Steeper Dose-response Curve Research Articles

Application of foamed additives to the surface of wet handsheets

We explored the application of foamed wet-end additives onto wet handsheets to qualify our method of application and to demonstrate the method’s usefulness for prescreening additives and foaming agents for packaging applications. We modified a laboratory drawdown coating machine to allow coating of wet handsheets with foamed additives. Initial sheet solids were adjusted to a target of 8%–25% by vacuum. Foam layer thickness was set mechanically. After application, the foamed additives were drawn into the sheet with vacuum. The additive dosage was adjusted by altering its concentration within the foaming formulation. We evaluated more than 100 foaming agents and 10 strength additives, comparing wet-end and foam-assisted addition with no addition on recycled linerboard and virgin linerboard furnish. Foam-assisted addition typically displayed a much steeper dose-response curve and much higher maximum strength levels than wet-end addition. Our results suggest potential target applications for this technology, such as lightweighting, and improved strength performance in mills with relatively closed water systems, where strength aids added into the wet end are adversely influenced by accumulation of inorganic ions and organic species.

Novel Application of Human Neurons Derived from Induced Pluripotent Stem Cells for Highly Sensitive Botulinum Neurotoxin Detection

Human induced pluripotent stem cells (hiPSC) hold great promise for providing various differentiated cell models for in vitro toxigenicity testing. For Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, several cell models currently exist, but none examine toxin function with species-specific relevance while exhibiting high sensitivity. The most sensitive cell models to date are mouse or rat primary cells and neurons derived from mouse embryonic stem cells, both of which require significant technical expertise for culture preparation. This study describes for the first time the use of hiPSC-derived neurons for BoNT detection. The neurons used in this study were differentiated and cryopreserved by Cellular Dynamics International (Madison, WI) and consist of an almost pure pan-neuronal population of predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western blot and quantitative PCR data show that these neurons express all the necessary receptors and substrates for BoNT intoxication. BoNT/A intoxication studies demonstrate that the hiPSC-derived neurons reproducibly and quantitatively detect biologically active BoNT/A with high sensitivity (EC(50) ∼0.3 U). Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A complex was demonstrated, and BoNT/A specificity was confirmed through antibody protection studies. A direct comparison of BoNT detection using primary rat spinal cord cells and hiPSC-derived neurons showed equal or increased sensitivity, a steeper dose-response curve and a more complete SNARE protein target cleavage for hiPSC-derived neurons. In summary, these data suggest that neurons derived from hiPSCs provide an ideal and highly sensitive platform for BoNT potency determination, neutralizing antibody detection and for mechanistic studies.

Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors.

Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.

Nociceptin/Orphanin FQ Receptor Activation Attenuates Antinociception Induced by Mixed Nociceptin/Orphanin FQ/μ-Opioid Receptor Agonists

Activation of brain nociceptin/orphanin FQ (NOP) receptors leads to attenuation of mu-opioid receptor (MOP receptor)-mediated antinociception. Buprenorphine, a high-affinity partial MOP receptor agonist also binds to NOP receptors with 80 nM affinity. The buprenorphine-induced inverted U-shaped dose-response curve for antinociception may be due to NOP receptor activation, given that, in the presence of the NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J113397), or in NOP receptor knockout mice, buprenorphine has a steeper dose-response curve and acts as a full agonist. To further explore the involvement of the direct activation of NOP receptors by buprenorphine and other compounds that activate both NOP and MOP receptors, the antinociceptive effects of 1-(1-(2,3,3alpha,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one. (SR16435), 3-ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), buprenorphine, pentazocine, and morphine, compounds with varying levels of MOP and NOP receptor affinity and efficacy, were assessed in mice using the tail-flick assay. The ability of the selective NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) to potentiate antinociception induced by the above compounds was examined to investigate whether activation of NOP receptors leads to attenuation of MOP receptor-mediated antinociception. SB-612111 potentiated antinociception induced by buprenorphine and the other mixed NOP/MOP receptor agonists SR16435 and SR16507. However, SB-612111 had no effect on pentazocine or morphine antinociception, two compounds with no NOP receptor-binding affinity. These results further support the hypothesis that activation of NOP receptors can lead to attenuation of MOP receptor-mediated antinociception elicited by mixed NOP/MOP receptor compounds such as buprenorphine, SR16435, and SR16507 and that, although buprenorphine has low efficacy in vitro, it has significant NOP receptor agonist activity in vivo.

