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S H Ip

Publications and source records attributed to S H Ip.

30 records · Page 2Linked to original sources
PubMed1982

Rapid enumeration of T lymphocytes by a flow-cytometric immunofluorescence method.

Combined with current advances in microprocessor-controlled flow cytometers, monoclonal antibodies provide a rapid means of phenotyping individual cell surface markers for a large number of clinical samples accurately and reproducibly, which may provide useful information in diagnosing disease and monitoring patients. We have developed a one-step flow-cytometric immunofluorescence procedure for enumerating E-rosette lymphocytes from whole blood by using the monoclonal antibody OKT11. This antibody recognizes the sheep erythrocyte receptor on the lymphocyte surface and can block sheep E-rosette formation. The flow cytometer we use, an Ortho Spectrum III, distinguishes lymphocytes from other leukocytes by measuring the narrow forward and right-angle light-scattering properties of the cells. The instrument further differentiates T lymphocytes frm non-T lymphocytes by measuring the green fluorescence signal of the OKT11-positive lymphocytes. In a typical sample, 1500--2500 lymphocytes are counted in 25 s. In a study of 158 patient samples, ranging from 1% to greater than 90% E-rosette-positive lymphocytes, the correlation coefficient between the manual E-rosette count and the flow immunofluorescence measurement is 0.943.

Antibodies, Monoclonal↗
PubMed1982

Light scatter as an adjunct to cellular immunofluorescence in flow cytometric systems.

Optical flow cytometry has provided a rapid method for the analysis of cell surface markers to accompany the recent proliferation of monoclonal antibodies specific to subsets of peripheral blood cells. This review presents the physical principles by which cell populations are distinguished in flow cytometry, thereby comparing the complementary roles of light scatter from the interior structure of cells, fluorescence from labeled antibody, and optical interactions with enzyme labels. The role of multiple labels is also discussed. Data handling and automation are reviewed with reference to state-of-the-art commercial systems with specific reference to techniques based on whole blood samples and rapid sample preparation and analysis.

Antibodies, Monoclonal↗
PubMed1982

Evaluation of E-rosetting human lymphocytes with OKT11 and other monoclonal antibodies.

Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette-positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

Antibodies, Monoclonal↗
PubMed1981

Inhibition of sterol and phospholipid synthesis in HL-60 promyelocytic leukemia cells by inducers of myeloid differentiation.

Myeloid differentiation is induced in HL-60 promyelocytic leukemia cells by dimethyl sulfoxide, retinoic acid, hypoxanthine, and a number of other chemical agents. We questioned whether the induction process was associated with changes in lipid synthesis. With [14C]acetate, a precursor for all cell lipids, a decrease in sterol and phospholipid synthesis (but not triglyceride synthesis) was observed within the first 5 hr after exposure to inducer, a time prior to inhibition of DNA synthesis or cessation of cell growth. Similarly, the membrane fraction of HL-60 cells exhibited decreased incorporation of newly synthesized lipid. Synthesis of phosphatidylcholine from choline as well as from the transmethylation of membrane phosphatidylethanolamine was also inhibited by myeloid inducers. In contrast, neither sterol nor phospholipid degradation was stimulated under these conditions. Both cholesterol and lanosterol were synthesized by growing HL-60 cells, but cholesterol esters were not. Synthesis of sterols was subject to feedback inhibition by cholesterol in the medium, but such feedback inhibition did not affect differentiation in the presence of myeloid inducers and did not alter the effect of myeloid inducers on phospholipid synthesis. Removal of dimethyl sulfoxide at 16 hr permitted a return to normal lipid synthesis and prevented differentiation, whereas removal of dimethyl sulfoxide at 40 hr was followed by continued inhibition of lipid synthesis and progressive differentiation. These studies demonstrate that the induction of myeloid differentiation is associated with an early inhibition of the synthesis of those lipids which are normally a part of cell membranes.

Acetates↗
PubMed1981

Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukemia cells (HL60) treated with chemical inducers.

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.

Animals↗
PubMed1977

Thermodynamic studies on subunit assembly in human hemoglobin. Temperature dependence of the dimer-tetramer association constants for oxygenated and unliganded hemoglobins.

Equilibrium constants for dimer-tetramer association of oxygenated human hemoglobin have been determined as a function of temperature, by analytical molecular sieve chromatography, yielding a van't Hoff enthalpy of 3.8 +/- 1.6 kcal/mol, and a unitary entropy of 48.4 +/- 5.7 e.u. Under the same conditions (0.1 M Tris/HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4) the equilibrium constants for dimer-tetramer association of unliganded hemoglobin have been determined as a function of temperature from kinetic studies of the forward and reverse rate constants. The derived enthalpy and unitary entropy for association of unliganded hemoglobin are 0deltaH2 = 28.9 +/- 0.5 kcal/mol and 0deltaS2 = -41.8 +/- 1.7 e.u., respectively. Thus the oxygenation-linked enthalpy of subunit association is 32.7 +/- 1.7 kcal. The corresponding entropic coupling is 90.2 +/- 5.9 e.u. The thermodynamic parameters are consistent with an increased role of hydrophobic interactions within the dimer-dimer contact region upon oxygenation, or a decreased role of hydrogen bonds and ion pair interactions. The opposite signs for association entropies in unliganded and oxygenated states suggest the existence of two distinct subunit interaction processes during the course of oxygenation with a reversal in sign at some particular binding step. The results provide a necessary basis for interpretation of calorimetric results on reconstitution of hemoglobin, described in an accompanying paper.

