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PubMed2001

Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells.

During apoptosis, cytochrome c is released from mitochondria to the cytosol to activate a caspase cascade, which commits the cell to the death process. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. To test this theory, we measured directly the dynamic re-distribution of green fluorescence protein (GFP)-tagged cytochrome c and morphological change of mitochondria within living HeLa cells during u.v.-induced apoptosis. We observed that mitochondria did not swell when cytochrome c was released from mitochondria to cytosol during apoptosis. Instead, mitochondria swelled to spherical shapes within 10 minutes of cytochrome c release. This finding strongly suggests that cytochrome c release in apoptosis was not caused by mitochondrial swelling. This conclusion was further supported in two separated experiments using an immunostaining method and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) treatment. In addition, we found evidence that cytochrome c was also released before mitochondrial swelling in apoptosis induced by other cell death-inducing treatments, including tumor necrosis factor (TNF) and actinomycin D.

Amino Acid Chloromethyl Ketones↗
PubMed1996

The inflammatory cytokines. New developments in the pathophysiology and treatment of septic shock.

Bacterial products [lipopolysaccharide (LPS) with Gram-negative bacteria and toxins, superantigens or cell wall fragments with Gram-positive bacteria] are the main activators of the septic shock cascade. These molecules interact with monocytes, macrophages and endothelial cells to produce inflammatory cytokines [tumour necrosis factor (TNF) and interleukins 1 and 6], and may activate other harmful pathways such as the coagulation system, complement cascade and lipid mediators. As a therapeutic strategy, antibodies directed against LPS have been well studied, although, on the whole, the clinical results have been disappointing. Other possible interventions that have not yet been tested clinically include natural intracellular antibacterial proteins (e.g. bacterial permeability-increasing protein) and high density lipoprotein (responsible for detoxifying LPS in the body). The stimulation pathway of responsive cells by bacterial products is also another possible target for intervention. Compounds under investigation include soluble CD14 and antibodies directed against CD14 or LPS binding protein. Antibodies directed against the cytokines are another option. Anti-TNF antibodies are currently being investigated, but conclusive evidence of their activity is still lacking. Soluble receptors (e.g. interleukin-1 receptor antagonist, or soluble TNF receptor) are another possibility; one soluble TNF receptor is still undergoing clinical investigation.

Animals↗
PubMed2003

Byssinosis among jute mill workers.

Although byssinosis in jute mill workers remains controversial, studies in a few jute mills in West-Bengal, India, revealed typical byssinotic syndrome associated with acute changes in FEV1 on the first working day after rest. The present study on 148 jute mill workers is reported to confirm the occurrence of byssinosis in jute mill workers. Work related respiratory symptoms; acute and chronic pulmonary function changes among exposed workers were studied on the basis of standard questionnaire and spirometric method along with dust level, particle mass size distributions and gram-negative bacterial endotoxins. The pulmonary function test (PFT) changes were defined as per the recommendation of World Health Organization and of Bouhys et al. Total dust in jute mill air were monitored by high volume sampling, technique (Staplex, USA), Andersen cascade impactor was used for particle size distribution and personal exposure level was determined by personal sampler (Casella, London). Endotoxin in airborne jute dust was analysed by Lymulus Amebocyte Lysate (LAL) "Gel Clot" technique. Batching is the dustiest process in the mill. Size distribution showed that about 70-80% dust in diameter of < 10 microm, 40-50%, < 5 microm and 10-20%, < 2 microm. Mean endotoxin levels found in hatching, spinning and weaving, and beaming were 2.319 microg/m3, 0.956 microg/ m3, 0.041 microg/m3 respectively and are comparable to the values obtained up to date in Indian cotton mills. Respiratory morbidity study reported typical byssinotic symptoms along with acute post shift FEV1 changes (31.8%) and chronic changes in FEV1 (43.2%) among exposed workers. The group with higher exposure showed significantly lower FVC, FEV1, PEFR and FEF25-75% values. The study confirmed the findings of the earlier studies and clearly indicated that the Indian jute mill workers are also suffering from byssinosis as observed in cotton, flask and hemp workers.