Comparison of the dose-dependent activity and paradoxical effect of caspofungin and micafungin in a neutropenic murine model of invasive pulmonary aspergillosis

The safety and concentration-dependent pharmacodynamic characteristics of the echinocandins make them ideal candidates for dosage escalation in the treatment of aspergillosis. However, paradoxical attenuation of antifungal activity with increasing doses has been reported for some echinocandins in experimental models of invasive pulmonary aspergillosis (IPA). We compared the activity of micafungin and caspofungin administered over a wide dosing range that encompasses clinical exposures (0.25-10 mg/kg) in a neutropenic murine model of IPA. Both echinocandins exhibited dose-dependent reductions in fungal burden; however, caspofungin displayed a relatively steeper dose-response curve with a modest paradoxical increase in fungal burden that was not seen in micafungin-treated animals. Equivalent activity was observed with both echinocandins at daily doses ranging from 4 to 10 mg/kg. Both micafungin and caspofungin exhibit dose-dependent pharmacodynamic activity in vivo in the treatment of neutropenic IPA. Both echinocandins were equivalent at dosages > or = 4 mg/kg day.

Age and Sex Differences in Malignant Mesothelioma After Residential Exposure to Blue Asbestos (Crocidolite)

Background:Blue asbestos was mined and milled at Wittenoom, Western Australia, from 1943 until 1966. Various public records were used to establish a cohort of residents of the nearby township. Mine tailings were distributed throughout the town.Aims:To report the incident number of malignant mesotheliomas that have occurred in residents of the town who did not work at the mine or mill; and to determine if female subjects are more susceptible to asbestos exposure than male subjects, and if children are more susceptible than adults.Subjects and methods:A total of 4,768 residents of the town of Wittenoom have been followed up in cancer and death registries.Results:There were 67 cases of mesothelioma, and 64 deaths with mesothelioma to the end of 2002. The mortality rate with mesothelioma increased with increasing residence duration, time since first exposure, and estimated cumulative exposure. The mesothelioma mortality rate was consistently lower for female subjects when compared with male subjects, but the dose-response curve was steeper for female subjects. The rate was lower in those first exposed as children compared with those first exposed at ≥ 15 years of age. The dose-response slope for asbestos exposure and mortality from mesothelioma was not different between those who were first exposed as children than those who were first exposed at ≥ 15 years of age.Conclusions:Former residents of a crocidolite mining town have a high rate of mesothelioma. The rate is higher in male subjects and those ≥ 15 years of age at first exposure, but women have a steeper dose-response curve.

Single-isomer drugs: true therapeutic advances.

Chirality is one of the main features of biology, and many of the processes essential for life are stereospecific, meaning that one out of two or more isomers may work best in a particular physiological situation. Could this be used in drug development and result in any clinical relevance and true therapeutic advance? There are occasions when the development of one of the isomers might be expected to be advantageous: for example, only one of the isomers may be active, only one of the isomers may cause adverse effects, or one of the isomers may have more advantageous pharmacological properties. As an example of the last, the successful development of esomeprazole will be described. Before the introduction of esomeprazole, the proton pump inhibitor omeprazole was the standard treatment for gastric acid-related diseases, such as gastro-oesophageal reflux disease. A serious type of gastro-oesophageal reflux disease is erosive oesophagitis, an increasingly common condition that may lead to life-threatening complications. Doubling the standard dose of omeprazole from 20 to 40 mg did not improve healing rates (74% versus 75%), and thus a substantial proportion of patients remained unhealed with standard treatment. The (S)-isomer of omeprazole, esomeprazole, was shown to heal more patients than omeprazole as a result of unique metabolic properties that clearly differentiates esomeprazole from omeprazole, the racemate. At comparable doses, these properties lead to several clinical advantages: higher bioavailability in extensive metabolisers (the majority of patients), lower exposure in poor metabolisers, less interindividual variation and a steeper dose-response curve at steady state resulting in a more pronounced inhibition of gastric acid secretion. Esomeprazole has been studied clinically for a variety of acid-related conditions, showing that the compound is as well tolerated and more effective with regard to healing and symptom relief than the recommended treatment with omeprazole. Thus, from this example it is clear that the exploration and development of single-isomer drugs may bring significant advances in treatment options.

