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Biomedical subjects

J A Cox

Publications and source records attributed to J A Cox.

At least 127 records · Page 7Linked to original sources
PubMed1986

Transtracheal repair of recurrent tracheoesophageal fistula.

A recurrent tracheoesophageal fistula is generally associated with considerable mediastinal induration and inflammation. The conventional operative approach may be formidable with considerable blood loss and a high complication rate. For two infants with recurrent fistulae, we have employed a simplified low cervical transtracheal approach through noninflamed tissues. This brief atraumatic procedure was followed by gratifying results. Details of the approach are presented.

Bronchoscopy↗
PubMed1986

Calcium-calmodulin interaction and cellular function.

Conformational and functional events in calmodulin (CaM) are directly governed by the degree of saturation by Ca2+, and do not depend on Ca2+-occupancy of any particular one of the four ion-binding sites. For instance, the exposure of a hydrophobicity at the surface of the protein coincides with the appearance of CaM X Can greater than or equal to 2. The species containing at least three Ca2+ is most often involved in target enzyme activation. The activation parameters explain the "on-off" behavior of the CaM-activated systems, in spite of the small amplitude of Ca2+-transients in stimulated cells. The strong energy coupling involved in activation is at the basis of a hysteretic behavior of the cell response: after the decay of free cytoplasmic Ca2+, the dissociation of the ternary complex between enzyme, CaM, and Ca2+ is delayed. Studying natural and synthetic model peptides that interact with CaM, we hope to obtain new insight into the CaM-binding domain in the different target enzymes. The minimal and sufficient requirement for high-affinity interaction of model peptides with CaM is a basic amphiphilic alpha-helix of at least 18 A. Preliminary data on the secondary structure of target enzymes indicate that their CaM-binding domains may have these structural requirements.

Animals↗
PubMed1985

Thermodynamics of the binding of calcium and strontium to bovine alpha-lactalbumin.

Microcalorimetry and equilibrium gel filtration were used to determine the thermodynamic functions delta H degrees, delta G degrees and delta S degrees guiding the interaction of Ca2+ and Sr2+ with bovine alpha-lactalbumin. Two methods of nearly complete metal removal from the protein gave identical results. The single Ca- and Sr-binding site, which has moderate affinity for these ions (KCa = 2.5 X 10(6) M-1 and KSr = 5.1 X 10(5) M-1), displays unusually large enthalpy changes of -118 kJ X mol-1 for Ca2+ and -75 kJ X mol-1 for Sr2+. The concomitant reaction entropies equal -273 and -142 J X K-1 X mol-1, respectively.

Animals↗
PubMed1985

The interaction of calmodulin with amphiphilic peptides.

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.

Amino Acids↗
PubMed1985

The design, synthesis, and characterization of tight-binding inhibitors of calmodulin.

Based on a consideration of the probable structure of calmodulin and some natural peptides known to interact with it, two calmodulin-binding peptides were designed. These peptides bind to calmodulin in helical conformations and are capable of forming electrostatic and hydrophobic interactions with calmodulin. Their dissociation constants for binding (less than or equal to 210 and 400 pM) place them as the tightest-binding inhibitors of calmodulin thus far reported. The study of the interactions of these and similar peptides with calmodulin will provide valuable insights into the mechanisms whereby calmodulin binds to target enzymes, and it also serves as an excellent model system for exploring the physical chemistry of protein-protein interaction.

Amino Acid Sequence↗
PubMed1985

Inguinal hernia of childhood.

The early recognition and treatment of the inguinal hernia in childhood should result in negligible morbidity and mortality with current anesthetic and recovery room support. The outpatient management of the postoperative patient has contributed to this objective as well as reduced the overall cost.

Child↗
PubMed1985

Relationship among calmodulin-, forskolin-, and guanine nucleotide-dependent adenylate cyclase activities in cerebellar membranes: studies by limited proteolysis.

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.

Adenylyl Cyclases↗
PubMed1985

Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C.

