Biomedical subjects

W Lin

Publications and source records attributed to W Lin.

At least 199 records · Page 11Linked to original sources

PubMed1996

Pharmaceutical fingerprinting: evaluation of neural networks and chemometric techniques for distinguishing among same-product manufacturers.

The present study was undertaken to evaluate several computer-based classifiers as potential tools for pharmaceutical fingerprinting by utilizing normalized data obtained from HPLC trace organic impurity patterns. To assess the utility of this approach, samples of L-tryptophan (LT) drug substance were analyzed from commercial production lots of six different manufacturers. The performance of several artificial neural network (ANN) architectures was compared with that of two standard chemometric methods, K-nearest neighbors (KNN) and soft independent modeling of class analogy (SIMCA), as well as with a panel of human experts. The architecture of all three computer-based classifiers was varied with respect to the number of input variables. The ANNs were also optimized with respect to the number of nodes per hidden layer and to the number of hidden layers. A novel preprocessing scheme known as the Window method was devised for converting the output of 899 data entries extracted from each chromatogram into an appropriate input file for the classifiers. Analysis of the test set data revealed that an ANN with 46 inputs (i.e., ANN-46) was superior to all other classifiers evaluated, with 93% of the chromatograms correctly classified. Among the classifiers studied in detail, the order of performance was ANN-46 (93%) > SIMCA-46 (87%) > KNN-46 (85%) = ANN-899 (85%) > "human experts" (83%) > SIMCA-899 (78%) > or = ANN-22 (77%) = KNN-22 (77%) > or = KNN-899 (76%) > SIMCA-22 (73%). These results confirm that ANNs, particularly when used in conjunction with the Window preprocessing scheme, can provide a fast, accurate, and consistent methodology applicable to pharmaceutical fingerprinting. Particular attention was paid to variations in the HPLC patterns of same-manufacturer samples due to differences in LT production lots, HPLC columns, and even run-days to quantify how these factors might hinder correct classifications. The results from these classification studies indicate that the chromatograms evidenced variations across LT manufacturers, across the three HPLC columns and, for one manufacturer, across lots. The extent of column-to-column variations is particularly noteworthy in that all three columns had identical specifications with respect to their stationary-phase characteristics and two of the three columns were from the same vendor.

Chemistry, Pharmaceutical↗

PubMed1996

Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.

Animals↗

PubMed1996

PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp.

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.

Bacterial Proteins↗

PubMed1996

An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys.

To explore the roles played by specific human immunodeficiency virus type 1 (HIV-1) genes in determining the in vivo replicative capacity of AIDS viruses, we have examined the replication kinetics and virus-specific immune responses in rhesus monkeys following infection with two chimeric simian/human immunodeficiency viruses (SHIVs). These viruses were composed of simian immunodeficiency virus SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rep. Virus replication was assessed during primary infection of rhesus monkeys by measuring plasma SIVmac p27 levels and by quantifying virus replication in lymph nodes using in situ hybridization. SHIV-HXBc2, which expresses the HIV-1 env of a T-cell-tropic, laboratory-adapted strain of HIV-1 (HXBc2), replicated well in rhesus monkey peripheral blood leukocytes (PBL) in vitro but replicated only to low levels when inoculated in rhesus monkeys. In contrast, SHIV-89.6 was constructed with the HIV-1 env gene of a T-cell- and macrophage-tropic clone of a patient isolate of HIV-1 (89.6). This virus replicated to a lower level in monkey PBL in vitro but replicated to a higher degree in monkeys during primary infection. Moreover, monkeys infected with SHIV-89.6 developed an inversion in the PBL CD4/CD8 ratio coincident with the clearance of primary viremia. The differences in the in vivo consequences of infection by these two SHIVs could not be explained by differences in the immune responses elicited by these viruses, since infected animals had comparable type-specific neutralizing antibody titers, proliferative responses to recombinant HIV-1 gp120, and virus-specific cytolytic effector T-cell responses. With the demonstration that a chimeric SHIV can replicate to high levels during primary infection in rhesus monkeys, this model can now be used to define genetic determinants of HIV-1 pathogenicity.

