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S T Hoffstein

Publications and source records attributed to S T Hoffstein.

At least 19 recordsLinked to original sources
PubMed1991

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

Animals↗
PubMed1990

Internalization of recombinant tissue-type plasminogen activator by isolated rat hepatocytes is via coated pits.

The uptake and internalization of tissue-type plasminogen activator (t-PA) by freshly isolated rat hepatocytes was investigated. Electron microscopic examination of the uptake of t-PA-colloidal gold conjugates (t-PA-gold) by isolated rat hepatocytes showed that t-PA-gold was internalized via coated pits. This was inhibited with excess t-PA. Uptake of 125I-t-PA by isolated rat hepatocytes was a rapid, saturable, and specific process. The initial rate of specific uptake was 0.1 fmol/10(6) cells per min. The specific uptake plateaued at 1.4 fmol/10(6) cells by 30 min and declined to 0.8 fmol/10(6) cells at 2 h. Depletion of cellular ATP by 85-90% did not affect the initial rate of specific uptake. However, specific uptake by ATP-depleted hepatocytes at 30 min was reduced by 37%. By 2 h specific uptake by ATP-depleted hepatocytes was only 5% lower than by untreated hepatocytes, suggesting that processing of t-PA and/or its receptor is ATP-dependent. Uptake of 125I-t-PA was temperature dependent. Specific uptake was reduced by approximately 20% at 22 degrees C and by 70% at temperatures below 16 degrees C. Finally, inhibition of coated pit formation by K(+)-depletion with nigericin decreased the uptake of 125I-t-PA. This inhibition was shown to be K(+)-specific since treatment with nigericin in the presence of K+ did not inhibit coated pit formation or 125I-t-PA uptake. A threshold K(+)-depletion level for inhibition of coated pit formation was also demonstrated since treatment under conditions that reduced cellular K+ by only 54% had no effect on coated pit formation or 125I-t-PA uptake. These data support our hypothesis that internalization of t-PA by isolated rat hepatocytes is via coated pits and suggest that uptake of t-PA is a receptor-mediated process.

Acridine Orange↗
PubMed1989

A new cytochemical method for ultrastructural detection of liposomes in tissues in vivo.

Multilamellar vesicles (MLVs) have been used as drug carriers to increase efficacy or decrease toxicity of a variety of therapeutic agents, including antineoplastics, antibiotics, and immunomodulators. Although analysis of the disposition of encapsulated materials is relatively simple using radiolabels or single enzymes, determining the cellular and subcellular disposition of intact MLVs, i.e., those that still retain their encapsulated materials, is much less straightforward. We have developed a technique that allows demonstration of the uptake of intact MLVs by Kupffer cells. The method is based on co-localization of paired enzymes, glucose oxidase (GO), and horseradish peroxidase (HRP). The rationale for the localization is that H2O2 generated from glucose and oxygen by GO acts as the substrate for the HRP-mediated oxidative polymerization of diaminobenzidine. Therefore, only sites of co-localization of GO and HRP should stain. Mice were injected IV with phosphatidyl choline MLVs encapsulating HRP and GO. Encapsulated enzymes were separated from non-encapsulated by passing the MLVs over a Sepharose 2B column. Control mice were injected with equivalent amounts of free GO. Mice were sacrificed 30 min after injection and liver tissue was fixed in 3% cacodylate-buffered glutaraldehyde for at least 18 hr. Tissues were washed in buffer, then stained in medium containing glucose, diaminobenzidine HCl, and dimethylsulfoxide in 0.1 M cacodylate buffer. In animals injected with MLV-encapsulated GO and HRP, vacuoles in Kupffer cells and some endothelial cells contained electron-dense reaction product. No other cell type, including polymorphonuclear leukocytes, was stained. In control animals no staining was seen. Our results indicate that encapsulation of paired enzymes may be a feasible method to demonstrate the cellular and subcellular disposition of intact liposomes.

3,3'-Diaminobenzidine↗
PubMed1988

Interleukin-1 stimulation of phospholipase activity in rat synovial fibroblasts. Possible regulation by cyclooxygenase products.