THE DISTRIBUTION OF THE MAXIMUM LIKELIHOOD ESTIMATOR IN UP-AND-DOWN EXPERIMENTS FOR QUANTAL DOSE-RESPONSE DATA

Standard maximum likelihood logistic or probit regression has been used in biopharmaceutical practice for inference about tolerance threshold distributions in situations where subjects (patients) have been allocated doses according to an up-and-down design. For example, a steeper dose-response curve than expected was reported in one such study. This article demonstrates that the maximum likelihood estimator systematically and considerably exaggerates the regression parameter with moderately large sample sizes. Thus a probable explanation for finding a steeper curve than expected is the method used to analyze the experiment, that is, the bias in the maximum likelihood estimator. An additional consequence of this bias is that the mean/median/ED50 are estimated with a misleading precision. In particular, confidence intervals are much too narrow. As a conclusion, we warn against conventional logistic or probit regression in combination with up-and-down designs.

Molecular chemotherapy for breast cancer.

Gene therapy for breast cancer initially involves local or systemic delivery. Local delivery may be intrapleural or via direct injection to lesions. However, systemic delivery remains the greatest challenge with targeting, although methods using antibodies or growth factor receptor ligands have been demonstrated in preclinical models. This review focuses on the next step of using tissue-specific promoters such as Muc-1, CEA, PSA, HER-2, Myc, L-plastin and secretory leukoproteinase inhibitor promoters. All of these have demonstrated differential upregulation in breast cancer and additional specificity may be obtained by using physiological stimuli that are more frequently expressed in cancers, such as glucose regulated promoters and hypoxia response elements or radiation inducible elements. Amongst the later are the EGR-1, p21 and tissue type plaminogen activator promoters. Potential therapy genes include the prodrug activation system 5-fluorocytosine and other analogues of antimetabolites, but all of these need gap junctions to transfer the phosphorylated metabolites. Other approaches involving more freely diffusible products include cyclophosphamide, ifosfamide and thymidine phosphorylase to activate 5-deoxy-5-fluoruridine to fluorouracil. The bystander effect is important both for cell killing and for immunological and antivascular effects. Breast cancer is one type of tumour where a major clinical research effort is underway using local delivery methods. For prodrug activation systems, the use of human enzymes is desirable to prevent an immunological response that would eventually eliminate cells producing the prodrug activation system. The use of alkylating agents has an advantage over antimetabolites in that they are cytotoxic to cycling and noncycling cells, and the cytotoxic products can diffuse across cell membranes without the need for gap junctions. They also have a much steeper dose response curve than antimetabolites.

Sensitizing potential of phenyl isocyanate in MDI products

Background:Blue asbestos was mined and milled at Wittenoom, Western Australia, from 1943 until 1966. Various public records were used to establish a cohort of residents of the nearby township. Mine tailings were distributed throughout the town.Aims:To report the incident number of malignant mesotheliomas that have occurred in residents of the town who did not work at the mine or mill; and to determine if female subjects are more susceptible to asbestos exposure than male subjects, and if children are more susceptible than adults.Subjects and methods:A total of 4,768 residents of the town of Wittenoom have been followed up in cancer and death registries.Results:There were 67 cases of mesothelioma, and 64 deaths with mesothelioma to the end of 2002. The mortality rate with mesothelioma increased with increasing residence duration, time since first exposure, and estimated cumulative exposure. The mesothelioma mortality rate was consistently lower for female subjects when compared with male subjects, but the dose-response curve was steeper for female subjects. The rate was lower in those first exposed as children compared with those first exposed at ≥ 15 years of age. The dose-response slope for asbestos exposure and mortality from mesothelioma was not different between those who were first exposed as children than those who were first exposed at ≥ 15 years of age.Conclusions:Former residents of a crocidolite mining town have a high rate of mesothelioma. The rate is higher in male subjects and those ≥ 15 years of age at first exposure, but women have a steeper dose-response curve.

