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Biomedical subjects

J L Weaver

Publications and source records attributed to J L Weaver.

At least 19 recordsLinked to original sources
PubMed1998

A semiautomated fluorescence-based cell-to-cell fusion assay for gp120-gp41 and CD4 expressing cells.

A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4+ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120-gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4+ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cell-to-cell fusion.

Antibodies, Monoclonal↗
PubMed1998

Nuclear magnetic resonance structural and ligand binding studies of BLBC, a two-domain fragment of barley lectin.

Plant lectins are useful targets for biophysical studies of protein-carbohydrate recognition, a process of general interest because of its many roles in human physiology. Here, nuclear magnetic resonance (NMR) based structural and carbohydrate binding data on a two-domain fragment of the normally four-domain barley lectin protein are presented. The structural data, while preliminary, clearly shows that the recombinantly produced simplified model system, called BLBC, retains a nativelike fold. However, unlike the full-length parent protein, which is dimeric, BLBC is shown by pulsed-field gradient NMR diffusion studies to be largely monomeric. Still, the fragment retains nativelike carbohydrate binding properties. These properties are examined in some detail using heteronuclear single quantum coherence (HSQC) NMR spectroscopy on a uniformly 15N-labeled sample. Ligand-induced chemical shift changes in the 1H-15N HSQC spectrum are monitored as 15N-labeled BLBC is titrated with increasing concentrations of the unlabeled carbohydrate, N,N',N"-triacetylchitotriose. Well-resolved resonances from the individual domains show that BLBC binds ligand at two distinct and independent ligand binding sites, one in each domain. Binding constants of (1.1 +/- 0.2) x 10(3) M-1 and (0.6 +/- 0.2) x 10(3) M-1 are determined for the B and C domain sites, respectively. These results are discussed in relation to ligand binding studies that have previously been carried out on a highly homologous protein, wheat germ agglutinin.

Amino Acid Sequence↗
PubMed1998

Hydrogen bonding geometry of a protein-bound carbohydrate from water exchange-mediated cross-relaxation.

We present heteronuclear two-dimensional methods for the analysis of the geometry of exchangeable protons on a protein-bound carbohydrate. By using a water-selective NOESY-HSQC, we observed cross-relaxation between carbohydrate hydroxyl protons and non-exchangeable ring protons in the complex of [13C6]-alpha-methyl-D-mannopyranoside with recombinant rat mannose binding protein. Using a simple kinetic model, we were able to explain the differences in the initial slopes of the resulting cross-relaxation buildup curves in terms of the geometry of the hydroxyl protons in the bound state. The hydroxyl rotamers consistent with our cross-relaxation data fit very well with predictions based on the crystal structure of MBP bound to a mannose-rich oligosaccharide. These methods should be applicable to other systems where both ligand exchange and water exchange are fast relative to the rate of cross-relaxation.

Animals↗
PubMed1997

Inhibition of HIV infection of H9 cells by chlorpromazine derivatives.

The binding between the HIV surface protein, gp120, and the CD4 coreceptor is known to be initiated by electrostatic interactions. Because of the ability of chlorpromazine to interact with proteins by charge transfer, we tested several derivatives for their ability to block binding of HIV to CD4+ cells. We have shown that 7,8-dioxo-chlorpromazine blocks binding of fluorescein isothiocyanate-labeled anti-Leu3a and rgp120 to peripheral human blood T4 cells and blocks syncytia formation between gp120- and CD4-expressing cells. We also found that 7,8-dioxo-chlorpromazine blocks HIV infectivity of H9 cells and acts synergistically with zidovudine.

Anti-HIV Agents↗
PubMed1996

Determination and application of vision standards in industry.

Job-related vision standards benefit both employee and employer. By comparing employees' vision to specific job requirements, workers can be better placed into positions commensurate with their visual abilities. For a company without a vision care program, a project was undertaken to determine visual standards, and the need for vision care services for employees to better meet these standards. The critical factors for performing specific visual tasks for 40 job classifications were identified through direct observation and measurement, and through worker interviews. Additionally, a sample of employees was screened to determine plant population characteristics, and to determine what percentage of employees met the newly formed standards. This study can serve as a model for the application of visual standards to the workplace and is pertinent, as "reduced vision" is increasingly defined by government as a disability.

Adult↗
PubMed1996

MDR1/P-glycoprotein function. I. Effect of hypotonicity and inhibitors on rhodamine 123 exclusion.