Acute Disease↗
PubMed1998

The endocrine system: an overview.

A plethora of hormones regulate many of the body's functions, including growth and development, metabolism, electrolyte balances, and reproduction. Numerous glands throughout the body produce hormones. The hypothalamus produces several releasing and inhibiting hormones that act on the pituitary gland, stimulating the release of pituitary hormones. Of the pituitary hormones, several act on other glands located in various regions of the body, whereas other pituitary hormones directly affect their target organs. Other hormone-producing glands throughout the body include the adrenal glands, which primarily produce cortisol; the gonads (i.e., ovaries and testes), which produce sex hormones; the thyroid, which produces thyroid hormone; the parathyroid, which produces parathyroid hormone; and the pancreas, which produces insulin and glucagon. Many of these hormones are part of regulatory hormonal cascades involving a hypothalamic hormone, one or more pituitary hormones, and one or more target gland hormones.

Endocrine Glands↗
PubMed1997

D5 dopamine receptors enhance Zn2+-sensitive GABA(A) currents in striatal cholinergic interneurons through a PKA/PP1 cascade.

Cholinergic interneurons have been implicated in striatally mediated associative learning. In classical conditioning paradigms, conditioned stimuli trigger a transient suppression of neuronal activity that is dependent upon an intact dopaminergic innervation. Our hypothesis was that this suppression reflected dopaminergic enhancement of sensory-linked GABAergic input. As a test, the impact of dopamine on interneuronal GABA(A) receptor function was studied by combined patch-clamp recording and single-cell reverse transcription PCR. Activation of D5 dopamine receptors reversibly enhanced a Zn2+-sensitive component of GABA(A) currents. Although dependent upon protein kinase A (PKA) activation, the modulation was blocked by protein phosphatase 1 (PP1) inhibition, suggesting it was dependent upon dephosphorylation. These results establish a novel mechanism by which intrastriatally released dopamine mediates changes in GABAergic signaling that could underlie the initial stages of associative learning.

Animals↗
PubMed2002

Comparison of innovator and generic salbutamol inhalers: a double-blind randomized study of efficacy and tolerance.

Preliminary feedback from physicians and pharmacists in Trinidad suggests that generic pressurized metered dose inhalers (pMDIs) of salbutamol are not as effective as Ventolin and that they have poor patient acceptance. This study was designed to compare the clinical efficacy and tolerance of two generic inhalers available in Trinidad (Asthalin and Salomol) with Ventolin in stable asthmatics. Twenty-one physician-diagnosed stable asthmatics were administered the inhalers in a Latin-square randomized double-blind study with 80% power to identify differences in forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC) and peak expiratory flow rate (PEFR) before and 0.25, 0.5, 1, 2 and 3 h after inhalation. Pulse and blood pressure were recorded at similar time points. Seventeen patients completed the study. Within 15 min basal respiratory function significantly increased following inhalation from all three inhalers with a gradual decline over the observation period. Asthalin produced the highest changes in FEV1, PEFR and the longest duration of effect (p < 0.001). Respiratory function tests did not differ between Ventolin and Salomol. Pulse was not affected by treatments and mean arterial blood pressure fell after Asthalin. Ventolin was not superior to the generic pMDIs in improving pulmonary function. Fifteen patents reported cough sensation after Asthalin. Throat irritation and cough sensation after inhaling Asthalin may negate patient compliance. We suggest that optimizing particle size and cascade impact in the Asthalin inhaler may improve patient tolerance and acceptance with enhanced treatment outcome with cost-efficacy.

Adult↗
PubMed1998

In vitro comparison of beclomethasone and salbutamol metered-dose inhaler aerosols inhaled during pediatric tidal breathing from four valved holding chambers.

