PubMed Health⌕ Search

Biomedical subjects

M A Lichtman

Publications and source records attributed to M A Lichtman.

At least 19 recordsLinked to original sources
PubMed1979

An ultrasensitive method for the measurement of human leukocyte calcium: lymphocytes.

Studies of the transport and distribution of calcium in leukocytes have been severely hampered by the inability to measure accurately and reproducibly the concentration of calcium in small numbers of cells. We have applied a recent development in analytical chemistry, the graphite furnace atomic absorption spectrophotometer, to this problem. The calcium content of human blood lymphocytes was determined by both graphite furnace and conventional flame atomic absorption spectrophotometry. The linearity, sensitivity and detection limits of the two techniques were compared. For measurement of calcium, the graphite furnace sensitivity was 55 times higher in aqueous samples and 60 times higher in cell samples than the flame technique. The detection limit of the graphite furnace was 800 times lower in aqueous samples and 1500 times lower in cell samples. The enhanced sensitivity of this technique allowed us to prepare samples with 20 times fewer blood cells. We have employed this graphite furnace technique to measure lymphocyte calcium content and its relationship to the calcium concentration and proportion of serum in the suspending medium. In the absence of serum, the lymphocyte calcium content approximately doubled as the medium calcium concentration was increased from 1 mumol/l to 0.5 mmol/l. At medium calcium concentrations of 0.5 mmol/l and above, the lymphocyte calcium content was 1.0 mmol/l cells. In medium adjusted to 2 mmol/l calcium, the lymphocyte calcium content approximately doubled as the medium serum concentration was increased from 0 to 2%. At medium serum concentrations of 2% and above, lymphocyte calcium content was 2 mmol/l cells. The exchangeable cell calcium, measured with 45Ca in the same samples, did not increase as serum was added to the medium.

Calcium↗
PubMed1979

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.

Cell Membrane↗
PubMed1979

Regulation of sodium and potassium transport in phytohemagglutinin-stimulated human blood lymphocytes.

Phytohemagglutinin (PHA) or concanavalin A treatment of lymphocytes causes an increase in membrane permeability so that the leak rates of Na and K increase 1.5- to 2-fold. Active Na and K transport increase proportionately in response to the increased membrane permeability. We have examined the role of lymphocyte Na concentration in sustaining the increased Na and K transport observed after PHA treatment. Cell Na concentration increases from 14.8 to 20.5 mmol/liter cell water in PHA-treated lymphocytes (P < 0.001). Four lines of evidence suggest that the 5-6 mmol/liter cell water increase in lymphocyte Na accounts for the increase in active Na and K transport in mitogen-treated lymphocytes. First, PHA does not increase directly the maximal Na, K-ATPase activity of isolated lymphocyte membrane vesicles. Second, when the Na concentration is increased by 6 mmol/liter cell water in unstimulated lymphocytes, Na and K transport increase nearly twofold. Third, the cell Na concentration (15 mmol/liter cell water) is near the K(m) for Na activation of the Na, K-ATPase in lymphocyte membranes. The ATPase activity thus, is capable of increasing as the cell Na rises above normal. Fourth, if lymphocytes are incubated in a medium containing a low Na concentration, K transport does not maintain the internal K concentration and the fall in cell K is accentuated in PHA-treated lymphocytes. These studies indicate that the adaptive acceleration of Na and K transport in mitogen-treated lymphocytes is mediated by a small increase in cell Na.

Biological Transport↗
PubMed1979

The exceptional responsiveness of certain human myeloid leukemia cells to colony-stimulating activity.

We have studied the marrow cells from a patient with acute myeloid leukemia (AML) for their responsiveness to colony-stimulating activity (CSA) in vitro. The AML cells were stimulated by CSA to rapid and extended growth in liquid culture. In the absence of CSA, the majority of cells died. CSA also stimulated the clonal growth of AML cells, and the minimum requirement for CSA was one-tenth to one-fiftieth that required to stimulate the growth of normal marrow CFU-C. CSA for AML cells was eluted from Sephacryl S-200 columns in fractions that represented an apparent molecular weight of 45,000 daltons. This fraction also produced optimal stimulation of normal human marrow. During remission, the patient's marrow cells did not grow in liquid culture and produced normal numbers of granulocytic and erythroid colonies in response to CSA and erythropoietin. Extended culture of the AML cells resulted in cell differentiation evidenced by decreasing proliferative capacity and by morphological and histochemical changes. These studies indicate that certain AML cells are extraordinarily responsive to CSA, an in vitro mediator of normal granulopoiesis.

Adult↗
PubMed1978

Parasinusoidal location of megakaryocytes in marrow: a determinant of platelet release.