Animals↗

PubMed1996

In vivo regional cerebral blood volume: quantitative assessment with 3D T1-weighted pre- and postcontrast MR imaging.

PURPOSE: To assess the use of pre-and postcontrast three-dimensional (3D) T1-weighted images to obtain quantitative regional cerebral blood volume (rCBV) maps and to delineate the vascular system of the brain. MATERIALS AND METHODS: Sagittal 3D pre-and postcontrast T1-weighted images were acquired in 10 patients and two volunteers. The images were processed with a knowledge-based segmentation algorithm to obtain independent blood volume maps of gray matter (GM), white matter (WM), and blood vessels and to quantitate rCBV in various cortical and deep cortical structures. Localized maximum intensity projection (MIP)-rCBV images were used to reveal the brain's venous system and vascularity in and around any lesions present. RESULTS: Group-averaged (n = 8) rCBV maps from both GM (4.80% +/- 0.37) and WM (2.02% +/- 0.14) yielded a ratio of GM-to-WM rCBV of 2.38 +/- 0.20. The ratio of rCBV between cortical and deep cortical GM was 1.15. rCBV maps showed better vascular contrast than did the postcontrast images in delineation of blood vessels and in the lesions present in two patients. A localized MIP-rCBV image had more than five times higher vascular contrast-to-noise ratio than that of the postcontrast MIP image. CONCLUSION: rCBV maps can be obtained in a standard clinical setting and can be used to reveal information on local blood volume.

Adult↗

PubMed1996

[The study of radioimmunoimaging on the nude mice bearing human ovarian carcinoma by labelling monoclonal antibody with 99mTc].

OBJECTIVE: To investigate the feasibility of radioimmunoimaging (RII) on the nude mice model by labelling anti-ovarian carcinoma monoclonal antibody (McAb, COC166-9) with 99mTc, and the possibility for clinical application. METHODS: McAb reduced by beta-mercaptoethanol was labelled directly with 99mTc. Imaging positive rate, negative rate and tumor/non-tumor (T/N) ratio were calculated. RESULTS: (1)McAb COC166-9 labelled with 99mTc reacted with ovarian carcinoma. (2) 8 nude mice models were injected with the labelled antibodies and were then imaged. In the experimental group the positive imaging rate of tumor was 100%, in the control group the negative rate was 100%. The results were the same as those of T/N ratios. (3)In two groups (10 nude mice), intraperitoneal (i.p.) or intravenous (i.v.) injection of McAb labelled with 99mTc, positive imaging rates of tumor 100%. The IV group was better than IP group in the radio-background. CONCLUSIONS: Immunoactivity of 99mTc-COC166-9 used for RII on the nude mice models was good, and the imaging effect was specific. Clinical application is promising.

Animals↗

PubMed1996

[Reasons for removing mammary prostheses in 27 patients after augmentation mammaplasty].

We have performed agumentation mammplasty on 867 patients since 1989. Twenty-seven of them had their prostheses removed for various reasons including infection, pain, rupture or exposure of the implant, capsular contracture, psychopath, etc. The authors think that the proper selection of good implants and submuscular insertion of the implant are important for reducing such complications.

Adult↗

PubMed1996

[Clinical study in epidural injection with lappaconitine compound for post-operative analgesia].