Hemoglobin A↗
PubMed1976

Kinetics of deoxyhemoglobin subunit dissociation determined by haptoglobin binding: estimation of the equilibrium constant from forward and reverse rates.

Deoxyhemoglobin tetramers dissociate into dimers very slowly, with half-times on the order of several hours. It is demonstrated that absorbance changes in the Soret region which accompany this dissociation and persist upon binding of haptoglobin 1-1 to the dissociated dimers can be used for accurate kinetic determinations over the necessarily long periods required for study. This method of study for the slow reactions depends upon long-term spectral integrity of the reaction mixtures and upon accurate measurement. The variation in rate constants determined by this procedure has been correlated with variations in structural constraints at the dimer-dimer contact region. In the presence of 2,3-diphosphoglycerate the rate constant is decreased, consistent with the role of this effector in binding to both beta chains and stabilizing the constrained deoxy tetramer against dissociation into alphabeta dimers. With hemoglobin specifically modified (des-Arg-141alpha) to eliminate half the constraining salt links within the dimer-dimer contact region, the dissociation rate is increased by approximately three orders of magnitude. In hemoglobin S where the amino acid substitution is not directly in the intersubunit contact region of interest, the dissociation rate is found to be approximately the same as that for hemoglobin A. Combination of the dissociation rate constants determined by haptoglobin binding with stopped-flow determinations of the rate constant for reassociation of dissociated dimers provides an estimate of the equilibrium constant, 0K2, for the deoxyhemoglobin dimer-tetramer equilibrium. This estimate is independent of any assumptions regarding other energetic quantities, and yields a value of 2.54 +/- 0.7 X 10(10)M-1 (heme) in 0.1 M Tris-HCl, 0.1 M NaCl, and 1 mM EDTA, pH 7.4, 21.5 degrees C. Thus the intersubunit contact energy is -14.0 +/- 0.2 kcal/mol of heme. The stabilization energy between deoxy and oxy tetramers is found to be approximately 6.4 kcal/mol, under these conditions.

Binding Sites↗
PubMed1976

Equilibrium gel permeation: a single-photon counting spectrophotometer for studies of protein interaction.

When a small column or flow cell packed with gel particles is completely saturated with a solution containing molecular species of interest, the average cross-sectional area occupied by the solute (partition cross section) is conveniently and precisely determined by direct optical scanning. For a mixture of interacting solutes this equilibrium gel permeation measurement yields the weight average of the species partition cross sections and the variation of this quantity with solute concentration permits determination of the solute interaction parameters (stoichiometry, equilibrium constants). We have developed a computer-controlled single-photon counting spectrophotometer for these measurements. The instrument exhibits high precision over a wide range of optical density. With counting times in the range of 10-1000 s the standard deviations on optical densities of protein solutions measured at 220 nm are typically 0.0006 at 1 OD, 0.002 at 2 OD, 0.005 at 4 OD. Beer's law tests show that deviations from linearity are less than these precision limits. Partition cross-section measurements for proteins can be made with an accuracy of better than 0.001 and information can be obtained with protein solutions at least as low as 1 mug/ml.

Binding Sites↗
PubMedDate not supplied

Monoclonal antibodies to T cell receptor gamma/delta complex react with human endometrial glandular epithelium.

We have tested a panel of four monoclonal antibodies to the T cell receptor (TCR) gamma/delta heterodimer (delta TCS-1, TS-8, TCR delta-1, anti- C gamma m 1) in pregnant and non-pregnant uteri. The TCR gamma/delta complex was not detected on stromal lymphocytes, but was localized in the cytoplasm of the endometrial glandular epithelium from most pregnant uteri. These antibodies also reacted with endometrial glandular epithelium in a majority of non-pregnant uteri; the reactivity was more consistent in secretory phase glands than that in the proliferative phase of menstrual cycle. However, three different monoclonal antibodies to CD3 (OKT3, leu 4, UCHT-1) failed to show any reactivity, suggesting that the presence of the TCR gamma/delta complex was not associated with CD3 complex. The TCR gamma/delta-positive glandular epithelial cells did not react with two monoclonal antibodies to the TCR alpha/beta complex, and they were also CD4- and CD8-negative. Together with the loss of the class 1 MHC antigens from these endometrial glandular epithelial cells in early pregnancy, these data suggest that these TCR gamma/delta-bearing endometrial glandular cells may undergo phenotypic alterations under local regulation of gene expression.

Antigens, CD↗