The MDR1 protein (P-glycoprotein) is a membrane ATPase whose expression results in resistance to several anti-tumor drugs. It has been proposed that the MDR1 protein, in addition to its pumplike properties, can function as (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54:618-622, 1994) or mediate the activity of (Hardy et al. EMBO J. 14: 68-75, 1995) a hypotonic stress-induced Cl- current. In addition, one study found that drug transport and Cl- channel-associated functions of MRD1 were separable and mutually exclusive and that, when cells were swelled, the MDR1 protein could not transport substrate. This hypothesis was tested in four pairs of isogenic cell lines with MDR1 transfectants expression 8,000-55,000 MDR1 antibody binding sites per cell. Cytoplasmic exclusion of rhodamine 123 was used as an indicator of MDR1 function to measure the effect of hypotonic stress, MDR1 inhibitors, and Cl- channel blockers on MRD1 transport function. It was found that MDR1 activity and its inhibition by cyclosporine A or flufenamic acid were unaffected by hypotonicity alone or in combination with Cl- channel blockers.

3T3 Cells↗
PubMed1996

MDR1/P-glycoprotein function. II. Effect of hypotonicity and inhibitors on Cl- efflux and volume regulation.

Resistance to anti-tumor drugs can be mediated by overexpression of the multidrug resistance 1 (MDR1) protein (P-glycoprotein). In three MDR1-transfected cell lines (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54: 618-622, 1994), a hypotonic stress-induced Cl- current has been demonstrated that can be inhibited by MDR1 substrates and Cl- channel blockers. We tested the hypothesis that MDR1 expression confers additional Cl- conductance by measuring regulatory volume decrease (RVD) in four pairs of isogenic cell lines and 36Cl efflux in two cell lines with and without hypotonic stress. The kinetics of RVD and response to Cl- channel blockers were indistinguishable in MDR and parental cells. Additionally, no significant difference was seen between 36Cl efflux rate constants under hypotonic conditions between NIH/3T3 and L1210 parental and MDR cells. We conclude that, in intact cells, the expression of MDR1 does not alter the rate of volume regulation or the rate 36Cl efflux under hypotonic conditions between parental and MDR cells.

3T3 Cells↗
PubMed1995

Cross-linking of CD4 in a TCR/CD3-juxtaposed inhibitory state: a pFRET study.

Instances when T cell activation via the T cell receptor/CD3 complex is suppressed by anti-CD4 Abs are generally attributed either to the topological separation of CD4-p56lck from CD3, or their improper apposition. Photobleaching fluorescence resonance energy transfer measurements permitted direct analysis of these alternatives on human peripheral blood lymphocytes. Distinction between changes of relative antigen densities or positioning was made possible by simultaneously recording donor and acceptor fluorescence in the energy transfer experiment performed on homogeneous populations of flow-sorted cells. We show here that CD4 stays in the molecular vicinity of CD3, while anti-CD3 stimulation is suppressed by anti-CD4 or cross-linked HIV gp120. Our data suggest that cross-linking of CD4 through particular epitopes is capable of inhibiting activation driven by Abs binding to specific sites on CD3 without major topological sequestration of the Ags, in such a way that additional positive signals will also be affected. Thus, these and other related cases of negative signaling via CD4 may be interpreted in terms of functional uncoupling rather than a wide physical separation of CD4 from the T cell receptor-CD3 complex.

Biophysical Phenomena↗
PubMed1995

Hematologic and serum chemistry values of captive Canadian lynx.

We present baseline values for 12 hematologic and 17 serum chemistry parameters collected from 22 captive lynx (Felis lynx canadensis) in December 1992, at Ronan, Montana (USA). There were no significant differences in hematologic parameters between yearlings and adults or between sexes. Lynx originally captured in the wild had significantly higher mean (+/- SE) counts of neutrophils (7.7 +/- 0.37 x 10(3) versus 7.2 +/- 0.35 x 10(3)) and lower counts of lymphocytes (1.1 +/- 0.05 x 10(3) versus 1.6 +/- 0.08 x 10(3)) compared to lynx born and raised in captivity. Yearling lynx had significantly higher values for alkaline phosphatase than adults (51.0 +/- 6.0 IU/l versus 17.5 +/- 0.8 IU/l.

Aging↗
PubMed1994

Cytochalasin D modulates CD4 crosslinking sensitive mitogenic signal in T lymphocytes.

It has previously been shown that crosslinking of the CD4 molecule, either with anti-Leu3a mAb or with gp120 (the HIV coat protein) plus anti-gp120 mAb, suppresses activation induced by wt31, a TcR/CD3-specific mAb. This suppression was associated with hindrance of the necessary association of the p56lck kinase bearing CD4 molecule with the TcR/CD3 complex. In this paper we demonstrate that this crosslinking-induced suppression can be bypassed by perturbing the microfilament system of CD4+ cells by pretreatment with 1 microM cytochalasin D. Using the fluorescence resonance energy transfer method, we have shown that the cytochalasin D-affected increase of mitogenesis is not due to changes in the TcR/CD3 to CD4 distance. Likewise, other membrane biophysical parameters, membrane potential and lateral diffusion of surface receptors, cannot be associated with these cytochalasin D-affected mitogenic changes. Cytochalasin D treatment elevates intracellular Ca2+ levels induced by wt31 mAb plus crosslinking and generates a TcR/CD3-dependent signal which is cyclosporin sensitive.