In this study, the effect and side-effect of epidural injection with lappaconitine compound for post-operative analgesia was observed. One hundred and twenty patients were randomly divided into 4 groups. Lappaconitine compound (LB) consisted of 12 mg of lappaconitine and 22.5 mg of bupivacaine, was given to group A (the group of observation), and lappaconitine 12 mg, bupivacaine 22.5 mg and morphine 2 mg to group B, C and D respectively for control. All were given by epidural injection with single blind method during post-operative pain of incision operation. Result showed that the initiating of analgesia was quicker in group A and C than that in group B and D, and the efficacy was group D > A > C > B. There was significant difference between group A and B in the above two parameters, P < 0.01 and P < 0.05. The analgisia maintenence time of single injection was D > A > B > C, that of group D was significantly longer than that of group A (P < 0.01). It indicated that the epidural injection with LB was more rapid and potent than that with lappaconitine alone in post-operative analgesia, and the former had no side-effect, it was safer than morphine.

Aconitine↗

PubMed1996

[Effect of silymarin on mouse liver damage, production and activity of tumor necrosis factor].

Tumor necrosis factor (TNF) has been well-characterized as a prominent mediator in the development of liver injury. Effects of silymarin (SB) on mouse liver damage, TNF production and activity were studied. Pretreatment with SB (25-50 mg.kg-1, i.p., bid x 3 d) before the lipopolysaccharides (LPS) injection markedly alleviated liver injury and diminished LPS-induced TNF production in Propionibacterium acnes (PA)-primed mice. SB (12.5-50 micrograms.ml-1) significantly inhibited LPS-induced TNF release from mouse peritoneal macrophage in a concentration-dependent manner. SB(12.5-100 micrograms.ml-1) was also shown to markedly reduce TNF cytotoxicity on human hepatic cell line GSG-7701 and mouse fibroblastic cell line L929 cells concentration-dependently. These results suggest that inhibition of TNF production and its actions may be involved in the mechanism of protective action of SB on liver damage.

Animals↗

PubMed1996

Effect of matrine on lipopolysaccharides/D-galactosamine-induced hepatitis and tumor necrosis factor release from macrophages in vitro.

AIM: To study the effects of matrine (Mat) on lipopolysaccharides (LPS)-induced fatal hepatitis in D-galactosamine (D-GalN)-sensitized mice and tumor necrosis factor (TNF) release from peritoneal macrophages (PMO). METHODS: Mice were pretreated with Mat (10, 50 mg.kg-1, i.p., bid x 3 d), and then injected i.p. LPS + D-GalN. Liver injury was assessed by quantifying plasma activity of alanine aminotransferase (ALT) and histopathological examination. The TNF activities in the supernatants of mouse PMO stimulated with LPS in the presence of Mat (32.5-500 mg.L-1) were monitored by the L929 target cells lytic assay. RESULTS: Mat pretreatment markedly diminished hepatic injury induced by LPS in combination with D-GalN. Mat inhibited LPS-induced TNF release from mouse PMO in vitro in a concentration-dependent manner. CONCLUSION: Mat protected the D-GalN-treated mice from the development of fatal hepatitis induced by LPS, and inhibited the LPS-induced TNF release from mouse PMO.

Alanine Transaminase↗

PubMed1996

Effects of matrine on mouse splenocyte proliferation and release of interleukin-1 and -6 from peritoneal macrophages in vitro.

AIM: To investigate the mechanisms of anti-inflammatory effect of matrine (Mat), its effects on mouse splenocyte proliferation, and release of interleukin-1 (IL-1) and interleukin-6 (IL-6) from mouse peritoneal macrophages. METHODS: Splenocyte proliferation was assayed by [3H] TdR incorporation. IL-1 and IL-6 activities were measured by thymocyte proliferation assay and B9 cell proliferation MTT colorimetric method, respectively. RESULTS: Mat (125-500 mg.L-1) obviously inhibited concanavalin A (Con A, 5 mg.L-1)- and lipopolysaccarides (LPS, 10 mg.L-1)-induced splenocyte proliferation and LPS-induced release of IL-1 and IL-6 from mouse peritoneal macrophages. CONCLUSION: Mat inhibited splenocyte proliferation and release of IL-1 and IL-6 in vitro.

