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Biomedical subjects

E D Clark

Publications and source records attributed to E D Clark.

14 recordsLinked to original sources
PubMed2001

Protein refolding for industrial processes.

Inclusion body refolding processes are poised to play a major role in the production of recombinant proteins. Improving renaturation yields by minimizing aggregation and reducing chemical costs are key to the industrial implementation of these processes. Recent developments include solubilization methods that do not rely on high denaturant concentrations and the use of high hydrostatic pressure for simultaneous solubilization and renaturation.

Disulfides↗
PubMed2000

Influence of MHC background on the antibody response to detergent enzymes in the mouse intranasal test.

The mouse intranasal test (MINT) was developed to assess the immunogenic potential of detergent enzymes. The BDF1 mouse (H-2(b/d)), a cross of C57Bl/6 (H-2(b)) x DBA/2 (H-2(d)) has been used for most of the development work. Preliminary data in the CB6F1(H-2(d/b)), a cross of Balb/c (H-2(d)) x C57Bl/6 showed that this strain was similar to the BDF1 in its response to enzymes. These data also showed that the parental strains responded differently to the enzymes. To understand better the influence the major histocompatibility complex (MHC) background has on immune responses to enzymes, 3 different enzymes were tested in 4 inbred strains (C57Bl/6, DBA/2, Balb/c, and C57Bl/10), 2 hybrid strains (BDF1 and CB6F1), and 2 congenic strains (Balb.B10 and B10.D2). BDF1 mice rank enzymes the same as the guinea pig, which in turn correlates with sensitization in occupationally exposed humans. The ranking is based upon the dose of enzyme needed to give one-half maximal IgG1 antibody response (ED(50)) where Termamyl is more potent than Alcalase, which is equipotent to Savinase. The H-2(d) strains ranked the enzymes the same as the BDF1 but generated ED(50)s for the proteases that were one order of magnitude greater than the BDF1 ED(50)s. The response to Termamyl was the same in the two F1 strains and the H-2(d) strains. The H-2(b) strains did not rank the enzymes the same as BDF1 but the ED(50)s for the proteases were similar to the ED(50)s in the F1 strains. The response to Termamyl in the H-2(b) strains was lower than the response in the F1 and H-2(d) strains. Initial data show that the inbred strains will make enzyme-specific IgE antibody to high doses of enzyme with DBA/2 > Balb/c > C57Bl/6 in terms of the robustness of the response. The IgG1 responses in the congenic strains were similar to the responses in the H-2 matched strains. In addition, the antibody response to enzymes was consistent regardless of the source of BDF1 mice. The responses to these enzymes are clearly MHC linked with a role for Class II I-E molecules indicated. The current data strongly support the use of the F1 hybrid as an appropriate strain for evaluating allergic responses to enzymes.

Administration, Intranasal↗
PubMed1997

Proteolytic detergent enzymes enhance the allergic antibody responses of guinea pigs to nonproteolytic detergent enzymes in a mixture: implications for occupational exposure.

A guinea pig intratracheal test was developed to assess the respiratory allergenicity of enzymes used in the detergent industry. Information gained from this test was used in a process for setting operational exposure guidelines to protect worker health. Mixtures of enzyme proteins were given to guinea pigs once per week for 10 weeks to determine whether there were interactions among enzymes that affected the induction of antibody responses to each enzyme in the mixture. Passive cutaneous anaphylaxis antibody titers against each enzyme were measured in sera. Mixtures of two or three enzymes always consisted of a protease (Alcalase, Savinase; Novo Industri A/S) with an alpha-amylase (Termamyl; Novo Industri A/S), a lipase (Lipolase; Novo Industri A/S), or both. Control animals were exposed to single enzymes. The antibody titers to Termamyl and Lipolase were significantly greater in animals dosed with the protease-containing mixtures as compared with control animals dosed with a single enzyme. Antibody titers to the protease were unchanged in the presence of additional enzymes in the mixture. Complete inactivation of protease activity abrogated the enhanced antibody response to Lipolase. Inhalation exposure of guinea pigs to a mixture of Alcalase and Lipolase also resulted in higher antibody titers to Lipolase as compared with animals exposed by inhalation to Lipolase alone, showing that the enhanced response was not due to intratracheal delivery of antigen to the respiratory tract. These results show that proteolytic enzymes in a mixture enhance antibody responses to other enzymes. This should be considered when defining exposure guidelines for protease-containing enzyme mixtures.

