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Biomedical subjects

W M Lee

Publications and source records attributed to W M Lee.

At least 109 records · Page 6Linked to original sources
PubMed1997

Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock, and myonecrosis.

OBJECTIVE: To quantitate gelsolin concentrations in serum of patients with a variety of conditions involving actin release into the circulation. DESIGN: Prospective evaluation of sera on consecutive patients. SETTING: Metropolitan county hospital. PATIENTS: Ninety hospital patients with a variety of well-characterized diseases. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Sera were studied from patients with acute liver failure (n = 18), chronic hepatitis (n = 17), cirrhosis of varying etiology (n = 17), pancreatitis (n = 10), acute myocardial infarction (n = 10), myonecrosis due either to polymyositis or crush injuries (n = 12), and septic shock (n = 6); results were compared with sera from healthy individuals (n = 25). Gelsolin was quantified by Western blotting with monoclonal anti-gelsolin and laser densitometry. Significant reductions in mean gelsolin concentrations compared with healthy controls were observed in patients with acute liver failure (47%), myocardial infarction (69%), sepsis (51%), and myonecrosis (66%). An inverse correlation was observed between gelsolin concentration and severity of illness, as indicated by the magnitude of serum enzyme concentrations. CONCLUSIONS: Gelsolin depletion occurs in a variety of tissue injury syndromes. Depletion of actin-scavenger capacity in the presence of continued actin release may affect outcome in situations of severe organ damage.

Actins↗
PubMed1997

Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event.

Animals↗
PubMed1997

Identification, isolation, and characterization of a 41-kilodalton protein from rat germ cell-conditioned medium exhibiting concentration-dependent dual biological activities.

In this report, we describe the purification of a novel protease with dual biological actions from germ cell-conditioned medium (GCCM) where germ cells were isolated from adult rat testes using a mechanical procedure. Using multiple HPLC columns and two sequential high performance electrophoresis chromatography steps in association with an [125I]-collagen film assay to detect protease activity, a 41-kDa polypeptide (41-kDa-P) was purified to apparent homogeneity from GCCM. Partial N-terminal amino acid sequence analysis of the purified protein revealed a sequence of NH2-KYEFYEIXLL that, when compared with the existing database at Protein Identification Resource (PIR), GenBank, and BLAST revealed that this is a unique protein. The purified protein, when incubated with [125I]-testin, a Sertoli cell secretory product that is localized at the intertesticular cell junction and is resistant to tryptic digest, was found capable of hydrolyzing testin dose dependently. The proteolysis of [125I]-testin by this 41-kDa protein was inhibited by alpha2-macroglobulin (a Sertoli cell secretory product) also in a dose-dependent manner. A study on the interactions between different classes of protease inhibitors and the purified 41-kDa protein revealed that it is a serine protease. At doses ranging between 0.5 and 50 ng/ml, 41-kDa-P induced a dose-dependent inhibition of Sertoli cell secretory function using testin and clusterin as markers without any apparent proteolytic activity. However, at doses greater than 0.5 microg/ml, 41-kDa-P was found to cleave [125I]-collagen and [125I]-testin at physiological pH, indicating that this 41-kDa protein has dual biological activities whose primary action is concentration dependent. In view of the biological activities of this protease, it is postulated that this protein may be involved in facilitating germ cell migration in the epithelium.

Animals↗
PubMed1997

Rabbit sex hormone binding globulin: primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli.

Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79.0, 68.1 and 63.2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1.6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1 lambda T for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5 alpha-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (Kd) for rabbit and human SHBGs produced in E. coli were 11.1 +/- 1.1 nM and 2.1 +/- 0.6 nM respectively, and rabbit SHBG formed a less stable protein-steroid complex (t1/2 = 5 min) than human SHBG (t1/2 > 60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5'-terminal half of SHBG from one species and 3'-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.

Amino Acid Sequence↗
PubMed1997

Lymphoepithelial carcinoma of the submandibular gland--a case report.

A case of lymphoepithelial carcinoma occurring in the left submandibular salivary gland is presented. Histologic features of this rare malignancy are described in detail. This unusual carcinoma has been described frequently in Eskimos and is usually located in the parotid gland. The raised serum IgG to Epstein-Barr virus capsid antigen suggests a causal relationship between Epstein-Barr virus and this type of salivary gland carcinoma.

