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Biomedical subjects

C W Jones

Publications and source records attributed to C W Jones.

At least 19 recordsLinked to original sources

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter.

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.

Amino Acid Sequence

Chemical compatibility of regional anesthetic drug combinations.

OBJECTIVE: To define the physical and chemical compatibilities of several classes of drugs that may be used in combination for peridural anesthesia. DESIGN: Morphine, fentanyl, bupivacaine, lidocaine, tetracaine, ketamine, and clonidine were admixed for one hour in five groups of three-drug combinations, plus one group of all seven drugs. The combinations were inspected macroscopically and microscopically to determine physical compatibility. The admixtures were evaluated by gas chromatography/mass spectroscopy (GC/MS) and compared with known standards to determine chemical compatibility. RESULTS: The admixtures showed no physical incompatibility on microscopic or macroscopic evaluation. Chemical compatibility of all mixtures was confirmed by GC/MS. Ion chromatograms of the drugs in admixtures were identical to previously established standards. CONCLUSIONS: The agents evaluated demonstrated physical and chemical compatibility under conditions that would be observed during the administration of peridural anesthesia. Combinations of these drugs therefore could be safely admixed for use in anesthesia.

Anesthesia, Epidural

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.

Culture Media

Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2.

Extracellular lipase was purified from a Tween 80-limited continuous culture of Pseudomonas aeruginosa EF2 by ultrafiltration of the culture supernatant followed by anion-exchange and gel-filtration FPLC. The lipase was composed of a single subunit (Mr 29,000, pI 4.9), which was capable of a variable degree of aggregation, and which exhibited both lipase activity, measured with the insoluble substrate olive oil (predominantly triolein), and esterase activity, measured with the soluble substrates p-nitrophenyl acetate and Tween 80. Lipase activity was approximately eight times higher than either type of esterase activity (kcat approximately 3000 s-1 for the hydrolysis of olive oil). The enzyme showed a marked regiospecificity for the 1,3-oleyl residues of radiolabelled triolein, was relatively stable at moderate temperatures (exhibiting a biphasic loss of activity with an initial t1/2 of 17.5 min at 60 degrees C) and was very stable to freezing and thawing. Lipase activity was only weakly inhibited by the serine-active reagent 3,4-dichloroisocoumarin, and was not inhibited by the chelating agent EDTA (1 mM). The N-terminal amino acid sequence of the Ps. aeruginosa EF2 lipase showed a marked similarity to those of several other bacterial lipases.

Amino Acid Sequence

The influence of calmodulin antagonists on calcium transport and uptake in the rat intestine.

The effects of calmodulin (CaM) antagonists on calcium transport, tissue retention and the cell-associated calcium space were determined in duodenal segments of intestine and isolated duodenal enterocytes from young male rats. The CaM antagonists trifluoperazine (TFP) and chlorpromazine (CPZ) which were added separately to the media bathing both the mucosal and serosal surfaces of duodenal segments decreased calcium transport and tissue uptake in a dose-related fashion over a concentration range of 0.1-1 mM. Both TFP and CPZ reduced active transport to a greater extent than calcium retention in the duodenal segments. In addition, TFP and CPZ produced a dose-related decrease in the observed intracellular calcium space in duodenal tissue and reduced calcium uptake into isolated enterocytes. The TFP-induced antagonism of calcium transport was greater when TFP was at the mucosal surface than at the serosal surface of the intestinal tissue. Although TFP and CPZ are not entirely specific CaM antagonists at the concentrations used in this study, the results indicate that CaM antagonism by TFP and CPZ decreases active calcium transport and calcium uptake in duodenal segments of rat intestine in a dose-related fashion. Therefore, the present study together with the reports of other investigators suggests that CaM is involved in the mediation of calcium translocation in intestinal tissue in a fashion which is similar to that reported in other biological tissue.

Animals

Kinetic vitreous fluorophotometry in experimental diabetes.

Kinetic vitreous fluorophotometry was used to measure dynamic alterations in blood-retinal barrier function. Normal hooded rats were compared with diabetic animals before and after insulin treatment. Rats with streptozocin-induced diabetes demonstrated significantly longer (P less than .001) half-periods of fluorescein loss from the vitreous when compared with controls, and insulin treatment significantly reduced (P less than .001) the mean half-period toward control values without normalization of serum glucose. These results suggest that kinetic vitreous fluorophotometry is a good indicator of blood-retinal barrier function and that sufficient amounts of insulin may be more important than normal blood glucose levels in recovery of altered barrier function in diabetes.

Animals

A study of the reaction of human tissue to proplast.

The femoral stems of Thompson prostheses coated with a polytetrafluoroethylene/carbon fiber composite (proplast) were studied using conventional histological examination, scanning electron microscopy and electron probe microanalysis in "successful" firm implants and in a "loose" clinically unsuccessful implant. Ingrowth was found throughout the coatings of the successful prostheses. In the most firmly fixed prosthesis the ingrowth consisted of fibrous tissue with abundant giant cells; however, no bone ingrowth was detected. There was less composite pore infilling in the unsuccessful implant. From both clinical, radiological and the studies described above, it is concluded that fibrous tissue ingrowth was a secondary stabilizing phenomenon in the proplast-coated prostheses studied.

