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M A Lichtman

Publications and source records attributed to M A Lichtman.

At least 19 recordsLinked to original sources
PubMed1992

Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.

Amino Acid Sequence↗
PubMed1991

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells.

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F-actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F-actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5-isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F-actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F-actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin A stimulated human neutrophils: superoxide production without a rise in intracellular free calcium.

Changes in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and sn-1,2-diacylglycerol (DAG) were determined in relation to agonist-induced human neutrophil superoxide (O2-) production. With 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2- production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP-induced O2- production was inhibited by pertussis toxin. In cytochalasin B-primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca]i but not IP3 proceeded O2- production, and pertussis toxin did not inhibit O2- production. EGTA inhibited the cytochalasin B/fMLP-induced increment in [Ca]i and O2- production by 75% and 50%, respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2- production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2- production. Second, pertussis toxin inhibited both fMLP-induced IP3 generation and O2- production but did not inhibit the rise in [Ca]i. Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2- production occurred in response to 0.015 microM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP-induced O2- production was not inhibited by EGTA. Taken together, these data suggest that 1) an increment in [Ca]i is not strictly essential for neutrophil O2- production, 2) unlike fMLP, Con A-induced O2- production does not proceed through a pathway involving the pertussis toxin-sensitive G protein, and 3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.

Alkaloids↗
PubMed1990

Control of actin conformation in AML myeloblasts: the effects of bryostatin and TPA.

Protein kinase C (PKC), an enzyme involved in signal transduction, is the receptor for both the tumor-promoting phorbol esters and the anti-neoplastic bryostatins. In many cells, phorbol esters and bryostatins cause similar effects; we have found that both agents increase actin polymerization in neutrophils. In some cells, however, the two agents result in different cell processes; we have found consistently different effects of these agents on actin conformation in myeloblasts obtained from leukemic patients. The patients tested all had increases in F-actin in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) and most had decreases in F-actin in response to bryostatin. The data suggests that leukemic myeloblasts have a different cytoskeletal response to a tumor promoter and an antineoplastic agent despite their common receptor.

Actins↗
PubMed1990

Activation of neutrophils: measurement of actin conformational changes by flow cytometry.

Actin, which comprises approximately 10% of the weight of cytoplasmic protein of neutrophils, is the principal component of the cytoplasmic microfilament lattice. It can exist in either of two physical states, G-actin, which is monomeric, or F-actin, which is polymeric or filamentous. Actin microfilaments support many forms of cell movement. Continuous remodeling of the microfilament lattice, which seems integral to sustained movement, is possible in part because of the ability of actin to change rapidly between its monomeric G-state and its filamentous F-state. Changes in the G- and F-actin equilibrium may be studied by flow analysis using a fluorescent probe which is specific for F-actin, 7-nitrobenz-2-oxa-1,3-diazole-(NBD)-phallacidin. Alterations in neutrophil F-actin have been measured in response to chemotactic agents (e.g., formyl peptides and leukotriene B4), inhibitors of cell movement (e.g., N-ethylmaleimide and cytochalasin B), agents that promote the oxidative burst (e.g., formyl peptides and phorbol esters), and priming agents [e.g., tumor necrosis factor (TNF)]. Measurements may be taken at intervals of a few seconds, allowing comparison of rapid changes in the F-actin content to other rapidly occurring changes, such as altered membrane ion permeability and activation of cellular enzymes. The use of metabolic inhibitors has allowed dissection of some of the biochemical pathways involved in actin assembly in living cells. Although clinical studies are few thus far, the technique has also been used to study basal and stimulated F-actin levels in circulating neutrophils in neonates and in family members of patients with neutrophil-actin dysfunction.

Actins↗
PubMed1989

Photoinhibition of 2-amino-2-carboxybicyclo[2,2,1]heptane transport by O-diazoacetyl-L-serine. An initial step in identifying the L-system amino acid transporter.