The Importance of Enzyme Inhibition Kinetics for the Effect of Thrombin Inhibitors in a Rat Model of Arterial Thrombosis

The relation between the antithrombotic effect in vivo, and the inhibition constant (Ki) and the association rate constant (k(on)) in vitro was investigated for eight different thrombin inhibitors. The carotid arteries of anaesthetized rats were exposed to FeCl3 for 1 h, and the thrombus size was determined from the amount of incorporated 125I-fibrinogen. The thrombin inhibitors were given intravenously, and complete concentration- and/or dose-response curves were constructed. Despite a 50,000-fold difference between the Ki-values comparable plasma concentrations of hirudin and melagatran were needed (0.14 and 0.12 micromol l(-1), respectively) to obtain a 50% antithrombotic effect (IC50) in vivo. In contrast, there was a comparable in vitro (Ki-value) and in vivo (IC50) potency ratio for melagatran and inogatran, respectively. These results can be explained by the concentration of thrombin in the thrombus and improved inhibition by the low-molecular-weight compounds. For all eight thrombin inhibitors tested, there was an inverse relationship between k(on)-values in vitro and the slope of the dose response curves in vivo. Inhibitors with k(on)-values of < 1 x 10(7) M(-1) s(-1) gave steep dose response curves with a Hill coefficient > 1. The association time for inhibition of thrombin for slow-binding inhibitors will be too long to give effective antithrombotic effects at low plasma concentrations, but at increasing concentrations the association time will decrease, resulting in a steeper dose-response curve and thereby a more narrow therapeutic interval.

Metabolism and storage ofp,p? dicofol in American kestrels (Falco sparverius) with comparisons to Ring Neck Doves (Streptopelia risoria)

Residues ofp,p′ dicofol,p,p′ dichlorobenzophenone (p,p′ DCBP),p,p′ dichlorobenzhydrol (p,p′ DCBH), 1,1-bis (4-chlorophenyl)-2,2-dichloroethanol (p,p′ DCD) and 1,1-bis (4-chlorophenyl)-2,2 dichloroethylene (p,p′ DDE) are reported in liver, fat, brain and oviducts of American kestrels exposed top,p′ dicofol. Breeding female kestrels were exposed to oral gavage dosages of 3.0 and 0.3 mg dicofol/kg body weight per day, equivalent to 1 or 10 mg dicofol/kg diet, for 39 days. Kestrels exhibited an eggshell thinning response similar to that produced by DDE, under these exposures. The major hepatic metabolite of dicofol in kestrels was DCD, but most organochlorine stored in tissues was in the form of dicofol. DDE was not a major metabolite of dicofol in kestrels. Compared with doves, kestrels had a significantly reduced metabolic capacity to transform dicofol to DCBP and DCBH, and had a steeper dose-response curve for dicofol-induced eggshell thinning.

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA)

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 × 10 9 to 2.2 × 10 10 1/mol. When mixed, these antibodies completely prevented the binding of rabbit and sheep polyclonal antibodies raised against erythrocyte Cu/Zn SOD. Whereas one antibody was directed against a common homology region of bovine and human Cu/Zn SOD, all the other antibodies reacted exclusively or preferentially with human Cu/Zn SOD. Only one epitope on the human Cu/Zn SOD molecule was accessible at two different sites as demonstrated in a homologous two-site assay with one and the same antibody used as both capture and indicator antibody. In the indirect two-site assay with unlabelled monoclonal antibodies, an additive effect with a steeper dose-response curve was obtained by mixing antibodies against different epitopes. A super-rapid one-step two-site enzyme immunoassay (overall duration 20 min) was established with antibodies against two different epitopes. Its detection limit was 0.5 μg SOD/1.

Value of a revised procainamide test in the Wolff-Parkinson-White syndrome

A shortest preexcited RR interval < 250 ms during atrial fibrillation identifies the patient with Wolff-Parkinson-White syndrome potentially at risk for ventricular fibrillation. Loss of preexcitation after infusion of up to 10 mg/kg of procainamide during sinus rhythm has been reported to correlate with a slow ventricular response during atrial fibrillation and has been proposed as a noninvasive test to establish risk of sudden death in these patients. Others have failed to establish this relation and have questioned the usefulness of the procainamide test. Such conflicting results were hypothesized to be a result of differing dosages and methodology. Consequently, this study tested the effect of incremental doses of procainamide (to a cumulative dose of 1 g) on the anterograde effective refractory period of the accessory pathway and related the reliability of the procainamide test to the dose at which preexcitation was lost. The effect of procainamide on the anterograde effective refractory period of the accessory pathway was dose dependent; patients who lost preexcitation had a steeper dose-response curve. Loss of preexcitation by a cumulative dose of 550 mg provided the best balance for sensitivity (60%) and specificity (89%) in identifying patients with preexcited shortest RR >250 ms. Specificity fell steeply after this dosage and higher doses were not useful. The diagnostic accuracy of the procainamide test is critically related to dosage and method of infusion.