Tritiated arachidonic acid (3H-AA)-labeled rat synovial fibroblasts stimulated with human recombinant interleukin-1 beta (rIL-1 beta) released incorporated radiolabel in a time-dependent and dose-dependent manner, with labeled prostaglandins representing 29% of the released radiolabel. Treatment of the cells with dibutyryl cAMP or prostaglandin E2 enhanced both spontaneous and rIL-1 beta-induced 3H-AA release; treatment with indomethacin or naproxen inhibited the response. The effects of these cyclooxygenase inhibitors on 3H-AA release were not reversed by the addition of prostaglandin E2. The activities of phospholipase A, phospholipase C, and diglyceride lipase were detected in the homogenates of rat synovial fibroblasts. Pretreatment of synovial cells with rIL-1 beta resulted in a threefold stimulation of phospholipase A activity and a slight increase in phospholipase C activity in cell homogenates. These data show that rIL-1 beta stimulates phospholipase activities in rat synovial fibroblasts and that at least one of these activities may be regulated by either prostaglandins or cAMP.

Animals↗
PubMed1988

FMLP-induced arachidonic acid release, phospholipid metabolism, and calcium mobilization in human monocytes. Regulation by cyclic AMP.

Radiolabeled human peripheral blood monocytes released [3H]arachidonic acid upon challenge with the calcium ionophore A23187 (10 microM), or f-Met-Leu-Phe (FMLP, 1 microM). Chromatographic analysis of [3H]arachidonic acid labeled phospholipids showed that stimulation by FMLP reduced the amount of labeled phosphatidylcholine exclusively. Treatment of the monocytes with 10(-3) M dibutyryl cyclic AMP (d-cAMP) or 5 X 10(-4) M isobutylmethylxanthine (IBMX) substantially inhibited [3H]arachidonic acid release (30%) and depletion from labeled phosphatidylcholine (PC) in FMLP--but not calcium ionophore--stimulated cells. Using the fluorescent probe Indo-1, the FMLP-induced cytosolic calcium increase was unaffected by 10(-3) M dibutyryl cyclic AMP. The results suggest that FMLP-stimulated phospholipase activity is regulated by cyclic AMP, but not by depressing receptor-medicated increases in cytoplasmic free calcium.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production.

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold increase in [3H]-AA release, a 7-fold increase in PGE2 production and a 2-fold increase in PLA2 activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1. A combination of the two cytokines had an additive effect on [3H]-AA release and PLA2 activity while PGE2 production was similar to that detected using interleukin-1 alone. [3H]-AA, was released in substantial amounts when sodium fluoride was used as a stimulus but PGE2 was not. These data show that tumor necrosis factor and interleukin-1 can both activate synovial cell PLA2 and induce generation of PGE2, but act in an additive rather than a synergistic fashion. Furthermore, the data show that PGE2 production is not always concordant with [3H]-AA release, suggesting that appropriate enzyme(s) must be activated.

Arachidonic Acid↗
PubMed1988

Cytochemical demonstration of constitutive H2O2 production by macrophages in synovial tissue from rats with adjuvant arthritis.

Generation of toxic oxygen metabolites by inflammatory cells is considered to be one of the mechanisms by which inflammation produces tissue injury. This concept is based on in vitro studies of purified leukocyte populations because it has not been possible to assess production of these metabolites in tissues. In order to determine whether or not inflammatory cells in tissue generate H2O2, the authors modified an earlier cytochemical method for the localization of H2O2. The incubation medium consists of 0.5 mM CeCl3 in a Hepes-buffered balanced salt solution with Cl- as the only anion. Synovial tissue from the knees of normal and 16-day adjuvant arthritic rats was incubated in this medium for 30 minutes and then fixed and processed for electron microscopy. No H2O2 reaction product was visible in normal synovium. In contrast, patchy deposits of H2O2 reaction product were seen adjacent to a subpopulation of synovial lining macrophages in synovium from inflamed knee joints. These data show that rat synovial macrophages are capable of generating H2O2 when appropriately stimulated and that such a stimulus is present in adjuvant arthritis.

Animals↗
PubMed1987

Recombinant tumor necrosis factor and interleukin-1 both stimulate human synovial cell arachidonic acid release and phospholipid metabolism.

Stimulation of [3H]-arachidonic acid labeled human synovial cells with 3.0 X 10(-10)M recombinant interleukin-1 or tumor necrosis factor resulted in the release of incorporated radiolabel (41.1% and 27.7% respectively). Analysis of [3H]-arachidonic acid labeled phospholipids showed that interleukin-1 and tumor necrosis factor diminished [3H]-arachidonic acid in phosphatidyl-choline phosphatidylserine and phosphatidylinositol. Treatment of [3H]-arachidonic acid labeled cells with 10(-3)M dibutyryl cyclic AMP or 10(-6)M PGE2 did not affect spontaneous or stimulated [3H] arachidonic acid release significantly. These data show that recombinant interleukin-1 and tumor necrosis factor stimulate human synovial cell phospholipase activity in a similar manner and that this activity is not affected significantly by agents that elevate cyclic AMP.