Administration, Inhalation↗
PubMed1995

Surface charge, fluidity, and calcium uptake by rat intestinal brush-border vesicles.

Biological membrane outer surfaces are negatively charged and interact with positively charged calcium ion during calcium uptake. Positively charged polycations such as polyarginine bind to membranes with high affinity, displacing bound calcium from the membrane. We tested the effect of polyarginine on uptake of calcium by brush-border membrane vesicles and examined the responses in terms of membrane fluidity by electron paramagnetic resonance (EPR). Polyarginine inhibited the saturable component of calcium uptake by a mechanism combining inhibition characteristics of strontium (competitive) and magnesium (non-competitive). Unlike the inhibition of non-saturable calcium uptake by strontium and magnesium, polyarginine increased kD, the rate constant for non-saturable calcium uptake, by a concentration dependent mechanism. These effects of polyarginine on calcium uptake were associated with decreased membrane fluidity at the uptake temperature. These findings are consistent with a role for surface negative charge in determining both saturable and non-saturable calcium uptake. Increased membrane fluidity is associated with decreased saturable and increased non-saturable calcium uptake. Although increased fluidity might be involved in the increased kD for non-saturable uptake, the concentration-specific stimulating effect of polyarginine suggests a gating mechanism.

Animals↗
PubMed1995

Effect of nitroblue tetrazolium on NO synthase and motor function of opossum esophagus.

Nitric oxide mediates neuromuscular events in the opossum esophagus. The NADPH diaphorase stain is used to localize nitric oxide synthase-containing enteric neurons. Cells stain by the NADPH diaphorase technique because they reduce nitroblue tetrazolium to the visible formazan. The effects of nitroblue tetrazolium on neuromuscular function and nitric oxide synthase of esophageal muscle were studied. The NADPH diaphorase stain was performed. Nitroblue tetrazolium inhibited lower esophageal sphincter relaxation, abolished the latency gradient of the off response, and inhibited nitric oxide synthase. The NADPH diaphorase technique stained myenteric plexus nerve cell bodies and nerve processes. Nitroblue tetrazolium is not a nonspecific muscle or nerve toxin, as nerve-mediated cholinergic responses, responses to exogenous nitric oxide, and responses to myogenic stimulation were maintained after nitroblue tetrazolium abolished the off response and lower esophageal sphincter relaxation. Nitroblue tetrazolium inhibits nitric oxide-mediated events and nitric oxide synthase. It stains neurons in the esophageal myenteric plexus.

Animals↗
PubMed1994

Characterization of nitric oxide synthase in the opossum esophagus.

BACKGROUND/AIMS: Nitric oxide mediates the nonadrenergic, noncholinergic neural control of esophageal motor function. The purpose of this study was to characterize the NO synthase found in the muscularis propria of the opossum esophagus and determine its distribution along the esophagus. METHODS: Esophageal muscle was homogenized in HEPES buffer and ultracentrifuged. The supernatant was exposed to [3H]L-arginine. The [3H]L-citrulline produced by NO synthase was separated from [3H]L-arginine with a Dowex AG 50 W-X8 column (Biorad, Hercules, CA). Assays were performed in the presence and absence of Ca2+ or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The distribution of NO synthase activity along the esophagus was determined. RESULTS: The apparent Michaelis constant and maximum velocity of NO synthase were 7.5 +/- 1.4 mumol/L L-arginine and 76.0 +/- 17.3 pmol.mg protein-1.min-1, respectively. The enzyme required both Ca2+ and NADPH for activity. Smooth muscle tissue from the lower esophageal sphincter and the esophageal body 1-2 cm or 5-6 cm above the lower esophageal sphincter differed little in enzymatic activity, ranging from 0.97 to 1.27 pmol.mg wet wt-1.min-1. Striated muscle had less activity with 0.40 pmol.mg wet wt-1.min-1. CONCLUSIONS: These data indicate the presence of a constitutive NO synthase in the esophagus of the opossum.