Alkaloids↗

PubMed1996

[Effect of matrine on mouse hepatitis and tumor necrosis factor production induced by Propionibacterium acnes/lipopolysaccharides].

The effect of matrine (Mat) on lipopolysaccharides (LPS)-induced fatal hepatitis and tumor necrosis factor (TNF) production in Propionibacterium acnes (PA)-primed mice were studied. Mice were injected i.p. LPS (10 micrograms/mouse) 7 d after i.p. PA (0.5 ml/mouse) to induce fatal hepatitis. After i.p. LPS, serum TNF activity rose to 1657 +/- 406 kU.L-1 at 1.5 h and ALT activity increased up to 1,496 +/- 890 U.L-1 at 5 h. Six of 8 mice died within 5 h and the massive hemorrhagic necrosis of the liver was observed in all mice. Administration of Mat (10, 50 mg.kg-1, i.p., bid x 3 d) before the LPS injection markedly reduced the elevation of serum TNF and ALT activity in a dose-dependent manner, and diminished the mortality induced by LPS. Liver congestion and necrosis induced by LPS in PA-primed mice were ameliorated markedly by Mat pretreatment. Mat (62.5-250 mg.L-1) inhibited LPS-induced TNF release from PA-primed mouse peritoneal macrophage in vitro in a concentration-dependent manner. These results seggest that Mat protected PA-primed mice from the development of fatal hepatitis induced by LPS due to inhibition of TNF production.

Alkaloids↗

PubMed1995

Quantitative analysis of Grb2-Sos1 interaction: the N-terminal SH3 domain of Grb2 mediates affinity.

Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.

3T3 Cells↗

PubMed1995

Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.

The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.

Amino Acid Sequence↗

PubMed1995

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins.

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

Amino Acid Sequence↗

PubMed1995

A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.

To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Cerebral arteriovenous malformations: flow quantitation by means of two-dimensional cardiac-gated phase-contrast MR imaging.

PURPOSE: To test two-dimensional cardiac-gated phase-contrast (2DGPC) magnetic resonance (MR) imaging in the evaluation of cerebral arteriovenous malformations (AVMs) and the alterations in flow that accompany embolization therapy. MATERIALS AND METHODS: A 2DGPC sequence was used to measure flow velocity in the feeding vessels and corresponding contralateral vessels (CCVs) of seven patients (six men and one woman, 21-55 years of age; mean, 37 years) with cerebral AVMs and in the vessels of four healthy volunteers (all men; mean age, 22 years). RESULTS: In the patients, the flow in the feeding vessels exceeded the flow in the CCVs by a margin far greater than the asymmetry in flow in the volunteers. The mean reduction in flow after embolization was 54.9% +/- 11.1 in embolized feeding vessels (n = 6) and 5.3% +/- 2.8 in nonembolized vessels (n = 3). Postembolization flow enhancement was observed in the CCV (n = 2), which suggests a degree of autoregulatory dysfunction. CONCLUSION: This technique can be used to evaluate the flow patterns of AVMs and the hemodynamic changes that occur with embolization.

Adult↗

PubMed1995

Cyclic ADP-ribose. A new component of calcium signaling.

Cyclic ADP-ribose is a recently discovered metabolite of NAD that appears to function in cellular calcium signaling. The discovery that NAD glycohydrolases are bifunctional enzymes that catalyze both the synthesis and hydrolysis of cyclic ADP-ribose raises many questions concerning the mechanisms by which these enzymes function in calcium signaling. Likewise, the identification of human lymphocyte antigen CD 38 as a bifunctional NAD glycohydrolase raises interesting questions concerning the involvement of cyclic ADP-ribose mediated calcium signaling in immune function. The dementia associated with niacin deficiency has been a long-standing curiosity. This signaling mechanism may resolve questions connecting this vitamin deficiency to central nervous system (CNS) function.

ADP-ribosyl Cyclase↗