A variety of phagocytosable and soluble agonists stimulate the human neutrophil respiratory burst enzyme, NADPH-oxidase, an activity required for normal microbicidal function. Of these agonists, the phorbol esters, which stimulate diverse systems by their ability to substitute for diacylglycerol to activate protein kinase C (the major phorbol ester receptor), have now been shown to directly stimulate NADPH-oxidase through this same receptor. Almost 90% of the specific receptors for phorbol 12,13-dibutyrate (PDBu) were found in the cytosol upon subcellular fractionation. The dissociation constant for [3H]PDBu was 1.2 nM. No significant difference was found in the distribution of the receptor between subcellular fractions from resting as compared with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils. On the basis of these binding studies, we were able to establish a reconstituted system in which PMA activated dormant NADPH-oxidase in a light membrane fraction when cytosol, NADPH, phosphatidylserine, or phosphatidylinositol and ATP were added. The calcium chelator, EGTA, inhibited the activation, which suggested a requirement for calcium at low concentrations. The half-maximally effective PMA dose was 1.1 nM, as predicted from the receptor content in these preparations. Reconstitution of oxidase activity was rapid, peaking within 1 min of incubation. Purified protein kinase C was able to substitute for the cytosol fraction, and accounted for 80% of the cytosol activity. These studies demonstrate that phorbol esters stimulate the neutrophil respiratory burst through activation of cytosolic protein kinase C, which in turn activates either a regulatory constituent or the NADPH-oxidase directly in the plasma membrane to generate an active O-2-generating system.

Caenorhabditis elegans Proteins↗
PubMed1984

Volvulus of the transverse colon in children--Report of a case and review of the literature.

Sigmoid, cecal and transverse colon volvulus have been responsible for approximately 3% of all large bowel obstruction in the United States. Transverse colon volvulus is much less frequent, occurring in less than 4% of all reported cases of colonic torsion. In the pediatric age group a review of the literature revealed seven cases only, to which we are able to add an eighth. Predisposing abnormalities implicated in the etiology of volvulus of the transverse colon include a redundant transverse colon, an elongated mesentery and close attachment or lack of fixation of the proximal and distal transverse colon. Chronic constipation and mental retardation are associated with this condition. The treatment is surgical intervention to untwist the volvulus and resect the redundant and often nonviable colon with primary colocolostomy or end-colostomy and mucous fistula as indicated.

Adolescent↗
PubMed1984

Sequential events in calmodulin on binding with calcium and interaction with target enzymes.

khe conformational and functional events in calmodulin (CaM) are disproportionate to the mean saturation by Ca2+. The enhancement of intrinsic tyrosine fluorescence closely follows the appearance of species CaM X Can greater than or equal to 1; the exposure of the hydrophobic patch at the surface of CaM coincides with the appearance of CaM X Can greater than or equal to 2. For the activation of four different target enzymes, i.e., brain phosphodiesterase and adenylate cyclase, red blood cell Ca,Mg-ATPase, and skeletal muscle phosphorylase b kinase, CaM X Can greater than or equal to 3 is required. The different enzymes have the same affinity for the active species. The direct interaction of CaM with Ca2+ and phosphorylase b kinase has been analyzed according to the theory of energy coupling: whereas the first two stoichiometric calcium-binding constants in the complex are not significantly different from those of free CaM, the third Ca2+ binds with an affinity at least 10(6)-fold higher to enzyme-bound CaM than to free CaM, which corresponds to a free energy coupling of -7 kcal/mol CaM. The similarities in the activation mechanism of different enzymes suggest the existence of one unique CaM-binding domain. The characteristics of the interaction between CaM and melittin, a small amphiphatic cytotoxin, led us to propose melittin as a model for such a CaM-binding domain.

Allosteric Regulation↗
PubMed1983

Free energy coupling in the interactions between Ca2+, calmodulin, and phosphorylase kinase.

