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J D Loike

Publications and source records attributed to J D Loike.

35 records · Page 2Linked to original sources
PubMed1992

Hypoxia induces glucose transporter expression in endothelial cells.

Endothelial cells in various tissues of the body are often exposed to hypoxic conditions. To examine the effects of sustained hypoxia on energy metabolism in endothelial cells, we have maintained bovine aortic and human umbilical vein endothelial cells in an atmosphere containing low oxygen concentrations (14 mmHg) for up to 96 h. We report here that endothelial cells maintained under these conditions upregulate their glucose transport activity, consume more glucose, and produce greater amounts of lactic acid than normoxic cells. Upregulation of glucose transport activity by hypoxic endothelial cells required several hours to occur, was associated with increased expression of mRNA and protein for the erythroid/brain form of the facilitative glucose transporter, and was not due to depletion of glucose from the medium. Prolonged treatment of endothelial cells with inhibitors or uncouplers of oxidative phosphorylation (antimycin, azide, dinitrophenol) under normoxic conditions also upregulated glucose transporter expression. These results suggest that reduced rates of oxidative metabolism may represent an important signal for cells to adapt metabolically to hypoxia. Furthermore, in our examination of endothelial cell energy metabolism, we discovered that endothelial cells contain phosphocreatine and express both the brain and muscle isozymes of creatine kinase.

Animals↗
PubMed1991

CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen.

Fibrinogen and fibrin serve as adhesive substrates for a variety of cells including platelets, endothelial cells, and leukocytes. Previously, we identified the C terminus of the gamma chain of fibrinogen as the region of the fibrinogen molecule that contains a ligand for CD11b/CD18 (complement receptor 3) on phorbol ester-stimulated polymorphonuclear leukocytes. In contrast, we report here that neutrophils stimulated with tumor necrosis factor adhere to fibrinogen-coated surfaces, but not to human serum albumin-coated surfaces, via the integrin CD11c/CD18 (p150/95). Monoclonal antibodies LeuM5 and 3.9, which are directed against the alpha subunit of CD11c/CD18, but not monoclonal antibodies OKM10 and OKM1, which are directed against the alpha subunit of CD11b/CD18, inhibit the adhesion of tumor necrosis factor-stimulated neutrophils to fibrinogen-coated surfaces. To identify the site on fibrinogen recognized by CD11c/CD18, we have examined the adhesion of tumor necrosis factor-stimulated neutrophils to surfaces coated with various fibrinogen fragments. Stimulated neutrophils adhere to surfaces coated with the N-terminal disulfide knot fragment of fibrinogen or fibrinogen fragment E. Moreover, peptides containing the sequence Gly-Pro-Arg (which corresponds to amino acids 17-19 of the N-terminal region of the A alpha chain of fibrinogen), and monoclonal antibody LeuM5, block tumor necrosis factor-stimulated neutrophil adhesion to fibrinogen and to the N-terminal disulfide knot fragment of fibrinogen. Thus, CD11c/CD18 on tumor necrosis factor-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the A alpha chain of fibrinogen.

Amino Acid Sequence↗
PubMed1988

Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy.

A deficiency of the protein dystrophin has recently been shown to be the probable cause of Duchenne's muscular dystrophy. We sought to determine the relation between the clinical phenotype and the status of dystrophin in muscle-biopsy specimens from 103 patients with various neuromuscular disorders. We found very low levels (less than 3 percent of normal levels) or no dystrophin in the severe Duchenne phenotype (35 of 38 patients), low concentrations of dystrophin in the intermediate (outlier) phenotype (4 of 7), and dystrophin of abnormal molecular weight in the mild Becker phenotype (12 of 18). Normal levels of dystrophin of normal molecular weight were found in nearly all the patients (38 of 40) with 20 other neuromuscular disorders we studied. These data show the clinical consequences of both quantitative alterations (in Duchenne's and intermediate dystrophy) in a single protein. The biochemical assay for dystrophin should prove helpful in delineating myopathies that overlap clinically with Duchenne's and Becker's dystrophies, and it shows promise as an accurate diagnostic tool.

Adolescent↗
PubMed1988

Complement receptor type three (CD11b/CD18) of human polymorphonuclear leukocytes recognizes fibrinogen.