Arachidonic Acid↗
PubMed1987

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP.

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils.

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.

Adult↗
PubMed1986

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver.

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.

5-Aminolevulinate Synthetase↗
PubMed1986

Structural requirements for chemotactic activity of leukotriene B4 (LTB4).

LTB4 (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB4 and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo-LTB4. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB4 was at 10(-7)M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB4 was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at c-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB4. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta6 double bond are all important structural features for chemotactic activity with delta6 stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Aggregation↗
PubMed1985

Neutrophils may directly synthesize both H2O2 and O2- since surface stimuli induce their release in stimulus-specific ratios.

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+----H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H2O2 should not form when cytochrome c is present to scavenge O2- before it can dismutate. Although H2O2 cannot be measured directly in the presence of cytochrome c because it is consumed in reoxidizing reduced cytochrome c, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochrome c. We found that the relative amounts of extracellular H2O2 and O2- that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochrome c from H2O2 oxidation when some stimuli were used but not with others. For example, the ratio of O2- to H2O2 produced by neutrophils exposed to PMA was about 2:1, the expected result if H2O2 were derived from O2-. However when cytochalasin B was added to the cells before the stimulus, the yield of H2O2 was reduced but not the yield of O2-. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O2- and H2O2. Coating the plastic with IgG doubled cytochrome c reduction without effecting H2O2. In contrast, coating with albumin reduced H2O2 without effecting cytochrome c reduction. Soluble IgG aggregates induced production of mostly O2- whereas immune complexes resulted in release of both metabolites. FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O2- than H2O2. The addition of catalase to the cytochrome c solution improved the yield of reduced cytochrome c when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H2O2 release is not a linear function of the amount of O2- generated and that either a variable fraction of O2- spontaneously dismutates to H2O2 or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H2O2 as well as O2-.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigen-Antibody Complex↗
PubMed1985

Surface contact inhibits neutrophil superoxide generation induced by soluble stimuli.

Human neutrophils in suspension undergo a metabolic burst and generate reactive O2- metabolites upon exposure to many soluble and particulate stimuli. They can also be stimulated to produce O2- when in contact with surfaces. We found that when neutrophils were allowed to settle into protein-coated surfaces the amount of O2- they generated varied with the nature of the protein: IgG greater than bovine serum albumin greater than plastic greater than gelatin greater than serum greater than collagen. However, when polymorphonuclear leukocytes were permitted to settle onto a surface and then were stimulated with either phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine the O2- response was greatly diminished compared to control cells that were exposed to the stimulus in suspension. In contrast, superoxide production in response to the particulate stimulus opsonized zymosan was similar in both suspended and settled neutrophils. The degree of inhibition was not related to the degree of adherence since the diminished response occurred with all of the surfaces tested and in the presence of cytochalasin B. Onset of inhibition was very rapid as was recovery when cells were resuspended. Whereas production of O2- was greatly inhibited by surface contact, release of lysosomal enzymes was only slightly affected. The effect of surface contact did not appear to be mediated via activation of adenylate cyclase since the combination of a phosphodiesterase inhibitor and exogenous dibuteryl cyclic adenosine monophosphate did not inhibit phorbal myristate acetate O2- production, but surface contact did. These data indicate that surface contact such as would occur during diapedesis and chemotaxis profoundly alters neutrophil behavior by an unknown mechanism and imply that observations made on polymorphonuclear leukocytes in suspension cannot be generalized to polymorphonuclear leukocytes in tissue.

Cell Adhesion↗
PubMed1982

Superoxide anion generation by human neutrophils exposed to monosodium urate.

We studied the capacities of naked and protein-coated monosodium urate (MSU) crystals to stimulate superoxide anion (O(2)) release by human polymorphonuclear leukocytes (PMN). Uncoated MSU estimated significant O(2) production by cytochalasin B-treated PMN. Precoating MSU with IgG caused an increase in mean O(2) production, whereas precoating heated MSU with serum or plasma inhibited O(2) release. Unheated MSU crystals, which activate complement to a greater extent than heated crystals, also provoked O(2) generation, an effect again abrogated by precoating with serum but not with plasma. Coincubation of unheated MSU and plasma resulted in an enhancement of O(2) generation. The results of these experiments support the hypothesis that adsorbed proteins modulate the phlogistic potential of MSU and that the surface activation of humoral mediators contributes to the local inflammatory response.

Adsorption↗
PubMed1982

Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils.

Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.

Cell Adhesion↗