Interactions between Ca2+, exogenous calmodulin, and white skeletal muscle phosphorylase kinase have been quantitatively studied by equilibrium gel filtrations and analyzed by means of the so-called "linked functions" theory (Weber, G. (1975) Adv. Protein Chem. 29, 1-83). Four moles of calmodulin, each saturated with at least 3 Ca2+ ions, bind to 1 mol of phosphorylase kinase with a Kdiss of 2.3 nM. The activation of the enzyme as a function of free [Ca2+] shows that the intrinsic Ca-binding properties of phosphorylase kinase do not change upon binding of exogenous calmodulin, and confirms that alpha beta gamma delta X Ca3 is the functional catalytic unit through which activation occurs. Direct binding studies reveal that the intrinsic Ca-binding properties of the enzyme remain the same in the presence of either 0.5 or 8 mM Mg2+, indicating that phosphorylase kinase is endowed with Ca-specific sites. Upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding: whereas its first two stoichiometric Ca-binding constants are not significantly different from those of free calmodulin, the third Ca2+ ion binds with an affinity at least 10(5)-fold higher than the corresponding one in free calmodulin. Calmodulin liganded with 1 or 2 Ca2+ displays the same low affinity for the enzyme as calmodulin depleted of Ca2+ (approximate Kdiss = 10(-4)-10(-3) M). The alpha beta gamma delta X calmodulin X Ca3 complex is strengthened by a free energy coupling of -8 kcal/mol upon complexation. The quantitative analysis of our results predicts that in spite of this high free energy barrier the dissociation of the complex (i.e. the inactivation of phosphorylase kinase) occurs rapidly upon lowering free [Ca2+].

Animals↗
PubMed1983

Structural changes in melittin and calmodulin upon complex formation and their modulation by calcium.

In the presence of Ca2+, calmodulin forms a 1:1 high-affinity complex (Kd = 3 nM) with melittin, a peptide from bee venom; in the presence of ethylenediaminetetraacetic acid, a second type of complex, of much lower affinity, is formed [Comte, M., Maulet, Y., & Cox, J. A. (1983) Biochem. J. 209, 269-272]. In this paper, these interactions were studied by tryptophan fluorescence and circular dichroism spectroscopy in near- and far-UV. Interaction between the two peptides in the presence as well as in the absence of Ca2+ leads to the shielding of the tryptophan residue of melittin from its aqueous environment and to an increase in the alpha-helical content of bound melittin; for instance the Ca2+-dependent high-affinity complex formation enhances the alpha-helical content of melittin from 5 to 72%. Provided Ca2+ is present, the interaction between the two peptides leads to significant changes in the environment of at least one tyrosine residue of calmodulin as measured by near-UV circular dichroism. In the absence of Ca2+, calmodulin binds two melittin molecules with a Kd of ca. 10 microM; at higher concentrations of free melittin, additional binding occurs (up to 5 mol of melittin/mol of calmodulin), with concomitant denaturation of calmodulin. In the presence of 4.0 M urea, the low-affinity complexes formed in the absence of Ca2+ dissociate, due to the denaturation of metal-free calmodulin, whereas the spectroscopic signals of the high-affinity Ca2+-dependent complex are not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Ca2+-dependent high-affinity complex formation between calmodulin and melittin.

The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.

Animals↗
PubMed1982

Calmodulin from neurospora crassa. General properties and conformational changes.

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.

Amino Acids↗
PubMed1982

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes.

The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlated with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+ calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be inferred from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin X Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.

Adenylyl Cyclases↗
PubMed1982

Changing treatment of pediatric splenic trauma.

A review of splenic injuries at Cincinnati Children's Hospital Medical Center from July 1978 to June 1980 revealed this form of injury in 29 patients. Treatment without surgery was successful in 21 patients. Seven patients required operation. One patient died shortly after admission of severe associated injuries. All patients admitted with blunt abdominal trauma were initially treated conservatively. If the clinical state improved, after transfusions if necessary, or remained stable and there were no objective signs of further blood loss, conservative therapy was continued. Liver-spleen scans were obtained on an urgent basis to confirm the diagnosis of splenic injury in patients who did not undergo surgery. No complications of treatment without surgery were recognized. The satisfactory outcome in these patients suggests that there is a place for treatment without surgery in some children with splenic injury.

Abdominal Injuries↗
PubMed1982

Tracheal agenesis: a clinical approach.

Tracheal agenesis, though seemingly rare and fatal to date, has been reported from multiple institutions with increasing frequency. Establishment of the diagnosis necessitates a high index of suspicion in an infant in respiratory distress at birth, without a cry and in whom intubation is difficult. Following definitive diagnosis by endoscopy, infants have been given a chance at survival on four occasions by prompt surgical intervention. The procedure proposed by Altman is adequate for initial stabilization. Postoperative management is aided by the use of continuous positive airway pressure (CPAP) and rigorous pulmonary toilet. Definitive correction should be considered at an early stage in order to prevent fatal complications.

Abnormalities, Multiple↗