STUDY OBJECTIVES: To determine the amount of salbutamol or beclomethasone delivered from metered-dose inhalers (MDIs) in particle sizes appropriate for inhalational treatment with four common holding chambers (AeroChamber, OptiChamber, Space Chamber, E-Z Spacer) when used under simulated pediatric tidal breathing conditions. DESIGN: Five devices of each type were tested with salbutamol (100 microg, Glaxo-Wellcome) and beclomethasone (50 microg, Glaxo-Wellcome) MDIs. Each device was connected to face replicas representative of 7-month-old (infant), 2-year-old (toddler), and 4-year-old (child) children and aerosol inhaled into a cascade impactor (Anderson) using a valve system and simulated tidal breathing patterns representative of children of these ages. MEASUREMENTS AND RESULTS: Amounts of drug inhaled in fine particles varied significantly between devices and ages (p < 0.01), eg, for beclomethasone, from 4 microg (infant, Space Chamber) to 8 microg (toddler, OptiChamber), and for salbutamol, from 18 to 36 microg. The AeroChamber and OptiChamber behaved quite similarly, delivering more drug in the fine particles than the other two devices, and having insignificant variations in these amounts with age (p > 0.05). CONCLUSIONS: Amounts of drug inhaled in fine particles during pediatric tidal breathing from valved holding chambers are dependent on the holding chamber model, the drug used, and the breathing pattern, although dependence on the breathing pattern is relatively small for some of the devices tested.

Administration, Inhalation↗
PubMed1994

Induction of an immune network cascade in cancer patients treated with monoclonal antibodies (ab1). II. Is induction of anti-idiotype reactive T cells (T3) of importance for tumor response to mAb therapy?

The antitumor effector functions of unconjugated monoclonal antibodies (mAb) in cancer therapy are not fully understood. Direct cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent cytolysis and apoptosis have been suggested. Induction of anti-idiotypic (ab2) and anti-anti-idiotypic (ab3) antibodies as well as the corresponding T cells (T2 and T3) has also been proposed to be of therapeutic significance. In this study induction of an immune network cascade in ten patients with colorectal carcinoma, treated with mAb 17-1A (ab1) was assessed. After treatment, all ten patients had anti-idiotypic antibodies and anti-anti-idiotypic antibodies with ab1-like binding specificity while only five of ten patients had T cells corresponding to ab3 (T3) as assessed by a proliferation assay (DNA synthesis), and an assay of interferon gamma production (ELISPOT) (Enzyme-linked immuno SPOT) in vitro or by a delayed-type hypersensitivity reaction in vivo. Purified T cells from four of the five patients with a positive T3 test responded with DNA synthesis after stimulation using human anti-mAb 17-1A anti-idiotypic monoclonal antibodies. These four patients had a clinical response showing a tumor reduction after therapy, while all six patients lacking a proliferative response failed to show tumor regression. Induction of a cell-mediated immune network cascade might accordingly be an important antitumor effector function of mAb and should be considered in the future design of mAb-based therapy protocols in cancer patients.

Adult↗
PubMed2000

In vitro anti-inflammatory activity of iridoids and triterpenoid compounds isolated from Phillyrea latifolia L.

Two iridoids, oleuropeoside and ligustroside, and two triterpenoid compounds, oleanolic acid and ursolic acid, have been isolated from the leaves of Phillyrea latifolia L. (Oleaceae). These compounds were tested for interactions with the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways of arachidonate metabolism in calcium ionophore-stimulated mouse peritoneal macrophages and human platelets, and for their effect on cell viability. Structure-activity relationships obtained for in vitro screening results were discussed. These compounds are capable of exerting inhibitory actions on enzymes of the arachidonate cascade. All compounds assayed showed a significant effect on prostaglandin E2 (PGE2)-release, with inhibition percentages similar to the reference drug indomethacin (IC50 = 0.95 microM). The IC50 values of the active compounds are: oleuropeoside 47 microM, ligustroside 48.53 microM, oleanolic acid 23.51 microM and ursolic acid 60.91 microM. In the leukotriene C4 (LTC4)-assay, only oleanolic acid showed a significant effect (IC50 = 16.79 microM). We also investigated the action of compounds on thromboxane B2 (TXB2)-release induced by calcium ionophore in human platelets. Of all the tested compounds, only ligustroside (IC50 = 122.63 microM) and ursolic acid (IC50 = 50.21 microM) showed a significant effect, although with less potency than the reference drug ibuprofen (IC50 = 1.27 microM). Thus, our compounds possess an array of potentially beneficial anti-inflammatory properties which may, alongside other constituents, contribute to the claimed therapeutic properties of the plant from which they are derived.