Amino Acid Oxidoreductases↗
PubMed1994

Induction of type I hypersensitivity in guinea pigs after inhalation of phthalic anhydride.

Guinea pigs were exposed through inhalation to phthalic anhydride (PA) dust at 0.5, 1.0, and 5.0 mg/m3, 3 hours/day for 5 consecutive days. Inhalation challenge with aerosolized phthalic anhydride-guinea pig serum albumin (PA-GPSA) conjugate elicited immediate-onset respiratory reactions in animals exposed to all three levels of dust. Inhalation challenge of a subgroup of animals with phthalic anhydride dust did not elicit an immediate response, as measured by changes in respiratory frequency and plethysmograph pressure. Serologic studies showed that these animals had allergic IgG1a antibody to PA-GPSA. There was a dose-dependent increase in specific IgG antibody activity, as measured by ELISA. Animals exposed to and challenged with 5.0 mg/m3 PA dust had significant numbers of hemorrhagic lung foci. Those animals with the greatest number of foci had high IgG antibody activity to PA, as measured by ELISA. This study showed that exposure to levels of PA dust as low as 0.5 mg/m3, below the current threshold limit value of 6.0 mg/m3, can sensitize animals to produce allergic antibody.

Administration, Inhalation↗
PubMed1993

Brush border calcium uptake in short-bowel syndrome in rats.

Resection of distal small intestine causes calcium malabsorption in humans and in a rat model of 50% distal resection. We tested the hypothesis that this calcium malabsorption is caused in the rat model by a brush border defect. We compared brush border membrane vesicles from the proximal small intestine of control (transection and anastomosis at mid-small intestine) with distally resected rats. Mucosal protein was 25% greater in the resected group and the vesicles were enriched 37-fold in sucrase activity when compared with homogenate. Kinetic constants Vmax (maximal initial rate of saturable calcium uptake at infinite concentration), kT (calcium concentration for saturable calcium uptake rate at half Vmax), and KD (rate constant for nonsaturable calcium uptake per unit concentration) were slightly but not significantly greater in the resected as compared with the transected group, ruling out the brush border as the cause for decreased transmucosal calcium transport.

Animals↗
PubMed1992

A tier approach for evaluating the respiratory allergenicity of low molecular weight chemicals.

A multi-level approach for evaluating low molecular weight chemicals as respiratory sensitizers is proposed. The approach involves four levels of testing that utilize both in vitro and in vivo methods. Tier 1 evaluates structure-activity information to determine if the chemical can covalently modify carrier molecules. It also includes a literature search to determine if the compound belongs to a family of chemicals that has been reported to induce hypersensitivity. Tier 2 tests the chemical's potential to haptenate carrier molecules (i.e., protein) under in vitro conditions. Positive results in Tiers 1 and 2 lead to testing in a guinea pig injection model to assess chemical immunogenicity (Tier 3). A positive result at this level leads to testing in a guinea pig inhalation model to address questions about relevant routes of chemical exposure and allergenicity (Tier 4). Tier 4 results are used in determining safe chemical exposure levels. We have evaluated three chemicals using this scheme: phthalic anhydride, reactive black b dye, and toluene diisocyanate. All three have reactive groups and haptenate protein in vitro. They induce a humoral immune response when injected into guinea pigs at equimolar concentrations, and they sensitize animals via inhalation exposure. The severity of the response (antibody titer and respiratory reactivity) can be used to rank-order the chemicals in terms of allergenic "potency." Our data indicate that this approach can detect chemical allergens and can be used to characterize them as moderate or strong respiratory sensitizers.