Antibodies, Monoclonal↗
PubMed1994

The L-selectin (Leu8) molecule is associated with the TcR/CD3 receptor; fluorescence energy transfer measurements on live cells.

Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-Leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecules.

CD3 Complex↗
PubMed1994

The interaction of immunosuppressive compounds in tandem stimulated peripheral human lymphocytes.

We have developed an in vitro system to model the interactions of drugs used to treat transplant rejection. This system consists of stimulation of human lymphocytes with a primary mitogen (anti-T-cell receptor complex antibodies (OKT3 or wt31)) and treatment with a primary immunosuppressive drug (ISD) (Cyclosporine A (CsA) or FK-506)). This is later followed by stimulation with a secondary mitogen (Interleukin-2 or anti-CD28), and treatment with a second ISD. This system allows a variety of concentrations and compounds to be rapidly tested. We have used this system to study the effect of various compounds when used as either primary or secondary ISDs. Our results show that when CsA is used as the primary ISD, further proliferation can be inhibited by rapamycin, mycophenolic acid, or suramin. When FK-506 is the primary ISD, inhibition of proliferation by rapamycin is variable depending on the primary and secondary mitogens. If rapamycin is the primary ISD, both CsA and FK-506 show antagonistic interactions. These results suggest that the order in which combinations of ISDs are administered in transplantation may have significant effects on the clinical outcome.

Antibodies, Monoclonal↗
PubMed1993

The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drug transporter.

The MDRI protein is an energy-dependent transport protein responsible for the multi-drug resistance seen in many tumors. A variety of drugs have been shown to inhibit the function of this pump, including compounds known to block various ion channels. The mouse lymphoma cell line L5178Y has been transduced with the human mdrI gene. Using this cell line, we have tested a number of compounds to determine whether there is a correlation between the ability to block a specific type of ion channel, or shift membrane potential, and the ability to act as an MDR-reversing agent using the fluorescent substrates Rhodamine 123 and daunorubicin as test compounds. Our results show no apparent correlation between the ability to block a specific ion channel and reversal of MDR transport ability. We have found active MDR inhibitors in compounds that affect K+, Na+, Ca++, H+, but not Cl- channels. Our data suggest that Cl- channel activity may be distinct from MDR activity. Several immunosuppressive compounds and analogs were also tested and found to be active reversing agents. Measurements suggest a significant difference in resting membrane potential between the L5178YvMDR line and the L5178Y parental cell line used in these experiments. No correlation was found between the ability of drugs to alter membrane potential and to inhibit MDR transport activity. Our results suggest that MDR transport function may be independent of the physiological movement of ions and show that a wide variety of compounds can inhibit MDR transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Specific disengagement of cell-bound anti-LAM-1 (anti-L-selectin) antibodies by aurintricarboxylic acid.

Brief treatment of human peripheral blood lymphocytes with the potential anti-HIV compound aurintricarboxylic acid (ATA) prompts the selective release of already bound L-selectin-specific anti-Leu8 and anti-LAM1-1 antibodies from the cells. Two other anti-LAM1 antibodies, anti-LAM1-3 and anti-LAM1-5 stay antigen-bound at the same time. Interestingly, the ATA-sensitive anti-Leu8 strongly competes with the ATA-resistant anti-LAM1-3 for binding. Photobleaching fluorescence resonance energy transfer (pFRET) measurements on flow-sorted cells suggests that these two antibodies compete for the same epitope, while anti-LAM1-5-FITC and anti-Leu8-PE bind to distinct sites, although they also compete for binding. Combining the data on competition, pFRET and ATA effect, we suggest that the ATA sensitive anti-Leu8 and resistant anti-LAM1-3 bind to overlapping but non-identical epitopes. This remarkably specific effect may be exploited for designing anti-inflammatory drugs that modulate leukocyte adhesion.

Antigen-Antibody Reactions↗
PubMed1992

CD4 changes conformation upon ligand binding.

Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with > or = 1 micrograms/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its Ag.

Antibodies, Monoclonal↗
PubMed1992

Prevention of binding of rgp120 by anti-HIV active tannins.

Several tannins with anti-HIV activity have been described previously (Nonaka et al., J Nat Prod 53: 587-595, 1990). We have shown that the tannins chebulinic acid and punicalin were able to block the binding of HIV rgp120 to CD4. These compounds were not toxic to stimulated human peripheral blood lymphocytes at concentrations ten times above their maximal effective concentration.

Antiviral Agents↗