Animals↗
PubMed2006

Beta-amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes.

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.

Alzheimer Disease↗
PubMed1988

Chronic in vivo ethanol administration alters the sensitivity of adenylate cyclase coupling in homogenates of rat brain.

The high activities of adenylate cyclase, phosphodiesterase and protein kinases in the synaptic terminals of the central nervous system makes these enzymes prime candidates for the in vivo actions of ethanol. Adult female rats were fed a liquid diet containing ethanol as 35% of the available calories for 6 days. This resulted in a decrease (22-45%) in the basal activity of adenylate cyclase, as determined by cyclic 3',5'-adenosine monophosphate (cAMP) production, in homogenates of all brain areas tested. In these homogenates the ability of guanosine triphosphate and noradrenaline to stimulate basal cyclase activity was severely reduced. These results suggest that ethanol administration causes an uncoupling of the beta-receptor/adenylate cyclase cascade and an interruption of the control of the synthesis of cAMP.

Adenylyl Cyclases↗
PubMed2004

Changes along the male reproductive axis in response to social context in a gonochoristic gobiid, Zosterisessor ophiocephalus (Teleostei, Gobiidae), with alternative mating tactics.

Sexual selection has given rise, in several taxa, to intrasexual variation in male phenotype. While evolutionary studies have provided explanations of the adaptive function of this dramatic male phenotypic diversity, the proximate control of its expression has still to be completely understood. Several observations, primarily from sex-changing species, indicated a major role of social interactions in reproductive axis regulation and consequently in the expression of alternative male phenotypes. Here we documented changes along the male reproductive axis in response to social context in a gonochoristic species, the grass goby Zosterisessor ophiocephalus, where fully functional alternative male mating tactics appear to be expressed as an ontogenetic gradient. In the grass goby, larger and older males dig a nest and perform parental care, while smaller males sneak fertilization during territorial male spawning. Territorial males are characterized by a higher number of gonadotropin-releasing hormone (GnRH) neurons in forebrain preoptic area, smaller testes, larger seminal vesicles, and viscous ejaculates that last longer and contain fewer sperm than those of sneakers. To experimentally investigate the role of social factors in inducing changes along the male reproductive axis, sneakers were tested in two different situations: nesting alone or with ripe females. Sneakers that mated and performed parental care showed dramatic changes in brain, reproductive apparatus morphology, and ejaculate traits. GnRH-immunoreactive cells in forebrain preoptic area increased in number, reaching values typical of wild-caught parental males. Testes size decreased while seminal vesicle size increased and ejaculates showed lower sperm densities. These results were discussed within the framework of the social transduction hypothesis, which predicts that social experience should mediate, through a cascade of internal processes, shifts between morphs throughout life.

Animals↗
PubMed2003

Evidence that nitric oxide-glutamate cascade modulates spinal antinociceptive effect of morphine: a behavioural and microdialysis study in rats.

