH receptor (hLHR) is a member of the G protein-coupled receptors characterized by the presence of seven-transmembrane (TM) helices. Inactivating mutations of the hLHR lead to Leydig cell hypoplasia (LCH), a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cell differentiation. We have identified three mutations of the hLHR in a patient with LCH: deletion of exon 8 (delta Exon 8), A872G transition resulting in Asn291Ser substitution in the extracellular domain, and C1847A transversion resulting in Ser616Tyr substitution in the seventh TM helix. Nucleotide sequencing, gene dosage, and allele-specific amplification analyses revealed that exon 8 deletion and the two missense mutations are present in different alleles of the hLHR. Constructs of mutated hLHR (hLHR-delta Exon8, hLHR-872/1847, hLHR-1847, and hLHR-872) were used to transfect 293 cells, and the properties of the hLHR expressed were examined. Ligand-binding assays failed to detect the expression of hLHR-delta Exon8. Transfectants expressing hLHR-872/1847 demonstrated greatly reduced ligand binding and ligand-induced cAMP accumulation in comparison to those expressing wild type hLHR. Similar reduction in cAMP accumulation was observed in transfectants expressing hLHR-1847, but not hLHR-872 alone. These findings suggest that, in addition to the 7-TM helices, the polypeptide encoded by exon 8 plays an important role in LHR expression and signal transduction. On the other hand, glycosylation of Asn291 may not be critical for these activities. These results also establish that LCH can result from impaired signal transduction due to compound heterozygous mutations. Implications of these mutations on structure-function relationship of the hLHR and the genotype-phenotype correlation in LCH are discussed.

Alleles↗
PubMed1995

Rapid identification and isolation of transcriptionally active regions from mouse genomes.

We report here the design, construction and testing of a self-inactivating (Sin) retrovirus promoter-trap vector suitable for identifying and isolating transcriptionally active regions from the mouse genome. When this vector, which contains the bacterial aph gene as its reporter, is integrated into a site downstream from an active host cell promoter, it expresses aph, whose product, aminoglycoside phosphotransferase, produces resistance to the antibiotic G418 in mammalian cells. The construct also contains a native aph promoter which functions in bacteria, but not in mouse cells, to express kanamycin (Km) resistance, plus an adjacent pBR322-derived replication origin. Thus, mammalian DNA segments containing actively transcribed regions flanking aph can be quickly isolated by restriction endonuclease treatment of total DNA from provirus-containing mouse cells, followed by self-ligation, transformation and Km selection of plasmids carried by bacteria transformed with this DNA. We tested this Sin retrovirus promoter-trap system by isolating eight DNA segments upstream to the provirus integration sites in the genome of virus-infected mouse F9 cells. We found that the Sin retrovirus vector produces a high yield of infectious virus particles carrying aph, and that the isolated genomic DNA fragments of F9 cells are transcriptionally active.

3T3 Cells↗
PubMed1995

Immunoaffinity purification of DNA polymerase delta.

A monoclonal antibody against human DNA polymerase delta (pol delta) was isolated with properties suitable for its utilization for immunoaffinity chromatography. The antibody was immobilized after periodate oxidation and coupled to a hydrazide-activated support. Starting from a partially purified preparation, calf thymus pol delta was purified about 200-fold in a single step. Further purification on ssDNA-cellulose resulted in isolation of a homogeneous preparation. The amount of enzyme isolated, ca. 0.3 mg of pure pol delta from 0.75 kg of calf thymus, is about 15-fold greater than can be achieved by conventional procedures. This procedure provides a significant advance in the isolation of pol delta in allowing its facile isolation from tissues in good yield. The isolated enzyme consisted of two subunits of 125 and 50 kDa. Characterization of the enzyme showed that these two subunits remained associated on glycerol gradient ultracentrifugation even in the presence of 2.8 M urea.

Animals↗
PubMed1995

Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty.