Megakaryocytopoiesis occurs in the hematopoietic (extravascular) compartment of marrow. Thus, platelets must traverse the wall of the vascular sinuses of marrow to enter the circulation. We have examined mouse and rat marrow, fixed by rapid immersion so as to maintain anatomical relationships as close to the natural state as possible. Quantitative transmission electron microscopy (TEM) of random transections of femurs established that megakaryocytes reside less than 1 mu from a marrow sinus wall with a probability unlikely to be the result of chance (P less than 0.001). An intimate relationship exists between the megakaryocyte periphery and the abluminal surface of the endothelial lining cell. At the time of platelet release megakaryocyte cytoplasm invaginates and penetrates the endothelial lining cell. The penetrating cytoplasm is detached and enters the marrow circulation. From their dimensions in comparison to circulating platelets, the released cytoplasm represents a packet of platelets that undergoes further fragmentation in the circulation. The parasinusoidal location of megakaryocytes and the process of sinus-wall penetration and platelet delivery was observed by TEM and scanning electron microscopy. These studies provided quantitative support for a specific anatomical arrangement of megakaryocytes in marrow. Moreover, the process of platelet release appears to be a physiological form of metastasis with invasion of vascular walls and vascular spread of cells, that are in this case amitotic.

Animals↗
PubMed1978

The relationships among arterial oxygen flow rate, oxygen binding by hemoglobin, and oxygen utilization in chronic cardiac decompensation.

We have examined the interrelationships among CaO2, blood flow, oxygen binding by hemoglobin, and VO2 in cardiac patients with and without chronic cardiac decompensation. We have quantified the role that decreased oxygen-binding to hemoglobin may play in maintaining VO2 in the presence of low systemic blood flow rates. The volume rate of oxygen delivery to tissues was expressed as the OFIa, the product of CO2 and blood flow. OFIa varied from 738 to 262 ml/min/m2, whereas VO2 varied from 170 to 117 ml/min/m2. Thus, in the patients with lowest OFIa (63% below the highest OFIa), VO2 was only down 19%. VO2 was maintained because the extraction of oxygen rose from about 20% to 50% in close association with the decrease in OFIa. Oxygen binding to hemoglobin was lower in patients with the lowest OFIa--and therefore, at in vivo conditions of pH, PCO2, and temperature, P50 in vivo was higher. The resulting facilitation of oxygen release at the PO2 of tissue capillaries could explain about one third of the observed increment in oxygen extraction in patients with low OFIa. An alternative interpretation is that a high P50 in vivo minimizes the reduction in PVO2 needed to maintain VO2 when increased proportional extraction of O2 compensates for decreased OFIa.

Aged↗
PubMed1978

Human cell lines that elaborate colon-stimulating activity for the marrow cells of man and other species.

We have established two human cell lines which elaborate colony-stimulating activity (CSA) for at least four species: man, mouse, rabbit, and dog. One, GCT, was isolated from a lung metastasis of a fibrous histiocytoma; the other, RC4, from a monocyte-enriched fraction of normal blood. Medium conditioned by either GCT or RC4 cells was more potent in stimulating human marrow growth in vitro than was monocyte-conditioned medium or human leukocyte feeder layers. Fractionation of cell-line-conditioned medium by Sephacryl S-200 chromatography indicated that the maximum activity of the CSA for human marrow cells is eluted within the range of 30,000-40,000 daltons. These cells lines provide a continuous source of large quantities of conditioned medium for purification of CSA. Moreover, the invariable growth-supporting activity for all species tested and the high potency of cell-line CSA facilitates studies of its elaboration and biologic effects.

Adult↗
PubMed1977

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes.

Human blood T-lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol.1 cell water-1.h-1) was half that in normal blood T-lymphocytes (20 mmol.1 cell water-1 h-1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K+ transport was 14 mmol.1 cell water-1.h-1 and treatment with PHA increased K+ transport only 30%. The intermediate values of basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data suggest that B-lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.

B-Lymphocytes↗
PubMed1977

The regulation of the release of granulocytes from normal marrow.

Figure 16 synthesizes the various aspects of marrow egress. The central anatomical relationship of the hematopoietic compartment to vascular sinus is shown above. The hatched blocks represent the sinus wall, capable of developing narrow migration channels. In the marrow, immature granulocytes alter their biophysical characteristics by developing motility, nuclear and cytoplasmic deformability and surfaces which facilitate egress. Humoral factors contribute to proliferation and maturation. Also, other humoral agents, releasing factors, may act on mature cells, for example, as cytoattractants, and on the sinus wall to reduce its adventitial cover and thereby to enhance egress. In the sinus, flow or discharge of sinus contents may be regulated by humoral agents or neural messages which may affect terminal sphincters or other structures. Although the marrow in situ is a difficult organ to study, future innovations are to be expected and our understanding of the delicate balance between hematopoietic cells, stroma and vasculature will be enhanced. Corrections of inferential errors due in part to the semi-quantitative and qualitative nature of much of our current data should be expected.