We evaluated the ability of spinally administered nitric oxide (NO) synthase inhibitor to modulate antinociceptive action of intrathecal (i.t.) morphine in rats by measuring the early and late phases of flinching and licking/biting in the formalin test. To determine the contribution of spinal NO and glutamate, we measured the release of NO metabolites (nitrite/nitrate) and glutamate from the spinal cord in rats, using a microdialysis probe placed in the lumbar space. The i.t. administration of NG-nitro L-arginine methyl ester (L-NAME) produced a dose-dependent reduction in the number of flinches during the late phase, whereas there were no significant alterations in the late phase licking/biting, and early phase flinching and licking/biting. Spinal administration of morphine at low doses produced a significant antinociceptive activity in the early and late phases of the flinching behaviour, whereas higher doses of morphine were required to obtain a significant effect in the licking/biting behaviour during both phases. Combination of L-NAME with morphine resulted in an enhanced reduction in the early and late phase flinching. Enhanced antinociceptive activity was observed in the late phase licking/biting by i.t. combined administration of L-NAME (400 nmol) and morphine (1.25 nmol). In the present study, we have confirmed our prior results that injection of formalin (5.0%) into the plantar surface of the paw evoked a biphasic spinal release of nitrite/nitrate and a transient release of glutamate. Formalin-evoked release of nitrite/nitrate and glutamate was also reduced markedly by i.t. combined administration of L-NAME and morphine. These behavioural and biochemical results suggest that i.t. administered L-NAME may enhance morphine-induced antinociception through an increased inhibition of nitrite/nitrate and glutamate releases evoked by formalin injection at the spinal cord level.

Analgesics, Opioid↗
PubMed2000

Antidepressants: past, present and future.

Since the discovery of first antidepressants in mid-1950's, the field has been intensively studied. Several new classes of compounds emerged and several hypotheses on the mechanism of their action were proposed. The novel antidepressants are either selective and reversible monoamine oxidase inhibitors, (e.g., moclobemide), or selective serotonin reuptake inhibitors (e.g., citalopram or paroxetine), or serotonin and noradrenaline reuptake inhibitors (e.g. , venlafaxine). Recently neuropeptides (e.g., thyrotropin-releasing hormone,TRH) or antagonists of neuropeptide receptors (e.g., tachykinin NK(1) receptor) undergo clinical tests. Several hypotheses proposed the predominant involvement of one or few neurotransmitter receptors in the mechanism of antidepressant action, but it is now assumed that several distinct receptor mechanisms' trigger different but converging intracellular signal cascades that activate transcription factors, which, in turn, promote the expression of genes encoding for proteins, that play a crucial role in restoring of neuronal functions involved in mood regulation.

Animals↗
PubMed2002

Implications for neuroprotective treatments.

Pharmacological neuroprotection against the consequences of seizures can be considered as primary neuroprotection where the object is to diminish the initial insult by suppressing the seizure activity or diminishing the associated ionic fluxes (of which the entry of Na+ and Ca2+ are the most significant), and secondary neuroprotection where the target is some later event in the chain linking ionic changes to altered brain morphology or function. Thus primary neuroprotection is provided by antiepileptic drugs and compounds acting on voltage-sensitive Na+ and Ca2+ channels or on glutamate receptors (NMDA, AMPA/KA or Group I metabotropic). Secondary neuroprotection may be a result of acting on the cascade leading to necrosis (e.g. free radical scavengers, NitricOxide synthase inhibitors, CycloOxygenase-2 inhibitors) or the cascades leading to apoptosis (e.g. MAP-kinase inhibitors, caspase-3 inhibitors). Other approaches may diminish the long-term morphological and functional effects of seizures (e.g. neurotrophin-related therapies). We need improved preclinical tests for identifying novel compounds with potential for providing secondary neuroprotection and antiepileptogenesis. Clinical trials of neuroprotective agents in chronic epilepsy in adults pose major practical difficulties but the severe childhood epilepsies provide opportunities for aggressive testing of novel compounds.

Animals↗
PubMed2003

Peroxynitrite generated in the rat spinal cord induces apoptotic cell death and activates caspase-3.