Human polymorphonuclear leukocytes (PMN) have previously been shown to bind to aggregates of fibrin and to fibrinogen-coated surfaces. During their interactions with fibrinogen-coated surfaces, PMN make such close contact with the surface that a portion of the secreted elastase activity is protected from macromolecular protease inhibitors in the surrounding medium. Here we show that the receptor on PMN that mediates this interaction is complement receptor type 3 (CR3; CD11b/CD18), a molecule previously identified as a receptor for the complement protein fragment C3bi. Monoclonal antibodies against CR3 that block the binding of C3bi also block the binding of PMN to fibrinogen-coated surfaces and the formation of a protected compartment. The region of fibrinogen recognized by CR3 lies at the carboxyl terminus of the gamma chain, since peptides based on this sequence effectively inhibit the binding of PMN to fibrinogen-coated surfaces. These peptides also block the binding of C3bi-coated erythrocytes to CR3, thus indicating that a single binding site is used for binding both C3bi and fibrinogen. Sequence analysis shows strong structural similarity between this region of fibrinogen and other known ligands of CR3. These studies thus indicate that CR3 functions as a receptor not only for C3bi but also for fibrinogen.

Antibodies, Monoclonal↗
PubMed1988

Extracellular creatine regulates creatine transport in rat and human muscle cells.

Muscle cells do not synthesize creatine; they take up exogenous creatine by specific Na+-dependent plasma membrane transporters. We found that extracellular creatine regulates the level of expression of these creatine transporters in L6 rat muscle cells. L6 myoblasts maintained for 24 hr in medium containing 1 mM creatine exhibited 1/3rd of the creatine transport activity of cells maintained for 24 hr in medium without creatine. Down-regulation of creatine transport was partially reversed when creatine-fed L6 cells were incubated for 24 hr in medium lacking creatine. Down-regulation of creatine transport occurred independently of amino acid and glucose transport. Furthermore, the down-regulation of creatine transporters by extracellular creatine was slowed by inhibitors of protein synthesis. These results suggest that creatine induces the expression of a protein that functionally inactivates the creatine transporters. Regulation of creatine transport by extracellular creatine also was observed in L6 myotubes and in cultures of human myoblasts and myotubes. Hence, the activity of creatine transport represents another site for the regulation of creatine homeostasis.

Animals↗
PubMed1986

Creatine uptake, metabolism, and efflux in human monocytes and macrophages.

The uptake of creatine in human monocytes and monocyte-derived macrophages can be divided into two components. One component of uptake is saturable (Km = 30 microM), specific for creatine, requires Na+ in the extracellular medium, and enables cells to take up creatine against a concentration gradient of creatine. The second component of uptake is nonsaturable and accounts for less than 10% of creatine uptake at physiological creatine concentrations (20-60 microM). Both monocytes and macrophages have similar Km values for creatine transport. However, macrophages exhibit a fivefold greater rate of creatine uptake than monocytes (3.6 vs. 0.7 pmol X micrograms protein-1 X h-1), indicating that monocyte differentiation into macrophages is associated with an increase in the number of creatine transporters in the membrane. Monocytes do not contain creatine kinase and do not phosphorylate creatine; monocyte-derived macrophages express creatine kinase and phosphorylate approximately 25% of intracellular creatine. These results and the finding that 2-deoxyglucose inhibits creatine phosphorylation by macrophages without affecting the transport of creatine indicate that phosphorylation of creatine is not required for net creatine accumulation.

Biological Transport, Active↗
PubMed1984

Creatine kinase expression and creatine phosphate accumulation are developmentally regulated during differentiation of mouse and human monocytes.

We have studied the expression of creatine kinase (CK) and the accumulation of creatine phosphate during the differentiation of human and mouse peripheral blood monocytes. Mouse monocytes cultured for 24 h do not contain detectable levels of CK and creatine phosphate. However, resident tissue macrophages and inflammatory elicited macrophages obtained from the peritoneal cavities of mice have 70 and 300 mU per mg protein of CK activity and contain 3 and 6 mol of creatine phosphate per mol of ATP, respectively. The major isozyme of CK in these cells has been identified as the brain form. These findings suggest that the differentiation of monocytes into macrophages is associated with the expression of CK and the accumulation of creatine phosphate. We have found a similar pattern in human monocytes. Human blood monocytes, maintained in culture for 24 or 48 h, do not contain detectable levels of CK or creatine phosphate. Monocyte-derived macrophages (monocytes maintained in tissue cultures for 1 to 2 wk) have up to 100 mU per mg protein of CK activity and contain 0.5 mol of creatine phosphate per mol of ATP. Human macrophages express multiple isozymes of CK including the brain (BB) and possibly the mitochondrial forms of this enzyme. Thus, the expression of CK and the accumulation of creatine phosphate in human monocytes is induced by their in vitro cultivation. The induction of CK during in vitro cultivation occurs independently of the concentration of creatine in the medium. However, the size of the creatine phosphate pool varies with respect to extracellular creatine concentration. Creatine phosphate and CK are not detectable in freshly isolated human lymphocytes, polymorphonuclear leukocytes or erythrocytes, but are found in freshly isolated human platelets.