Neutral amino acid uptake into mammalian cells occurs predominantly through the L, A, and ASC carrier-mediated transport systems. The proteins responsible for transport by these systems have not been isolated, and the three pathways presently are defined by their amino acid specificity and physiologic parameters. We have found that the amino acid derivative, O-diazoacetyl-L-serine (azaserine), is a potentially useful probe for identification of the L-(leucine-favoring) system transporter in human T-lymphocytes. Uptake of azaserine competitively inhibits the uptake of the prototype L-system amino acid, 2-amino-2-carboxybicycloheptane (BCH). Azaserine undergoes photolytic cleavage with 365 nm incident light to yield a highly reactive carbene intermediate and free N2. Following photolysis of [14C]azaserine in a suspension of lymphocytes, the 14C label is detectable within a crude cytoplasmic membrane preparation, and this process is inhibited by a 50-fold excess of unlabeled azaserine or 2-amino-2-carboxybicycloheptane, suggesting that the 14C-product is associated with the membranes at or near the L-system transport site. Furthermore, photolysis of azaserine in the presence of lymphocytes results in specific irreversible inhibition of L-system transport. Thus, photolysis of azaserine provides an initial step toward the identification of the L-system transporter.

Amino Acid Transport Systems↗
PubMed1989

Characterization of human marrow stromal cells: role in progenitor cell binding and granulopoiesis.

Adherent cell layers and their associated extracellular matrices form when human marrow is incubated in cultures containing hydrocortisone and horse serum. These stromal layers contain cells positive for alkaline phosphatase; secrete collagens types I and III and fibronectin, bind the anti-actin monoclonal antibodies (MoAbs) HHF and CGA-7; stain with oil red O, and express the acetylated LDL receptor. Highly purified CD34 (My10)-positive progenitor cells attach to these stromal layers, and a 16-fold enrichment of CFU-GM in both stromal attachment and semisolid agar assays was observed. Granulopoiesis persisted up to 40 days (mean duration 25 days) after passaged stroma were recharged with stromal cell-depleted target cells in a two-stage liquid marrow culture system. Although equal to marrow fibroblasts in their ability to bind CD34+ myeloid progenitors, stromal layers were better at supporting granulopoiesis. This system provides an in vitro model to characterize the components of stroma and stroma-cytomatrix that enhance marrow progenitor cell localization and maintenance.

Antigens, CD34↗
PubMed1989

Diacylglycerol and calcium induce rapid enhancement of A-system amino acid transport by independent mechanisms in human T-lymphocytes.

Sn-1,2-diacylglycerols (DAG) and ionized-free calcium can act as intracellular second messengers for cell activation. Traditionally, T-lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T-lymphocytes demonstrate rapid enhancement of A-(alanine-favoring) system amino acid uptake when treated with DAG or ionomycin. A 30-40% increase in the initial velocity of uptake (vi) of the synthetic A-system specific amino acid, methylamino-isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The vi was enhanced 60% from 12 to 19 mumol/liter cell water per min after 30 min exposure of T-cells to optimal concentrations of dioctanoylglycerol (30 microM), oleoylacetylglycerol (30 microM), or ionomycin (5 microM) (P less than .01 for each agent). A 50-fold excess of non-radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier-mediated on treatment with these agents. Cycloheximide, 100 micrograms/ml, inhibited protein synthesis but did not block the A-system amino acid transport enhancement induced by DAG or ionomycin. The DAG-induced increase in the vi was blocked 40% with 100 microM H-7, an inhibitor of protein kinase C. H-7 treatment did not inhibit the ionomycin-induced A-system enhancement. A marked increase in cytoplasmic free calcium was measured when T-lymphocytes were exposed to ionomycin but not on DAG exposure, and the A-system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A-system amino acid uptake in T-lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A-system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation.

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL-60 cells (Costa-Casnellie et al.: Journal of Biological Chemistry 263:11851-11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL-60 cells produce superoxide in response to active phorbol esters, whereas immature HL-60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) was observed in mature neutrophilic HL-60 cells but not in their immature counterparts. Inactive 4-alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: 1) its apparent Km for Na+ transport increased 2-fold; 2) its Vmax increased 2.6-fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.

Carrier Proteins↗
PubMed1988

Protein kinase C modulates actin conformation in human T lymphocytes.

We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes.

Actins↗
PubMed1988

The Na+/H+ exchanger in immature and mature granulocytic HL-60 cells. Property changes induced by intracellular acidification and cell maturation.