We previously demonstrated that the peroxynitrite concentration increases after impact spinal cord injury. This study tests whether spinal cord injury-elevated peroxynitrite induces apoptotic cell death. Peroxynitrite was generated at the concentration and duration produced by spinal cord injury by administering S-morpholinosydnonimine through a microdialysis fiber into the gray matter of the rat spinal cord. Fragmented DNA was visualized by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling. Transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons were quantitated by counting the transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and neuron-specific enolase double-stained neurons along the fiber track in the sections removed at 6, 12, 24 and 48 h post-peroxynitrite exposure. Peroxynitrite significantly increased transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons at all time points examined (P< or =0.001) compared with artificial cerebrospinal fluid controls (Two-way analysis of variance followed by Tukey test), peaking at 24 h post-exposure. Electron microscopic observation of characteristic features of apoptosis confirmed peroxynitrite-induced neuronal apoptosis. Total transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive cells were counted in areas near and 0.2 mm away from the fiber track. The counts both peaked at 24 h with no significant difference between the two areas. However, at 6 and 12 h post-exposure the counts were significantly higher near than away from the fiber track (P=0.03 and P=0.007 respectively, paired t test). Immunohistochemical staining indicates caspase-3 was activated by peroxynitrite; this activation peaked at 6 h post-exposure, suggesting that activation of caspase-3 might be an early event in the apoptotic cell death cascade. We conclude that 1) peroxynitrite generated in the cord at the level produced by spinal cord injury induces neuronal apoptosis, indicating a role for peroxynitrite in secondary spinal cord injury; 2) caspase activation might be involved in peroxynitrite-induced neuronal apoptosis; 3) therefore removal of peroxynitrite should reduce secondary cell death after spinal cord injury.

Animals↗
PubMed1998

Influence of flow rate on aerosol particle size distributions from pressurized and breath-actuated inhalers.

Particle size distribution of delivered aerosols and the total mass of drug delivered from the inhaler are important determinants of pulmonary deposition and response to inhalation therapy. Inhalation flow rate may vary between patients and from dose to dose. The Andersen Sampler (AS) cascade impactor operated at flow rates of 30 and 55 L/min and the Marple-Miller Impactor (MMI) operated at flow rates of 30, 55, and 80 L/min were used in this study to investigate the influence of airflow rate on the particle size distributions of inhalation products. Total mass of drug delivered from the inhaler, fine particle mass, fine particle fraction, percentage of nonrespirable particles, and amount of formulation retained within the inhaler were determined by ultraviolet spectrophotometry for several commercial bronchodilator products purchased in the marketplace, including a pressurized metered-dose inhaler (pMDI), breath-actuated pressurized inhaler (BAMDI), and three dry powder inhalers (DPIs), two containing salbutamol sulphate and the other containing terbutaline sulphate. Varying the flow rate through the cascade impactor produced no significant change in performance of the pressurized inhalers. Increasing the flow rate produced a greater mass of drug delivered and an increase in respirable particle mass and fraction from all DPIs tested.

Aerosols↗
PubMed1995

Purification and partial characterization of an early pregnancy factor-induced suppressor factor (EPF-S1).

PROBLEM: The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF-S1 and EPF-S2 (previously estimated M(r) 15,000 and 55,000 respectively). The activity of the former is MHC-restricted while the latter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF-S1 and EPF-S2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF-S1 to homogeneity. METHOD: Congenic mouse strains were used to map the genetic restriction of EPF-S2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF-S1 induces EPF-S2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF-S1, from immune ascites (from growth in athymic mice of the anti-EPF-S1 producing rat-mouse hybridoma R2T gamma, in which EPF-S1 is complexed to antibody). Techniques used were acidification followed by application to Sep-pak C18 cartridges, high performance cation-exchange chromatography and two reversed-phased HPLC steps on a C3 column. Purified material was analyzed by SDS-PAGE and Edman degradation. RESULTS: Approximately 10 micrograms EPF-S1 were isolated fom 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS-PAGE, where it ran as a single band of approximate M(r) 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N-blocked. CONCLUSION: Definitive primary characterization of EPF-S1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.

Animals↗