Genomic DNA from 32 unrelated families with male-limited precocious puberty was examined for the previously described Asp-578-->Gly, Met-571-->Ile, and Thr-577-->Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR). Twenty-eight families had the inherited form of the disorder, and of these, 24 were found to have the Asp-578-->Gly mutation. Four additional mutations were found among the remaining four families with the inherited form and in four sporadic cases of the disorder: an A-->C transversion resulting in substitution of leucine for Ile-542 in the fifth transmembrane helix, an A-->G transition resulting in substitution of glycine for Asp-564 in the third cytoplasmic loop, a G-->T transversion resulting in substitution of tyrosine for Asp-578 in the sixth transmembrane helix, and a T-->C transition resulting in substitution of arginine for Cys-581 in the sixth transmembrane helix. Human embryonic kidney cells transfected with cDNAs for each of the mutant hLHRs, created by PCR-based mutagenesis of the wild-type hLHR cDNA, exhibited increased levels of basal cAMP production in the absence of agonist, indicating constitutive activation of the mutation hLHRs. Three of the additional mutations had specific features: Ile-542-->Leu and Cys-581-->Arg appeared ligand-unresponsive, whereas Asp-578-->Tyr appeared to correlate genotype with phenotype. We conclude that the region spanning nt 1624-1741 of exon 11 is a hotspot for heterogeneous point mutations that constitutively activate the hLHR and cause male-limited precocious puberty.

Amino Acid Sequence↗
PubMed1995

A nonsense mutation of the human luteinizing hormone receptor gene in Leydig cell hypoplasia.

Leydig cell hypoplasia (LCH) is a form of male pseudohermaphroditism in which Leydig cell differentiation and testosterone production are impaired. This report describes the first case of a nonsense mutation (A1635C) in exon 11 of the human luteinizing hormone receptor (hLHR) gene in two sisters with LCH. This mutation causes loss of function of the receptor by introducing a stop codon at residue 545 in transmembrane helix 5 of the hLHR. Surface expression of the truncated hLHR (hLHR-t545) in human embryonic kidney cells stably transfected with cDNA encoding hLHR-t545 was diminished compared to the wild-type hLHR and hCG-induced cAMP accumulation was impaired. These results establish that single base mutations in exon 11 of the hLHR gene can produce inactivation as well as activation of the hLHR. Furthermore, they demonstrate that functional domains between transmembrane helix 5 and the C-terminal cytoplasmic tail of the hLHR are required for normal cell surface expression of the receptor and signal transduction.

Base Sequence↗
PubMed1995

Inverse relationship between peroxisomal and mitochondrial beta-oxidation in HepG2 cells treated with dehydroepiandrosterone and clofibric acid.

A transformed human hepatoma cell line was examined to determine if it was an appropriate model system for studying the mechanism of action of two peroxisome proliferators that lower blood lipids. Cultures of HepG2 cells were exposed to four different concentrations of either the hypolipidemic drug, clofibric acid (CLO), or the adrenal steroid, dehydroepiandrosterone (DHEA). Activities of two peroxisomal enzymes, palmitoyl-CoA oxidase and catalase, and two mitochondrial enzymes, carnitine palmitoyl-CoA transferase and succinate-INT-reductase, were measured in CLO- and DHEA-treated cells. In general, as the concentration of these hypolipidemic agents increased from 0 to 1000 microM, the specific activities of peroxisomal palmitoyl-CoA oxidase and catalase increased, and mitochondrial carnitine palmitoyl-CoA transferase and succinate-INT-reductase decreased. The activity of lactate dehydrogenase was significantly higher in the medium of cultures exposed to the 500 and 1000 microM concentration of DHEA compared with the control cultures, indicating the cytotoxic effects of this steroid at millimolar levels in vitro. In summary, the peroxisomal proliferators, DHEA and CLO, inversely altered peroxisomal and mitochondrial beta-oxidation in HepG2 cultures, but not to the extent reported for rat hepatocytes in vitro. In vitro concentrations of DHEA greater than 500 microM adversely affected the viability of HepG2 cells. The results of this study suggest that beta-oxidation in this human hepatoma cell line may not be as sensitive to hypolipidemic agents as are primary cultures of rat hepatocytes.

Analysis of Variance↗
PubMed1994

Pregnancy-specific glycoprotein gene expression and the induction by 5-bromo-2'-deoxyuridine.

