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Biomedical subjects

T Richardson

Publications and source records attributed to T Richardson.

At least 55 records · Page 3Linked to original sources
PubMed1989

Cloning and sequencing of a complementary deoxyribonucleic acid coding for a bovine alpha s1-casein A from mammary tissue of a homozygous B variant cow.

A cDNA clone for bovine alpha s1-casein variant A was isolated from a mammary gland cDNA library using a synthetic degenerate oligonucleotide probe. The largest Pst I insert containing an EcoR I site was sequenced. It contained 1090 base pairs, 47 in the 5' noncoding region, 603 in the coding region and 440 in the 3' noncoding region. The nucleotide sequence was compared with three published cDNA sequences for alpha s1-casein variant B. The most obvious difference was the absence of the 39 bases encoding the 13 amino acids that are present in the B variant but absent from the A variant. In addition, five other single base positions differed within individual codons among the four sequences at the third base for each codon, but this did not change the amino acids encoded. There were, however, a number of differences found in the 3' noncoding region. The isolated cDNA was subjected to site-directed mutagenesis to replace a Val-Ile dipeptide with Phe-Phe to increase the chymosin sensitivity of the protein. When the milk proteins from mammary gland tissue extracts were typed, the alpha s1-casein A gene product was not detected.

Amino Acid Sequence↗
PubMed1989

New contract.

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Aged↗
PubMed1988

Effects of cyclocryotherapy on aqueous humor dynamics in cats.

The effects of graded cyclocryotherapy were studied using pneumatonometry, fluorophotometry, and light and electron microscopy. Cats were treated with either 90 degrees, 180 degrees, or 270 degrees of cyclocryotherapy and were followed up for six weeks. The response of the eyes to treatment was evaluated in terms of intraocular pressure (IOP) response, aqueous humor flow rate, and permeability of the blood-aqueous barrier compared with the untreated fellow eye. The average IOP response was determined to be -20%, -44%, and -47% for the 90 degrees, 180 degrees, and 270 degrees treated eyes, respectively. The aqueous humor flow rate was estimated to be -14%, -44%, and -52% for each of the groups, respectively. Finally, the permeability of the blood-aqueous barrier, measured 60 minutes after injection of fluorescein, was estimated to be a percent increase of 22%, 332%, and 285% for each of the groups, respectively. Histologically, the maximally treated eyes (270 degrees) demonstrated greater disruption of the ciliary body compared with the control eyes. Thus, graded cyclocryotherapy causes graded destruction of the ciliary epithelium and proportionally related changes in IOP and aqueous humor dynamics.

Animals↗
PubMed1988

Molecular cloning and expression of bovine kappa-casein in Escherichia coli.

A cDNA library was constructed using poly(A) +RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using 32P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein.

Amino Acid Sequence↗
PubMed1987

Phenytoin infusion in severe pre-eclampsia.

Intravenous phenytoin sodium was given as a high-dose infusion (10 X 8-18 mg/kg) for anticonvulsive prophylaxis to 2 eclamptic patients and to 24 patients with moderate to severe pre-eclampsia. There were no major maternal or neonatal side-effects. Plasma phenytoin levels were within the therapeutic range (7-20 mg/l) at 30 min and 6 h after the infusion in all patients, and remained at a therapeutic level in 21 patients after 12 h. After a second dose of phenytoin in 19 patients, drug levels were within the therapeutic range at 24 h.

Female↗
PubMed1987

Cloning and sequence analysis of bovine beta-casein cDNA.

A bovine beta-casein cDNA clone was isolated from a cDNA library prepared from mammary gland mRNA. Sequence analysis revealed 25 nucleotides (nt) of the 5' noncoding region, 672 nt of the complete sequence coding and a 3' region of approximately 500 nt. When the nucleotide sequence of bovine beta-casein cDNA is compared to rat beta-casein cDNA (5), a high degree of homology is observed in the first 100 nt corresponding to the signal peptide of the pre-beta-caseins.