Adult↗
PubMed1983

A fluorescence quenching technique using trypan blue to differentiate between attached and ingested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages.

A fluorescence quenching method, using trypan blue, is described for quantifying the ingestion of either glutaraldehyde-fixed sheep red blood cells or antibody coated glutaraldehyde-fixed sheep red blood cells by murine peritoneal macrophages. This method is based on the observations that glutaraldehyde-fixed red blood cells fluoresce at about 585 nm when excited at 490 nm and that when trypan blue is in intimate contact with fixed red blood cells, the fluorescence is converted from a chartreuse to a red color. Thus, the ingestion of fixed erythrocytes by murine macrophages can be monitored by fluorescence microscopy after the addition of 1 mg/ml of trypan blue at the end of the assay. Extracellular glutaraldehyde-fixed red blood cells fluoresce a red color whereas the intracellular particles continue to fluoresce a chartreuse color. This method offers a simple and convenient technique for rapidly distinguishing between intracellular and extracellular glutaraldehyde-fixed red blood cells in macrophages.

Animals↗
PubMed1982

VP16-213 and podophyllotoxin. A study on the relationship between chemical structure and biological activity.

VP16-213, a semi-synthetic derivative of podophyllotoxin, is an effective antitumor agent in the treatment of a variety of leukemias and solid tumors. A comparison of the mechanism of action of VP16-213 and podophyllotoxin has revealed that although both drugs inhibit the uptake of nucleosides into HeLa cells, they exhibit other biological properties which are quite distinct. Podophyllotoxin is a potent inhibitor of microtubule assembly in vitro, while VP16-213 has no effect in this system. VP16-213 induces single stranded breaks in HeLa cells DNA, an effect which may be related to its antitumor activity. In contrast to VP16-213 treated cells, podophyllotoxin-treated cells maintain DNA integrity. Structure-activity relationship studies have identified some of chemical sites of VP16-213 and podophyllotoxin responsible for each of their biological properties. These studies illustrate that chemical modification of podophyllotoxin can generate derivatives which possess new and unique biological properties.

Animals↗
PubMed1981

Application of differential scanning microcalorimetry to the study of cellular processes: heat production and glucose oxidation of murine macrophages.

Differential scanning microcalorimetry provides a noninvasive method for studying heat evolution in living cells. We used this technique to measure the heat evolved by thioglycollate broth-elicited mouse macrophages, and the effects of NaF, KCN, cycloheximide, and cytochalasins B and D on this parameter. The total heat evolved in the interval 10--37 degrees C scanned at 1 degree C min-1 ranged from 300 to 2500 X 10(-12) cal (1 Cal = 4.184 J) per cell, depending on cell density, glucose concentration, and the presence or absence of various drugs.

Animals↗
PubMed1979

Increased ATP and creatine phosphate turnover in phagocytosing mouse peritoneal macrophages.

Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.

Adenosine Triphosphate↗
PubMed1978

Structure-activity study of the inhibition of microtubule assembly in vitro by podophyllotoxin and its congeners.

This study investigates the inhibition of microtubule assembly in vitro by podophyllotoxin and its derivatives, which include in part the antitumor compounds 4'-demethylepipodophyllotoxin ethylidene beta-D-glucoside (VP-16-213) and 4'-demethylepipodophyllotoxin thenylidene beta-D-glucoside (VM-26); the cyclic ethers, cyclic sulfides, and cyclic sulfones of podophyllotoxin and deoxypodophyllotoxin; epipodophyllotoxin; picropodophyllotoxin; and several 4'-demethyl compounds. The inhibitory activity of these derivatives is sensitive to the configuration and size of substituents at position 4 in ring C and to steric features of substituents at position 12 in ring D. Decreasing activity correlates with the increasing size of the substituent at position 12, as indexed by their van der Waals radii. These results suggest that rings C and D of these drugs are involved in their interaction with the podophyllotoxin-binding site in tubulin.