We have compared the properties of the Na+/H+ exchanger in two cell populations: growing promyelocytic HL-60 cells (immature) and HL-60 cells induced to mature into granulocytes by dimethyl sulfoxide. The exchanger was activated by intracellular acidification from pH 7.25 to pH 5.5. In both immature and mature granulocytic cells, this type of activation resulted in the expected increase in Vmax for Na+ uptake but also in an increase in KmNa. Maximum acidification caused an increase in Vmax of approximately 10-fold in both types of cells. The increase in KmNa was influenced by cell maturation. In immature cells, the KmNa was higher than in mature cells at all pH values tested, and this difference increased with acidification. Maximum acidification increased the KmNa from 15 +/- 4 to 124 +/- 17 mM in immature cells and from 10 +/- 3 to 43 +/- 20 mM in mature cells. Intracellular pH also influenced the pattern of inhibition of 22Na uptake by dimethylamiloride, a specific inhibitor of the exchanger. At intracellular pH 7.0 dimethylamiloride inhibition was mostly competitive in immature and competitive in mature cells. At lower intracellular pH, 5.9, the inhibition was mixed in both types of cells. Thus, the properties of the exchanger in granulocytic cells are influenced by the cell maturation stage and the intracellular pH.

Amiloride↗
PubMed1988

Trans-stimulation of L-system amino acid transport in normal and chronic leukemic human lymphocytes: phorbol ester restores function in CLL.

Chronic lymphocytic leukemia (CLL) B-lymphocytes have a unique and specific diminution of L-system (leucine favoring) amino acid uptake; the maximal velocity is approximately 10% of normal B-lymphocytes. Treatment of CLL B-cells with the maturational agent, tetradecanoyl phorbol acetate, results in restoration of L-system amino acid uptake to normal velocity. To further characterize the effect of phorbol ester on the L-system of CLL B-cells, we have examined the ability of normal and CLL lymphocytes to exchange intracellular for extracellular amino acids by the L-system (trans-stimulation). A 60% increase in L-system uptake was noted in normal B- and T-lymphocytes in the presence of a high intracellular concentration of 2-amino-2-carboxy-bicycloheptane (BCH), a largely L-system-specific substrate. L-system transport was not trans-stimulated in CLL B-lymphocytes. Phorbol ester treatment restored L-system uptake in CLL to a normal Vmax of 900 mumol/liter cell water per minute in the absence of BCH loading. The Vmax could be increased further to 2,400 if phorbol ester-treated CLL cells were loaded with BCH. Hence, phorbol esters result not only in a normalization of L-system uptake in CLL B-cells but the transport system demonstrates exchange rates comparable to normal lymphocytes.

Amino Acids↗
PubMed1988

Upper and lower time limits in the decision to recommend marrow transplantation for patients with chronic myelogenous leukaemia.

Long-term survival of patients with chronic myelogenous leukaemia (CML) requires marrow transplantation from a histocompatible donor. The optimal timing of the transplant is difficult to determine because of the high peritransplant mortality of 20-35% and the existence of a group of patients who can have the disease controlled by drug treatment for prolonged periods. We have developed a mathematical model implemented with a computer program which calculates lower and upper time limits for the timing of marrow transplantation. The lower time limit for transplantation is derived from the loss of life expectancy with delay, and the upper time limit is calculated by comparing the transplant survival probability with the probability of surviving an additional year without a transplant. Thus, an objective basis is provided for bracketing the most appropriate time for transplantation. This analysis suggests that the decision to transplant can be postponed in some patients for periods longer than may generally be recommended.

Adult↗
PubMed1988

Phorbol ester restores L-system amino acid transport of B lymphocytes in chronic lymphocytic leukemia.

L (leucine-favoring)-system amino acid transport is uniquely and selectively diminished in chronic lymphocytic leukemia B lymphocytes: the maximal velocity of transport is 10% of normal B lymphocytes. We examined L-system transport in chronic leukemic B lymphocytes after incubation with tetradecanoyl phorbol acetate to determine if the transport abnormality can be corrected by the apparent cell maturation induced by this agent. Amino acid uptake was measured using 2-amino-2-carboxy-bicycloheptane, an L-system specific synthetic amino acid. Marked enhancement of L-system transport occurred in each of 12 leukemic cell populations; the initial velocity of transport in phorbol ester-treated cells increased 8-fold and 14-fold at 16 and 40 h, respectively, compared with untreated cells. The Vmax of the L-system in phorbol ester-treated leukemic cells was similar to that of phorbol ester-treated normal B lymphocytes. The L-system enhancement of the leukemic cells paralleled the development of plasmacytoid features at 40 h. Uptake of leucine, a naturally occurring L-system amino acid, was also increased by tetradecanoyl phorbol acetate. Cycloheximide, 100 micrograms/ml, which inhibited over 90% of protein synthesis in phorbol ester-treated chronic leukemic cells, blocked completely the phorbol ester-induced L-system enhancement. Phorbol ester treatment restores the selective L-system transport defect in chronic lymphocytic leukemia B lymphocytes, and this process coincides with in vitro maturation of the leukemic cells.