The pregnancy-specific glycoproteins (PSGs) of the placenta, members of the immunoglobulin superfamily, are encoded by multiple linked genes located on chromosome 19. To study the control of PSG expression, we have immortalized differentiated human placental cells (HP-A1) temperature-sensitive for transformation by a recombinant adenovirus-(ori-)-SV40 tsA mutant virus. We now show that expression of the PSG gene in HP-A1 cells is temperature-sensitive. At the permissive temperature (33 degrees C), these cells expressed low levels of PSG mRNA and synthesized a 64-kDa PSG. Shifting HP-A1 cells to a nonpermissive temperature (39.5 degrees C) increased PSG mRNA expression and biosynthesis with preferential increase in the synthesis of a 54-kDa and a low level of a 72-kDa PSG. Moreover, PSG expression was greatly induced by 5-bromo-2'-deoxyuridine (BudR), which selectively increased synthesis of PSGs of 72 and 54 kDa. In the presence of BudR, HP-A1 synthesized PSGs of 72, 64, and 54 kDa, similar to the pattern seen with placental PSGs. Ribonuclease protection assays demonstrated that HP-A1 cells express the majority of PSG mRNAs and BudR stimulated expression of PSG1 and PSG1-like transcripts. Reverse transcription and polymerase chain reaction analysis using PSG gene-specific primers demonstrated that untreated HP-A1 cells expressed primarily PSG1, PSG2, PSG4, and PSG5 mRNAs. BudR stimulated the expression of all PSG transcripts except PSG4. Moreover, in transient expression assays, BudR increased chloramphenicol acetyltransferase (CAT) expression directed by PSG1-I, PSG4, PSG5, PSG6, and PSG11 promoter-CAT fusion genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Miniature excitatory postsynaptic currents in bipolar cells of the tiger salamander retina.

The synapse between photoreceptor and bipolar cell is important for at least three reasons: (1) it is the first synapse in the visual pathway; (2) it is the best-known tonic chemical synapse; and (3) it has perhaps the most complex and highly organized synaptic morphology in the entire brain. Yet little is known about how neurotransmitter is released from this synapse. We present in this report evidence which suggests that the release of photoreceptor neurotransmitter, presumably glutamate, is probably mediated by clusters of synaptic vesicles which give rise to discrete miniature excitatory postsynaptic currents (MEPSCs) in bipolar cells. The MEPSCs are Ca(2+)-, osmotic- and CNQX-sensitive, and they share the same reversal potential (near -3 mV) as the glutamate-induced postsynaptic current. The frequency of MEPSCs increases upon presynaptic depolarization, and the mean peak conductance is about 54 pS. MEPSCs exhibit wide variations of amplitudes and durations, probably resulting from random variations of number of synaptic vesicles and the degree of synchronization in individual release clusters.

Ambystoma↗
PubMed1994

Compensation equity between men and women in academic medicine: methods and implications.

BACKGROUND: Compensation inequity by gender is a problem across occupations in the United States. Most compensation-monitoring efforts in academic medicine have been informal. The authors developed an analytic method for formal, ongoing evaluation of compensation equity in academic medicine. METHOD: A historical cohort study was conducted at Michigan State University College of Medicine using data from 1990, 1991, and 1992 to (1) evaluate methods for monitoring compensation equity, (2) test the feasibility of compensation-equity monitoring as part of administrative information systems, and (3) determine whether compensation inequity existed in a case study of faculty salaries. Internal market adjustments for specialty, clinical or basic science "type," and calendar- or academic-year appointments were made before establishing a male cohort for each female faculty member. RESULTS: The method developed appears feasible for routine administrative monitoring of compensation equity. When the compensations of women of each type and rank were compared with the compensations of their male cohorts, inequities appeared to exist for basic scientists, but not clinicians, based on a criterion of the groups' compensations being 4% or more below those of their cohorts for two successive years. CONCLUSION: The authors suggest that formal monitoring of compensation equity is an important and feasible administrative undertaking to correct historical inequities. This is an area in which leadership by U.S. medical colleges is needed.

Cohort Studies↗