Animals↗
PubMed1976

Effects of podophyllotoxin and VP-16-213 on microtubule assembly in vitro and nucleoside transport in HeLa cells.

VP-16-213, a semisynthetic derivative of podophyllotoxin, is an active antitumor agent. In this paper, the effects of VP-16-213 and podophyllotoxin on microtubule assembly in vitro and nucleoside transport in HeLa cells are compared. At 100 muM, VP-16-213 does not inhibit microtubule assembly in vitro, while 5 muM podophyllotoxin completely prevents the formation of microtubules. The presence of the glucoside moiety in VP-16-213 is responsible for the inactivity of VP-16-213 in this system because 4'-demethylepipodo-phyllotoxin, the nonglucoside congener of VP-16-213, inhibits microtubule assembly. In HeLa cells, VP-16-213 and podophyllotoxin share a common biological property; both agents inhibit the uptake of thymidine and uridine into cells by inhibiting the facilitated diffusional component of nucleoside transport. The conncentrations of drug necessary to inhibit thymidine and uridine uptake into HeLa cells by 50% are 10 and 5 muM, respectively, for podophyllotoxin, and 25 and 20 muM for VP-16-213. The action of podophyllotoxin on nucleoside transport appears unrelated to its effect on microtubule assembly, since VP-16-213, which does not inhibit microtubule assembly, inhibits nucleoside transport.

Adenosine↗
PubMed1976

Effect of VP-16-213 on the intracellular degradation of DNA in HeLa cells.

The effect of VP-16-213 on cellular DNA was studied by following the sedimentation profiles of radioactive DNA in HeLa cells on alkaline sucrose gradients. In VP-16-213 treated cells, high-molecular-weight DNA is converted to a lower molecular-weight form in a dose-dependent, temperature-dependent reaction. The effect of VP-16-213 on cellular DNA is reversed after the drug has been removed from the growth medium for 150 min. These results suggest that VP-16-213 induces single-stranded breaks in DNA in HeLa cells and that HeLa cells can repair these breaks within 150 min. The nonglucoside derivative of VP-16-213, 4'-demethylepipodophyllotoxin, also induces the cleavage of cellular DNA but podophyllotoxin has no effect on DNA. A structure-activity relationship study, in which the effects of various VP-16-213 and podophyllotoxin congeners were tested for their ability to cleave cellular DNA,revealed that an hydroxyl group at the C-4' position is required for activity and that the configuration of the C-4 carbon influences the activity of a congener. These results may offer insights into the mechanism of action of VP-16-213 as an antitumor agent.

Adenoviruses, Human↗
PubMedDate not supplied

Microglia, scavenger receptors, and the pathogenesis of Alzheimer's disease.

The senile plaque is the pathological hallmark of Alzheimer's disease. Senile plaques are composed of beta amyloid fibrils, associated with activated microglia, astrocytes, and dystrophic neurons. We have recently identified class A scavenger receptors as the main receptors mediating the interaction of microglia with beta amyloid fibrils. Adhesion of microglia to beta amyloid fibrils leads to immobilization of these cells on the fibrils, and induces them to produce reactive oxygen species. We propose that interactions of microglial scavenger receptors with fibrillar beta amyloid may stimulate the microglia to secrete apolipoprotein E and complement proteins, which may further contribute to neurotoxicity and neuronal degeneration. Therefore, microglial scavenger receptors may be novel targets for therapeutic interventions in Alzheimer's disease.

Alzheimer Disease↗
PubMedDate not supplied

A comparison of the mechanisms of action of VP-16-213 and podophyllotoxin.

A comparison of the mechanism of action of VP 16-213 and podophyllotoxin has revealed that although both drugs inhibit nucleoside uptake in HeLa cells, they exhibit other biological properties which are quite distinct. Podophyllotoxin inhibits microtubule assembly in vitro while VP-16-213 has no effect in this system. VP-16-213 induces single-stranded breaks in DNA in HeLa cells, an effect which may explain the cytoxicity of VP-16-213. In contrast to VP-16-213-treated cells, cells treated with podophyllotoxin maintain DNA integrity and show no evidence of single-stranded breaks.

Biological Transport↗