Aminoisobutyric Acids↗
PubMed1987

Characterization of the Na+/H+ exchanger during maturation of HL-60 cells induced by dimethyl sulfoxide.

We have studied the activity of the Na+/H+ exchanger during dimethyl sulfoxide (Me2SO)-induced maturation of the human promyelocytic leukemia cell line HL-60. 22Na uptake was measured in cells preloaded with Li+ or NH+4 in order to specifically activate the Na+/H+ exchanger. Measurement of the rate of uptake as a function of sodium concentration revealed a decrease in Km for Na+ from 38 +/- 3 to 13 +/- 1 mM after 20-24-h treatment with Me2SO. Vmax was not changed significantly. Inhibition of the exchanger by dimethylamiloride (DMA) and by acidic external pH was similar in treated and untreated cells. Thus it is unlikely that the Na+ binding site is altered. A change, however, was observed in the regulation of the exchanger by intracellular pH. In control cells maximal stimulation of the Na+ uptake was observed when the intracellular pH decreased from 7.25 to 7.00. In Me2SO-treated cells the 22Na uptake at intracellular pH 7.00 was greater than in the control and continued to increase as the intracellular pH was adjusted below 7.00, down to 6.75. This suggests that the Na+/H+ exchanger in Me2SO-treated cells is altered structurally in its allosteric H+ binding site. The appearance of this modified exchanger preceded by a period of days the appearance of a functional property characteristic of mature granulocytes, that is, the capability to produce superoxide, suggesting that the modified exchanger may be required for the expression of the mature phenotype. A second modification, a decrease in the Vmax of the 22Na uptake, occurred after 2 days treatment with Me2SO. This reduction may reflect a decrease in the number of functioning exchangers per cell.

Amiloride↗
PubMed1987

The effects of sulfhydryl inhibitors and cytochalasin on the cytoplasmic and cytoskeletal actin of human neutrophils.

To better understand the changes that occur in cytoplasmic actin during cell movement, we studied the effect of inhibitors of cell movement on the molecular conformation of actin and its incorporation into the Triton-insoluble cytoskeleton of human neutrophils. The sulfhydryl reactive compound N-ethylmaleimide caused an increase in cellular F-actin as measured by uptake of the F-actin specific fluorescent probe 7-nitrobenz-2-oxadiazole-phallacidin. However, N-ethylmaleimide reduced the amount of actin associated with the Triton-insoluble cytoskeleton. Dithiobisnitrobenzoic acid, a sulfhydryl reagent that does not cross cell membranes efficiently, did not alter the F-actin content of neutrophils. The effect of N-ethylmaleimide was blocked by the presence of dithiothreitol, a donor of sulfhydryl groups. N-ethylmaleimide did not affect the polymerization of actin in a cell-free system. Cytochalasin B did not alter F-actin content of neutrophils but did decrease actin in cytoskeletons of resting neutrophils. Cytochalasin inhibited the increase in F-actin initiated by the chemoattractant N-formylmethionylleucylphenylalanine. We propose that N-ethylmaleimide blocks the stabilization of G-actin in cytoplasm, interferes with the incorporation of F-actin polymer into the cytoskeleton, and depolymerizes the cytoskeleton. In contrast cytochalasin stabilizes G-actin in the presence of chemotactic peptide. These data suggest that reversible conversion of G-actin to F-actin and incorporation of F-actin into the Triton-insoluble cytoskeleton are important for neutrophil movement.

Actins↗
PubMed1987

Maturation-associated changes in the peripheral cytoplasm of human neutrophils: a review.

The process of hemopoietic stem cell differentiation and proliferation leads to a large number of precursor cells committed to the neutrophil lineage. These precursor cells undergo a further limited degree of proliferation but, most importantly, undergo a dramatic process of maturation which alters the phenotype of the cell from a myeloblast, which is incapable of normal circulation and function into a segmented neutrophil capable of chemokinesis, chemotaxis, particle ingestion, microbicidal action, and other functions required to subserve the inflammatory process. This review describes the changes in the cell surface and cell cytoplasm that occur during precursor cell maturation and, to the extent possible, correlates molecular and macromolecular changes during maturation with the development of functional capacity.

Antigens, Surface↗