Animals↗
PubMed1987

Preparation and characterization of immobilized beta-lactamase for destruction of penicillin in milk.

beta-Lactamase I (Bacillus cereus) was covalently bound to cyanogen bromide-activated, crosslinked agarose. An initial 5.00 mg of soluble beta-lactamase were used in the immobilization reaction for each preparation, and average coupling yield was 80.5%. Of the enzyme immobilized on the matrix, an average 53.4% remained active. To minimize diffusional effects on immobilized enzyme activity, reaction mixtures were rotated at 250 rpm throughout the study. The shape of the pH activity curve of the immobilized enzyme was identical to that of the soluble enzyme; both exhibited optimum pH around 7.0. In general, only 2-fold differences in Michaelis constant and maximum volume were observed between native and immobilized enzyme when penicillin G was used as the substrate. However, the Michaelis constant of the immobilized enzyme increased up to 22-fold that of the native enzyme when cephaloridine was used as the substrate. The immobilized enzyme exhibited enhanced stability in the acidic pH region in contrast to the native enzyme, which had superior stability in the alkaline pH region. The heat stability of the immobilized enzyme was about twice that of native enzyme after heat treatment at 60 degrees C for 30 min. Approximately a 10% increase of storage stability on immobilization of beta-lactamase was observed when stored at room temperature (23 +/- 1 degree C) for up to 6 d in the absence of antimicrobial agents. Little loss of activity (less than 2%) was noted after repeated use of the immobilized enzyme up to seven times each in 10.0 ml of skim milk containing .5 U/ml penicillin G.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Enhancement by sodium salicylate of the blood glucose lowering effect of chlorpropamide-drug interaction or summation of similar effects?

The ability of sodium salicylate (3 g) to enhance the blood glucose lowering action of chlorpropamide (200 mg) has been confirmed in healthy male volunteers who received an oral test dose of 50 g glucose. Salicylate raised the plasma concentration of insulin and lowered that of cortisol but did not alter the concentration of chlorpropamide. The area under the blood glucose concentration-time curve was used as the measure of drug response and the significance of drug effects was assessed by analysis of variance. In one study on five volunteers the effect of combining salicylate and chlorpropamide was additive. In a second study on six volunteers 200 mg chlorpropamide, 3 g sodium salicylate and 100 mg chlorpropamide + 1.5 g salicylate were equi-effective. The enhancement of chlorpropamide action by salicylate in this single dose study is consistent with the summation of similar effects. It is not necessary to postulate an interaction.

Adult↗
PubMed1985

Use of microbial beta-lactamase to destroy penicillin added to milk.

A simple method is described for destruction of penicillin residues in bulk milk to an undetectable amount (less than .003 U/ml) with commercially available crude beta-lactamase enzyme. Milk containing .1 or .5 U/ml penicillin G was treated with .01 to 1.0 mU/ml of beta-lactamase (Bacillus cereus) for up to 96 h. The Bacillus stearothermophilus var. calidolactis assay was used to quantify penicillin in milk between .003 to 1.0 U/ml. The .5 U/ml of penicillin G was reduced to an undetectable amount within 18 h at 4 degrees C by 1.0 mU/ml of beta-lactamase. The development of titratable acidity over 5 to 6 h in contaminated milks treated with beta-lactamase and inoculated with Streptococcus thermophilus GH, Streptococcus cremoris, Streptococcus lactis, or a commercial starter culture was the same as for control milk samples containing no additives or only enzyme. Pilot-scale manufacture of Swiss and Cheddar cheeses from contaminated milks treated with beta-lactamase yielded cheeses of comparable quality, to control cheeses produced from penicillin-free milk. There were no delays in acid production as judged from pH measurements during production and ripening of the cheeses. About 50% of beta-lactamase activity added to milk remained after pasteurization at 63 degrees C for 30 min. The safety for human consumption of cheese containing small quantities of penicillin degradation products from milk treated with beta-lactamase remains to be established.

Animals↗
PubMed1985

Chemical modifications and genetic engineering of food proteins.