Administration, Inhalation↗
PubMed1990

ELISA for human IgE antibody to subtilisin A (Alcalase): correlation with RAST and skin test results with occupationally exposed individuals.

An ELISA was developed to detect specific IgE antibody to the Bacillus subtilis-derived proteolytic detergent enzyme, subtilisin A (Alcalase), in sera from exposed detergent workers. Workers in the detergent industry are exposed via inhalation to low levels of the enzyme dust in the presence of detergent dust. Chemically inactivated Alcalase was used as the test antigen. Significant binding of IgE antibody to the immobilized enzyme was detected in the ELISA. The binding of allergic antibody to Alcalase was specifically inhibited in a dose-dependent manner by preincubating sera with 0.1 to 100 micrograms of inactivated Alcalase. Binding of IgE antibody to Alcalase could not be inhibited by two other inactivated bacterial proteases, Savinase and Esperase, derived from different Bacillus species. ELISA and skin test results demonstrated total agreement for 27 of 31 samples (87%), whereas RAST and skin test results demonstrated total agreement for only 24 of 31 samples (77%), indicating that the ELISA was more sensitive than the RAST. We conclude that the ELISA is a sensitive, fast, alternative to the RAST for detection of Alcalase-specific IgE antibody in detergent enzyme-exposed workers.

Antibodies↗
PubMed1976

Effect of first part of duodenum on gastric emptying in dogs: response to acid, fat, glucose, and neural blockade.

Five dogs were prepared, each with a gastric and duodenal fistula (5 cm distal to the pylorus), to study the inhibitory role of the first 5 cm of the duodenum on gastric emptying. The basic design of the experiments was to instill the test meal (300 ml at 37 degrees C, containing phenol red 40 mg 1(-1)) into the stomach and collect it at 1- or 2-min intervals for 10 or 20 min from the duodenal fistula. As the test meal emptied from the stomach it bathed the first 5 cm of duodenum and thus stimulated the appropriate receptor. A Foley catheter with an inflated balloon prevented passage into the second part of the duodenum. Test meals of hypertonic glucose (15%, 865 milliosmoles kg-1) or 20 and 80 mM of sodium oleate emptied at the same rate as water when allowed to bathe the first 5 cm of duodenum, whereas test meals of 100 mM of HCl were slowed. In further studies using neural blocking agents, the emptying of water meals was slowed with subcutaneous atropine sulfate (0.03 and 0.15 mg kg-1), intravenous hexamethonium chloride (10 mg kg-1), and norepinephrine bitartrate (0.04 mg kg-1). The emptying of 100 to 120 mM HCl meals was slowed by subcutaneous atropine sulfate (0.03 and 0.15 mg kg-1), intravenous norepinephrine bitartrate (0.04 mg kg-1), and the intravenous alpha-receptor blocking agents phenoxybenzamine HCl (2 mg kg-1) and phentolamine (2 mg kg-1), was unaffected by intravenous hexamethonium chloide (10 mg kg-1), and was unchanged (1.0 mg kg-1) or slightly slowed (2.0 mg kg-1) by the beta-receptor blocker propranolol. In contrast, acid test meals were emptied at the same rate as water when treated with intravenous guanethidine monosulfate (2 mg kg-1) or intramuscular reserpine (1 mg kg-1), indicating that the acid inhibition was mediated by an adrenergic mechanism. The emptying of water meals was unchanged by these two drugs. The authors suggest that the first 5 cm of duodenum contain receptors for inhibition of emptying of acid but not for fat or hypertonic glucose. Furthermore, the neural blocking studies indicate that the inhibitory effect of acid in the first part of the duodenum is an adrenergic mechanism which appears to be neither alpha nor beta-receptor-mediated.

Animals↗