Adolescent↗
PubMed1996

Rhinovirus enters but does not replicate inside monocytes and airway macrophages.

Potential interactions between rhinovirus (RV) and both the airway macrophage and its precursor cell, the blood monocyte, were investigated in terms of direct binding, intracellular replication, cell survival, and cytokine production. When HeLa cell suspensions are inoculated with RV as a positive control, virus titer increases by 100-fold in the first 24 h, confirming intracellular replication. In contrast, RV titer in monocyte and macrophage suspensions steadily decreased. Despite a lack of productive RV replication, cell-associated RV RNA was detectable using a biotin-labeled cDNA probe as early as 6 h after inoculation. Direct binding of RV16 to macrophages was confirmed using radiolabeled virus, although preincubation with anti-ICAM-1 mAb did not block this interaction. Synthesis of RV RNA, as indicated by [3H]uridine incorporation in actinomycin D-treated cells, was detected in HeLa cells but not macrophages, suggesting that the viral RNA detected inside macrophages was from input virus and was not newly synthesized. RV inoculation did not adversely affect monocyte or macrophage viability. Finally, RV caused macrophage activation, as indicated by the induction of TNF-alpha secretion. These in vitro findings suggest that macrophages interact with major group RV in vivo, and raise the possibility that there is a second cellular receptor for these viruses. Furthermore, macrophages do not serve as permissive host cells during in vivo RV infection, but instead may be active participants in anti-RV immunity and RV-induced airway inflammation.

Antibodies, Monoclonal↗
PubMed1996

Management of acute liver failure.

Acute liver failure (ALF) constitutes a medical emergency requiring the prompt response of experienced clinicians. As it is relatively infrequent, and tends to evolve rapidly, decisions concerning care and prognosis must be made promptly. Determining etiology is vital since prognosis is largely determined by the cause of the illness, and the use of antidotes may be lifesaving. Estimating the severity of the liver failure is also important, because if liver transplantation is necessary, it must be undertaken quickly. No treatment thus far is better than good general care of the comatose patient, with attention to the special problems associated with acute liver failure: cerebral edema, infection, circulatory collapse. A number of issues have been debated recently, including use of prostaglandins and N-acetylcysteine for treatment of all forms of acute liver failure, and the use of extracorporeal liver assist machines, however, the efficacy of non-specific treatments for this complex syndrome has not been proven.

Drug-Related Side Effects and Adverse Reactions↗
PubMed1996

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

Amino Acid Sequence↗
PubMed1996

Identification of sequences in c-myc mRNA that regulate its steady-state levels.

The level of cellular myc proto-oncogene expression is rapidly regulated in response to environmental signals and influences cell proliferation and differentiation. Regulation is dependent on the fast turnover of c-myc mRNA, which enables cells to rapidly alter c-myc mRNA levels. Efforts to identify elements in myc mRNA responsible for its instability have used a variety of approaches, all of which require manipulations that perturb normal cell metabolism. These various approaches have implicated different regions of the mRNA and have led to a lack of consensus over which regions actually dictate rapid turnover and low steady-state levels of c-myc mRNA. To identify these regions by an approach that does not perturb cell metabolism acutely and that directly assesses the effect of a c-myc mRNA region on the steady-state levels of c-myc mRNA, we developed an assay using reverse transcription and PCR to compare the steady-state levels of human myc mRNAs transcribed from two similarly constructed myc genes transiently cotransfected into proliferating C2C12 myoblasts. Deletion mutations were introduced into myc genes, and the levels of their mRNAs were compared with that of a near-normal, reference myc mRNA. Deletion of most of the myc 3' untranslated region (UTR) raised myc mRNA levels, while deletion of sequences in the myc 5' UTR (most of exon 1), exon 2, or the protein-coding region of exon 3 did not, thus demonstrating that the 3' UTR is responsible for keeping myc mRNA levels low. Using a similar reverse transcription-PCR assay for comparing the steady-state levels of two beta-globin-myc fusion mRNAs, we showed that fusion of the myc 3' UTR lowers globin mRNA levels by destabilizing beta-globin mRNA. Surprisingly, fusion of the protein-coding region of myc exon 3 also lowered globin mRNA steady-state levels. Investigating the possibility that exon 3 coding sequences may play some other role in regulating c-myc mRNA turnover, we demonstrated that these sequences, but not myc 3' UTR sequences, are necessary for the normal posttranscriptional downregulation of c-myc mRNA during myoblast differentiation. We conclude that, while two elements within c-myc mRNA can act as instability determinants in a heterologous context, only the instability element in the 3' UTR regulates its steady-state levels in proliferating C2C12 cells.