Relationships of structure to function of proteins can be studied using chemical modifications of amino-acid side chains and, more recently, recombinant deoxyribonucleic acid techniques to alter primary sequences. A wide array of chemical modifications are available to the food chemist for manipulating the functionality of food proteins. The esterification of side-chain carboxyl groups in proteins to yield polycationic polymers is emphasized in this review as an example of changing the functionality of a protein via chemical derivatization. However, chemical modifications of proteins generally suffer from a lack of control in the extent of derivatization attainable, oftentimes yielding polydisperse products. Recent advances in recombinant deoxyribonucleic acid technology offer the opportunity to relate systematically well-defined alterations in the primary sequence to changes in protein functionality. Using oligonucleotide-directed mutagenesis, one can now use synthetic oligodeoxynucleotides to prepare semisynthetic genes coding for specific changes in the primary sequence of proteins. Incorporation of the altered genes into an appropriate host can lead to the production of the modified protein for structure-function relationship studies. These recombinant deoxyribonucleic acid techniques may eventually provide the means to engineer proteins and enzymes.

Circular Dichroism↗
PubMed1985

Protein-lipid interactions in concentrated infant formula.

Radiolabeled milk proteins ([carbon-14] beta-lactoglobulin or [carbon-14] kappa-casein) were added to raw skim milk used to prepare concentrated humanized infant formula. Ultracentrifugation of the sterilized product allowed separation of three fractions: lipids and the proteins associated with them; free casein micelles and other dense particles; and the fluid phase. Distribution of radiolabeled tracer proteins or of protein measured by chemical methods among these three phases varied significantly with differences in processing conditions (time and temperature of sterilization) or amount of certain additives (potassium hydroxide or urea). In the range of 0 to 8 meq/L of potassium hydroxide added to the formula after homogenization but before sterilization, the lipid layer content of carbon-14 from [carbon-14] kappa-casein in the sterilized product decreased by 4.7% for each 1 meq/L of added potassium hydroxide. Lipid layer content of protein decreased by 2 g/L (of a total of 32 g/L) for each 1 meq/L potassium hydroxide. Such differences in the structure of the product, related to interactions of protein with lipid, protein, or calcium phosphate, may correlate with physical properties and stability of milk-based lipid-rich products.

Chemical Phenomena↗
PubMed1985

Selected functionality changes of beta-lactoglobulin upon esterification of side-chain carboxyl groups.

Free carboxyl groups of bovine beta-lactoglobulin were esterified with methanol, ethanol, and n-butanol. The modified proteins showed increased positive charge as the number of ionizable carboxyl groups was reduced. The methyl, ethyl, and butyl esters of beta-lactoglobulin showed enhanced surface activity, determined with surface and interfacial tension measurements at an air/water and oil/water interface, respectively. The methyl ester showed the largest enhancement in surface activity relative to the native protein. The ethyl and butyl esters were less effective in lowering the surface and interfacial tension but were more surface active than the native protein. The hydrophobic probe, 1,8-anilinonaphthalene sulfonate, showed enhanced fluorescence in the presence of native and modified beta-lactoglobulin. The largest enhancement in fluorescence of the hydrophobic probe was noted in the presence of the methyl ester of beta-lactoglobulin. The ethyl ester also effectively enhanced the fluorescence of the probe. The slopes of the curves of the fluorescent response for the native and modified proteins were graphically related to the interfacial tension at an oil/water interface for the corresponding protein. A large positive correlation was noted between the effectiveness of the proteins in reducing the interfacial tension and the enhancement in fluorescence of ANS with hydrophobic binding by the proteins. A general method to quantify carboxyl groups of proteins, employing the formation of a colored hydroxamate-ferric ion chelate, was also developed. Modification of the reaction for proteins in general, and carboxyl-modified proteins in particular, appears to have potential. Use of the hydroxamic acid reaction made it possible to estimate the apparent extent of carboxyl modification of beta-lactoglobulin through esterification with methanol, ethanol, and n-butanol.

Amino Acid Sequence↗
PubMed1984

Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers.

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.

Animals↗