Aged

Intubation and other experiences in cardiac surgery: the consumer's views.

Recently publications have advocated earlier weaning and early extubation of the trachea in patients after cardiac surgery. Greater comfort of the patients is one of the advantages claimed for this policy. One hundred consecutive adult patients were questioned on the fifth postoperative day to assess the relative comfort or discomfort of the patients during tracheal intubation and ventilation. Overnight tracheal intubation and ventilation were easily tolerated by over 90 per cent of patients who had received morphine and diazepam. There was a high incidence of amnesia. Some of the other claimed advantages appear inconclusive and further study is desirable to elucidate the effect of early removal of the tracheal tube upon the indicence of pulmonary complications.

Adult

Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families.

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stage-specific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.

Animals

Evolution of two major chorion multigene families as inferred from cloned cDNA and protein sequences.

Complete or partial sequences are reported from six chorion cDNA clones of the silkmoth Antheraea polyphemus. The proteins encoded belong to the two major chorion protein classes, A and B, each of which is encoded by a multigene family. The sequence comparisons define some major features of the families and suggest how these genes may be evolving. Deletions and insertions might be involved in expanding or contracting internally repetitive regions. Sequence divergence is localized, thus defining sequence domains of distinct evolutionary properties and presumably distinct functions.

Amino Acid Sequence

Interaction of pyridoxal 5-phosphate with apo-serine hydroxymethyltransferase.

The interaction of pyridoxal 5-phosphate with beef liver serine hydroxymethyltransferase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) has been investigated using sedimentation velocity, kinetic and equilibrium techniques. No evidence for an aggregating system could be found in sedimentation velocity experiments in the presence or absence of pyridoxal 5-phosphate. Reassociation of pyridoxal 5-phosphate with apoenzyme and reacquisition of enzymic activity follow identical kinetics. An initial fast step is followed by a second order process with a rate constant of 66 M-1. s-1. A dissociation constant of 27.5 micrometer was obtained from equilibrium studies. No interaction of binding sites was exposed by altering pH or in the presence of glycine or folate. Maxima observed in pH profiles with both binding and reactivation are interpreted as the composite fo two overlapping processes, one of which is ionization of the pyridinium nitrogen of pyridoxal 5-phosphate and the other a functional group on the apoenzyme. Evidence is presented to indicate the necessity for the formation of an enzyme . pyridoxal 5-phosphate Schiff's base complex during catalytic turnover.

Apoenzymes

The properties of adenosine triphosphatase from exponential and synchronous cultures of Alcaligenes eutrophus H16.

The properties of Alcaligenes eutrophus ATPase (adenosine triphosphatase) were investigated by using subcellular fractions prepared from cells growing in exponential and synchronous cultures. Both the soluble and membrane-bound forms of the ATPase were inhibited non-competitively (K(i) 142mum) by Nbf-Cl (4-chloro-7-nitrobenzofurazan), whereas only the membrane-bound enzyme was inhibited (non-competitive; K(i) 750mum) by NN'-dicyclohexylcarbodi-imide. Neither the activity of the ATPase nor its sensitivity to these two inhibitors varied during exponential growth. However, marked variations in ATPase activity were observed during synchronous growth, which were characterized by maxima at approx. 0.4 and 0.9 of a cell cycle and minima at approx. 0.1 and 0.6 of a cycle. Sensitivity to Nbf-Cl and NN'-dicyclohexylcarbodi-imide also varied during the cell cycle; maximum inhibition by the former occurred at approx. 0.4 and 0.9 of a cell cycle, whereas maximum inhibition by the latter was located at approx. 0.1 and 0.6 of a cell cycle. Proton conductance by whole cells was also periodic during the cell cycle, the lowest rates occurring at approx. 0.15 and 0.55 of a cycle and the highest rates at approx. 0.4 and 0.9 of a cycle, but -->H(+)/O quotients for the oxidation of endogenous substrates remained relatively constant and indicated the presence of four proton-translocating respiratory segments throughout the cell cycle. These results are discussed in terms of ATPase and respiratory-chain structure and function during the cell cycle of Alcaligenes eutrophus.

4-Chloro-7-nitrobenzofurazan

Serine transhydroxymethylase. Equilibrium binding of folate analogs as active site probes.

Formation of a quinoid-like structure within the glycyl-pyridoxal phosphate moiety of serine transhydroxymethylase (5,10-methylenetetrahydrofolate: glycine hydroxymethyltransferase, EC 2.1.2.1) is dependent upon the dissociation of the 2-S hydrogen of glycine which in turn requires the presence of tetrahydrofolate or analogs thereof. Equilibrium binding studies with the series folate, dihydrofolate, and tetrahydrofolate showed that reduction of the pteridine ring enhances both quinoid formation and binding. A 5,8-deazafolate series showed that modifications in the 4 position, 10 position and the glutamyl position yield interrelated alterations of quinoid formation which could not be correlated with binding.

Binding Sites