Animals↗
PubMed1976

Adaptive enhancement of amino acid uptake and exodus by thymic lymphocytes: influence of pH.

Entry of certain free amino acids (alpha aminoisobutyric acid (AIB), alanine and proline), but not of leucine into rat thymic lymphocytes increased progressively when the cells were incubated in amino acid deficient medium. Actinomycin D, cycloheximide, or a high concentration of AIB abolished the time-related increase in AIB accumulation, whereas exposure to a high concentration of leucine had no effect. This phenomenon could not be attributed to a progressive alteration in the nature of the incubation medium nor to reduced transinhibition of AIB uptake. The exodus of AIB also increased with time, but to a smaller degree than AIB entry. Initial rates of AIB entry and exodus increased with increases in the pH of the incubation medium over the range 6.5-8.0. The effects of pH on entry and exodus were time-related, increasing progressively oveb nullified the magnified time related increments in AIB transport caused by prolonged incubation at pH 8.0. The influence of a given pH on transport of AIB decreased rapidly when the cells were transferred to medium of another pH, but this tendency diminished the longer the cells were exposed to the initial pH. pH influenced the entry of alanine and proline in the same fashion as that of AIB, but did not affect leucine entry. These results indicate that thymic lymphocytes exhibit adaptive enhancement in the accumulation of free amino acids that are transported largley by the A or alanine-preferring system, and that the adaptive process involves both entry and exodus. Moreover, alterations in pH modify entry and exodus of these same amino acids, profoundly affect the magnitude of time-released increases, and may induce fundamental changes in the mechanism(s) serving amino acid transport.

Aminoisobutyric Acids↗
PubMed1976

The use of a single venous blood sample to assess oxygen binding in haemoglobin.

The measurement of pH, PO2, PCO2 and SO2 in a single venous blood sample can be used to determine the P50 at standard or at in vivo conditions. This technique makes it feasible for a physician, firstly, to make an assessment of the net adaptation of the red cell to reductions in blood oxygen content or flow and, secondly, to make an initial assessment of whether a haemoglobin with altered affinity for oxygen is present in subjects with polycythaemia or anaemia.

Acidosis↗
PubMed1976

Detection of mutant hemoglobins with altered affinity for oxygen. A simplified technique.

The detection of high- or low-affinity hemoglobins in subjects with polycythemia or anemia is difficult for most physicians because of the requirement for special equipment to do oxygen-hemoglobin dissociation curves. Measurement of the pH, oxygen tension, and oxygen saturation of antecubital venous blood with instruments present in most clinical chemistry laboratories permits an estimate of the strength of oxygen binding to hemoglobin. An equation can be used to convert the venous oxygen tension (standardized to pH 7.4) and the oxygen saturation to the P50 of the oxygen-hemoglobin dissociation curve on which the observed point falls. The data indicate that this method is a reliable initial step in the identification of a hemoglobin with abnormal affinity for oxygen and may be applied to population studies, since reliable results are obtained with venous blood stored at 4 degrees C for up to 24 hours.

Hemoglobins, Abnormal↗
PubMed1975

Acidification of plasma by the red cell due to radiographic contrast materials.

The effect of water-soluble radiographic contrast material on pH when added to blood in clinical dosages in vitro or when used in vivo for diagnostic purposes was examined. Contrast material caused a reduction of blood pH. The mechanism of this occurence was found to be the balancing of the negative charge of intracellular organic anions by the extracellular anionic contrast material molecules. The normal negative potential of about 10 mV across the red cell membrane was reduced, nullified, or reversed depending on the concentration of contrast material added to blood. As the inside of the cell became more positive with respect to the outside, protons were, in effect, repelled into plasma, although the apparent exodus of protons occurs by the generation and outward diffusion of carbon dioxide. Since the acidemia is dependent on rehydration of carbon dioxide in plasma, a reaction measured in seconds, the site of injection and transit time of dye will contribute to the pH of the plasma during passage through a regional capillary bed. We speculate that an alteration in membrane potential and/or the acute acidemia may contribute to the adverse effects of contrast material, particularly on tissues dependent on membrane electrical rhythmicity such as the myocardium.

Acidosis↗