Immunoradiometric assay of carcinoembryonic antigen with use of avidin-biotin labeling.

In evaluating an enzyme-linked immunoassay of carcinoembryonic antigen (CEA) we found that the IgG fraction of polyclonal anti-CEA antibodies (DAKO) bound very well to the walls of polypropylene test tubes. We therefore developed an immunoradiometric CEA assay based on this binding of polyclonal anti-CEA antibody. We biotinylated a commercially available monoclonal antibody (Hybritech) and bound this to the CEA-anti-CEA bound to the tube wall. For detection we used 125I-labeled streptavidin. In comparison with several immunoassays for CEA this system offered several advantages such as greater linearity of the standard curve (from 0 to 74 micrograms/L), a steeper dose-response curve, and smaller coefficients of variation in the clinically useful range. This assay system may be used for other large molecules, so that only one tracer, the 125I-labeled streptavidin, has to be labeled; thus the technique seems suited for several different assays.

Corticotropin-Releasing Activity in the Rat Gastric Antrum

Rat gastric antrum, duodenum, pancreas, and spleen were extracted in acetic acid, treated with acetone, and purified on a C-18 cartridge. These extracts, in a dose equivalent to one respective organ, were examined for CRF bioactivity in vitro using rat half pituitaries, with gastric antrum extract showing a significant CRF activity. The antrum extract showed a dose-related CRF activity in vitro using rat pituitary cell culture, and the dose-response curve appeared to be parallel with that of synthetic rat hypothalamic CRF. Subsequent ion-exchange chromatography on a SP-Sephadex column showed that antrum CRF coeluted with basic materials (SP-III fraction), while rat hypothalamic CRF coeluted with weakly basic materials (SP-II fraction). The SP-III fraction was further purified by gel filtration on Sephadex G-50. CRF activity was eluted in two areas: large mol wt fraction (10,000-15,000) and small mol wt fraction (1500-2000). Hypothalamic CRF was eluted between them. The CRF activities of the two fractions were completely abolished by trypsin digestion, suggesting a peptide nature. The large molecular weight fraction exhibited a steeper dose-response curve than the hypothalamic CRF in vitro using cell culture, and the response to a dose equivalent to two antra exceeded the maximum response exhibited by the hypothalamic CRF. However, the fraction failed to increase serum corticosterone when injected in pharmacologically blocked rats. On the other hand, the small molecular weight fraction showed a lesser CRF activity and a similar dose-response curve to that of the hypothalamic CRF as tested in vitro. This fraction significantly stimulated corticosterone secretion in vivo as well. The small molecular weight activity did not appear to be due to other peptides or amines which have been reported as causing ACTH release. Although the physiological roles of the small molecular weight antrum CRF are unknown, it is possible that this CRF plays a role during stress as a tissue CRF.

Toxic effects of some xanthine derivatives with special emphasis on adverse effects on rat testes

Testicular toxicity and effects on thymus and body weights of 4 xanthine derivatives (D4026: 1,8-dimethyl-3-phenylxanthine, D4152: 8-methyl-3-phenylxanthine, D4160: 1,8-dimethyl-3-(2-methylbutyl)-xanthine, D4173: 8-methyl-3-(2-methylbutyl)-xanthine) were studied in Sprague-Dawley rats and cellular toxicity in human embryonal cells. The effect on toxicity by variation of substituent at positions 1 and 3 was tested. The compounds were administered orally to the rats once a day for 1 month. Mortalities were noted only with D4160. Dose related decreases in body weight gain were found for all substances, but only marginally with D4152. A significant decrease in thymus weight relative to control was observed with all substances, D4152 being the least potent. No effects on testes weights were found with any treatment but histological examination disclosed degeneration of germ producing epithelium of all rats given 100 μmol/kg of D4026 but not at 25 μmol/kg. One rat out of 5 showed testicular damage at 400 μmol/kg of D4173 or D4152. Plasma analysis for unchanged compounds showed significantly higher plasma concentrations at the high dose compared with the low dose with the exception for D4152 showing unexpectedly low levels. In the cellular toxicity test, D4160 was the most potent while D4152 was the least potent. D4026 had a steeper dose-response curve than the others but was less potent than D4160. The 1-methylated xanthine derivatives seemed to be more toxic than the two in position 1 unsubstituted analogues. Mechanisms for testicular toxicity of xanthine derivatives in the rat and clinical relevance of animal data are discussed.

