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M K Walsh

Publications and source records attributed to M K Walsh.

26 records · Page 2Linked to original sources
PubMed1996

Investigating the use of the chymosin-sensitive sequence of kappa-casein as a cleavable linker site in fusion proteins.

The chymosin-sensitive sequence of bovine k-casein A (kappa-CN A) was investigated as a cleavable linker site between the two domains of a streptavidin-chloramphenicol acetyltransferase fusion protein. Two DNA sequences were synthesized which encode the amino acids from 101 to 107 and from 97 to 113 of bovine kappa-CN A. These sequences were separately cloned in-frame to a streptavidin expression vector used for fusion protein construction. The gene for chloramphenicol acetyltransferase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensitive linker vector forming plasmids pStCL1CAT and pStCL2CAT. The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western blot analysis of chymosin-treated cell lysates showed a pH-dependent cleavage of the fusion proteins. Fusion proteins were also bioselectively immobilized onto biotinylated controlled-pore glass beads and treated with chymosin. CAT was specifically released by chymosin treatment and was identified by SDS-PAGE.

Amino Acid Sequence↗
PubMed1990

The effect of platelet-activating factor (PAF), histamine, and ethanol on vascular permeability of the guinea pig conjunctiva.

Increased vascular permeability, one of the characteristic features of immediate hypersensitivity (Type I), is mediated through a variety of compounds, including histamine and platelet-activating factor (PAF), a phospholipid inflammatory mediator. The effects on vascular permeability of histamine, PAF, and ethanol, the solvent for PAF, were compared in the guinea pig conjunctiva. Permeability at 30 min was investigated by evaluation of conjunctival edema and Evans blue extravasation (clinically estimated and colorimetrically measured). Doses of PAF from 1 to 10 nmol produced an increase in vascular permeability, with a peak effect at 10 nmol. Ethanol had no effect on vascular permeability below 40 X 10(3) nmol; above this concentration, however, permeability increased, reaching a maximum at 175 X 10(3) nmol. At low doses of PAF and ethanol, the effects were additive, whereas at 20-80 nmol of PAF with high concentrations of ethanol there was no additive effect of PAF, producing a decrease in the net effect of PAF. Histamine increased vascular permeability, with a minimum effect at 10 nmol and a maximum effect at 450 nmol. The slopes of the dose-response curves for all three compounds were linear and parallel, with statistically different potencies. The potencies for each compound were identical by all three methods of evaluation. Therefore, we conclude that PAF is a potential mediator in hypersensitivity reaction in the guinea pig conjunctiva, and that its effect is similar to but much more potent than that of histamine or ethanol. Since ethanol alone has a significant effect on vascular permeability, studies on PAF effects using control solutions without ethanol may be difficult to interpret.

Animals↗
PubMed1987

Trichloroacetic acid (TCA) precipitability of 125I in the blood of mice fed 125I.

A recent study showed that after feeding 125I-labelled proteins or free 125I to mice, as much as 40% of total radioactivity in the circulation precipitated upon mixing whole blood with 10% trichloroacetic acid. We examined this potential limitation to the use of radiolabelled tracers for studies on intestinal digestion of proteins and protein uptake, and identified its mechanism. BALB/c mice were gavage-fed or injected intravenously (i.v.) with Na125I. Blood obtained at 15 min was added directly to 10% trichloroacetic acid (TCA) or was processed to obtain serum or plasma. On mixing with 10% TCA, 25-33% of the radioactivity in whole blood was precipitated; less than 2% of the radioactivity in plasma or serum was precipitated. In vitro studies identified hemoglobin as the primary carrier protein participating in this reaction. If hemoglobin was replaced by methemoglobin or cyanomethemoglobin, then the reaction with 125I did not occur, suggesting that iron in the heme group may be the site for 125I binding and that iron must be in its reduced or ferrous form (Fe2+). The administration of non-radioactive NaI in vivo or its addition to reaction mixtures in vitro completely inhibited the precipitation of 125I by hemoglobin in the presence of TCA. Thus the addition of non-radioactive iodide to TCA stock solutions may effectively prevent non-specific binding of 125I to free hemoglobin released unintentionally during venipuncture or at other stages of blood processing.

Animals↗
PubMed1986

Transfer of protein antigens into milk after intravenous injection into lactating mice.

We investigated the transfer of bovine serum 125I-albumin (125I-BSA), bovine 125I-gamma-globulin (125I-BGG), 125I-ovalbumin (125I-OVA), and 125I-beta-lactoglobulin (125I-BLG) from the blood into the milk of lactating mice. Equal amounts (by weight) of the radiolabeled proteins were injected intravenously into mice 1 wk postpartum. Total radioactivity, trichloroacetic acid-precipitable radioactivity, and specifically immunoprecipitable radioactivity were measured in serum, mammary gland homogenate, and milk. Clearance of immunoreactive OVA (iOVA) and iBLG from the circulation was more rapid than iBSA and iBGG. The radioactivity in mammary tissue associated with BSA and BGG was greater than 70% immunoprecipitable throughout the 4-h test interval; 125I-OVA and 125I-BLG were less than 12% precipitable 1 and 4 h after injection. In milk obtained at 4 h, there was an approximately 10-fold greater accumulation of iBSA or iBGG than of iOVA or iBLG. These experiments demonstrate that protein antigens differ in their ability to transfer from maternal circulation into milk. The transfer into milk appeared to be in proportion to persistence of the antigens in the maternal circulation.

Animals↗
PubMed1986

Influence of circulating maternal antibody on the transfer of dietary antigen to neonatal mice via milk.

125I-BSA injected intravenously into lactating mice persisted in the circulation for 4 h, entered the mammary gland, and was transferred to the neonate via the milk. When injected with anti-BSA antibodies, it was rapidly cleared from the circulation. Although animals receiving anti-BSA antibodies transferred more radioactivity to the neonate via the milk, the radioactivity was not TCA-precipitable or bound to staphylococcal protein A, nor was it immunoreactive. These findings suggest that circulating maternal antibody can limit the transfer of specific protein antigen from mother to newborn.

Animals↗