Animals↗
PubMed1996

Steroid therapy of chronic hepatitis: characteristics associated with response in anti-hepatitis C virus-positive and -negative patients.

OBJECTIVES: The goals of this study were to examine responses to corticosteroid-containing therapy in non-B chronic hepatitis patients with different anti-hepatitis C virus (HCV), autoantibody, and biochemical test results and to determine what factors correlate with response. METHODS: Patients with a prior or current history of steroid therapy for putative autoimmune or chronic non-A, non-B hepatitis were assessed. Responses during the first 6 months of therapy were categorized as "complete" (normal aminotransferases for > or = 1 month), "partial" ( > 50% reduction), or "no response." RESULTS: Sufficient data available to permit evaluation in 32 patients. Complete responses were noted in 17, partial responses in 12, and no response in three subjects. By multivariate analysis, only absence of anti-HCV and presence of cirrhosis were independent predictors of response. Nonresponders were found to have lower scores in a proposed autoimmune hepatitis scoring system, but scores of complete and partial responders were not significantly different. Despite a lower likelihood of a complete response, 80% (12/15) of patients with multiantigen positive anti-HCV tests had either partial or complete initial responses to corticosteroid-containing therapy, and, in nine patients, aminotransferases fell to < 2 times the upper limit of normal. All 15 anti-HCV-negative patients, but only three of 15 anti-HCV-positive patients, entered complete responses that were sustained (aminotransferases < twofold abnormal) on regimens containing < 20 mg/day or prednisolone or prednisone. CONCLUSIONS: Although anti-HCV-positive patients frequently exhibit partial initial responses to immunosuppressive therapy, the absence of specific anti-HCV antibodies was better as a predictor of completeness of response than assessment of autoantibodies or degree of biochemical abnormalities.

Adrenal Cortex Hormones↗
PubMed1996

Isolated duplications of the cystic duct: case report and implications in laparoscopic cholecystectomy.

During laparoscopic cholecystectomy on an elderly patient, we were alarmed to find another "cystic" duct after division of the cystic duct, and the operation was converted to open laparotomy. This is the first documented case in the English literature of duplication of a cystic duct with two ducts joining the common hepatic duct at the same level. This and other anatomic variations are important considerations in the safe performance of laparoscopic cholecystectomy.

Cholangiopancreatography, Endoscopic Retrograde↗
PubMed1995

B7-1 and interleukin 12 synergistically induce effective antitumor immunity.

Enhanced host rejection of tumor cells is the primary goal of cancer immunotherapy and, in many murine tumor models, has been accomplished by engineering cells to express B7 costimulatory molecules or creating an environment rich in certain cytokines. We examined the effect of tumor cell B7-1 expression and administered recombinant interleukin 12 (IL-12) on the syngeneic host response to rapidly growing, poorly immunogenic SCK mammary carcinoma cells and to more slowly growing, immunogenic K1735 melanoma cells. Whereas B7-1 expression induced rejection of K1735 cells in 78% of mice, and IL-12 induced rejection in 38%, B7-1 expression induced rejection of SCK cells in only 28% of mice, and IL-12 induced rejection in none. The relative ineffectiveness of either B7-1 or IL-12 alone to induce rejection of SCK cells led us to combine the two manipulations. This resulted in rejection of SCK cells in 74% of mice and dramatically delayed tumor development in the remainder. Tumor rechallenge studies indicated that the surviving mice developed specific immunity to wild-type SCK cells. Lymphocyte subset ablation and IFN-gamma depletion studies indicated that rejection of SCK tumor cells brought about by the synergistic effects of B7-1 and IL-12 is mediated by a rapidly developing, systemic antitumor immune response that is dependent on the presence of both CD8+ and CD4+ T cells and involves IFN-gamma. Additionally, the synergistic effect of B7-1 expression and IL-12 administration is capable of inducing rejection of control SCK tumors simultaneously established in the opposite flank. The efficacy of B7-1 and IL-12 in inducing protective immunity against a poorly immunogenic, aggressive murine tumor indicates that this combination is particularly effective at producing a potent antitumor immune response that may be of therapeutic benefit.

Animals↗