The effect of leukotrienes C 4 and D 4 on the microvasculature of guinea-pig skin

The effects of chemically-synthesised leukotrienes C 4 and D 4 (5(S) hydroxy-6(R)-δ-glutamylcysteinylglycinyl-7,9,11,14-eicosa- 4tetraenoic acid, LTC 4; 5(S) hydroxy-6(R)-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, LTD 4) on the microvasculature have been measured in guinea-pig skin using [ 125I]-albumin accumulation to measure plasma exudation and 133Xe clearance to measure blood flow changes. As previously shown using biosynthetic material, LTD 4 caused vasoconstriction resulting in reduced blood flow. Similarly, LTC 4 was found to have vasoconstrictor activity but was more potent and had a steeper dose-response curve than LTD 4. There was no evidence of conversion of exogenous arachidonic acid to vaso-constrictor activity in the skin in vivo (in the absence of another stimulus): intradermally injected arachidonic acid produced vasodilatation, but induced little change in blood flow in animals pretreated with indomethacin. The vasodilator effect of arachidonic acid is presumed to be due to conversion to either PGE 2 or PGI 2. These results suggest that cyclo-oxygenase is normally active in the skin, whilst lipoxygenase requires activation in some way. As reported in a previous study, LTD 4 induced plasma exudation when injected into the skin, but pronounced responses could only be induced by LTD 4 mixed with a vasodilator prostaglandin such as PGE 2. In contrast, LTC 4 induced no exudation when tested alone and little when PGE 2 was added. However, evidence was obtained that LTC 4 has some permeability-increasing activity which is marked by its potent vasoconstrictor activity.

Serum of lipopolysaccharide-treated mice contains two types of colony-stimulating factor, separable by affinity chromatography.

Serum from mice treated with bacterial lipopolysaccharide (LPS) was fractionated by Con A-Sepharose affinity chromatography, and assayed in vitro for colony-stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A-Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte-macrophage and macrophage colonies. The fraction eluted from the Con A-Sepharose column with alpha-methyl-D-glucopyranoside (pool B) had a steeper dose-response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and poolA. The apparent molecular weights of the two types of CSF were determined by two different gel-filtration procedures. Sephacryl S-200 gel-filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel-filtration on Sepharose CL-6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony-forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6--6.2 mm/hr) compared to the CFC responding to mouse-lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0--4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6--5.2 mm/hour). A time-course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proportion of different colony types depends significantly on the incubation period and suggested that pool tb csf induced an early commitment of CFC towards macrophages differentiation.

Age-Dependence of the X-Ray-Induced Deficiency in DNA Synthesis in HeLa S3 Cells during Generation 1

The radiation-induced deficiency in DNA synthesis in Generation 1 was studied as a function of the age of HeLa S3 cells at the time of exposure to 220 kV x rays in the previous generation (Generation 0). The amount of DNA synthesized is dependent on the stage in the generation cycle at which cells are irradiated. The smallest deficiency (20 to 35 percent after a dose of 500 rad) is observed in cells irradiated in early G1 or early G2, while the greatest deficiency (55 to 70 percent after 500 rad) is found in cells irradiated at mitosis or at the G1/S transition. The high sensitivity of cells at G1/S is also manifested by a steeper dose-response curve. Cells irradiated in late G2, past the point where their progression is temporarily blocked by x rays, synthesize a normal amount of DNA in Generation 1, while cells that are held up in the G2 block exhibit deficient synthesis in the next generation. The extent of the deficiency in early G1 cells can be enhanced by treatment with 1 mM hydroxyurea for several hours immediately following irradiation. The possibility that deficient DNA synthesis is related to cell killing, and the relation betweenmore » the G2 block and deficient synthesis, are discussed. (auth)« less