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Biomedical subjects

M C Young

Publications and source records attributed to M C Young.

At least 37 records · Page 2Linked to original sources
PubMed1996

The kinetic mechanism of formation of the bacteriophage T4 DNA polymerase sliding clamp.

DNA replication in bacteriophage T4 requires the assembly of a structure called the "sliding clamp" near the 3' end of the DNA strand that is to be extended. This structure is a trimer ring of the T4 gene 45 product (gp45) and serves to regulate the processivity of the DNA polymerase within the T4 DNA replication system. The placement of this ring is performed by an ATPase complex of the products of T4 genes 44 and 62 (gp44/62) that consists of four gp44 subunits and one gp62 subunit. In an effort to understand the role of ATP hydrolysis in processes occurring during the formation of the phage T4 DNA sliding clamp, we have performed direct substrate and product binding experiments and steady-state and presteady kinetic experiments on the gp44/62-gp45 system. Substrate (ATP) and product (ADP) binding studies show that the gp44/62 complex binds 4(+/-1) ATP molecules with a Kd of 34(+/-12) microM, and 3.7(+/-0.3) ADP molecules with a Kd of 14(+/-7) microM. The binding of the other reaction product (inorganic orthophosphate) could not be detected. Presteady-state kinetic analysis of ATP hydrolysis during the sliding-clamp-loading process indicates a biphasic progress curve, consisting of an initial rapid "burst" phase with an amplitude of four ATP molecules per gp44/62 complex and a rate of 15 s(-1), followed by a second slower phase corresponding to the steady-state rate of ATP hydrolysis by this complex. The rate of the burst phase is kinetically consistent with the previously observed rate of T4 DNA polymerase holoenzyme formation. The burst amplitude depends solely on the concentration of gp44/62 ATP binding sites present. These results suggest that the formation of a single T4 sliding clamp requires the hydrolysis of four ATP molecules by one gp44/62 complex in a process requiring 0.5 to 1 second. A model describing the clamp-loading process is discussed in the context of these results.

Adenosine Diphosphate↗
PubMed1995

Simultaneous acute cerebral and pulmonary edema complicating diabetic ketoacidosis.

OBJECTIVE: To report on a child with diabetic ketoacidosis (DKA) who developed simultaneous acute cerebral edema (CE) and pulmonary edema (PE), required extracorporeal membrane oxygenation (ECMO), and yet survived without significant neurological or pulmonary handicap. CASE SUMMARY: A 3-year-old girl with DKA as the first manifestation of insulin-dependent diabetes mellitus (IDDM) sustained coincident acute CE and PE 9 h into therapy. The former responded to mannitol, but the latter matured into adult respiratory distress syndrome (ARDS) resistant to conventional management and requiring ECMO. CONCLUSIONS: CE, PE, and ARDS can complicate DKA. Survival without sequelae is possible with aggressive treatment.

Adult↗
PubMed1994

Kinetic theory of ATP-driven translocases on one-dimensional polymer lattices.

A theory is presented to describe the coupling of the directional movement of ATPase-containing translocases (such as helicases) along polymeric lattices to the steady-state kinetic parameters of the ATPase activity that drives this movement. This theory was developed to explain the results of an experimental investigation of one such enzyme, the bacteriophage T4 gene 41 protein helicase. The salient feature of the theory is that it correctly predicts the dependence of the rate of ATP hydrolysis by ATP-driven translocases on the length of polymer lattices along which they move. In the steady-state rate equation: [formula: see text] either Vmax, or K(act), or both, may depend on the lattice length. Two translocation models are considered. The first is a simple mechanism of the type E<-->E-Lat-->E, where the E-Lat-->E step represents the sum of the translocation steps of the enzyme along, and enzyme release from, the lattice. In the second model this mechanism is expanded to add an additional kinetic step, either before or after the translocation process. Variants of this second model can be used to represent the most simple translocase mechanisms. Another method of measuring the lattice length dependence of an ATP-driven translocase, which is applicable particularly to ATPases moving along DNA lattices, involves the use of lattice-binding proteins (such as single-stranded DNA binding proteins) that can block the movement of the translocases and therefore simulate lattice ends. In this protocol the dependence of the ATPase kinetics of the translocase on lattice length can be studied by experiments on long lattices complexed with lattice-binding proteins to various binding densities. This method is not always as unambiguous as direct measurement of ATPase activity on lattices of defined length, but can help to discriminate between mechanisms. The significance of the steady-state kinetic parameters obtained in such experiments is discussed in terms of the mechanistic rate constants that define the models we have investigated.

Adenosine Triphosphatases↗
PubMed1994

Kinetic parameters of the translocation of bacteriophage T4 gene 41 protein helicase on single-stranded DNA.

The bacteriophage T4 gene 41 helicase protein (gp41) carries a single-stranded DNA-dependent ATPase activity that is essential to its helicase activity. This ATPase activity can be stimulated by a wide variety of single-stranded DNA cofactors, including homo-oligomers and homopolymers 8 to approximately 10,000 nucleotide residues in length, and by natural single-stranded DNA, such as bacteriophage M13 DNA. The steady-state ATPase activity of gp41 on single-stranded homopolymeric cofactors is dependent on the length of the cofactor, in that the kinetic parameters Vmax and K(act) (or KmDNA) have a characteristic length dependence. Vmax values for different DNA lengths show a hyperbolic dependence on DNA length, while K(act) values are independent of DNA lengths exceeding approximately 20 nucleotide residues. Use of the detailed theoretical analysis developed in the preceding paper reveals that: (1) these results support the earlier proposal that gp41 translocates on single-stranded DNA in an ATP-dependent manner; (2) translocation is undirectional; (3) translocation is processive to an extent that depends on the base composition of the DNA employed, with the average distance translocated per binding event ranging from 60 to 700 nucleotide residues; and (4) the detailed translocation mechanism of gp41 includes an obligatory slow step before or after the ATP-driven translocation process. Defined lengths of natural and homopolymer single-stranded DNA have also been created as gaps of known length distribution between clusters of gene 32 protein (gp32) bound along long single-stranded DNA molecules. ATPase data obtained with cofactors of this type also show unidirectional ATP-driven translocation of gp41 on both natural and homopolymeric single-stranded DNA. Direct binding studies of gp41 to short dT oligomers reveal two further features of the interaction of gp41 to single-stranded DNA: (1) nucleoside triphosphate binding is necessary for the formation of stable gp41-ssDNA complexes; and (2) the DNA binding site size of gp41 is between 12 and 20 nucleotide residues per protein monomer. Possible translocation mechanisms for gp41 are discussed within the context of these results.

Adenosine Triphosphatases↗
PubMed1993

Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription.

Pre-steady state kinetics of misincorporation were used to investigate the addition of single nucleotides to nascent RNA by Escherichia coli RNA polymerase during transcription elongation. The results were fit with a branched kinetic mechanism that permits conformational switching, at each template position, between an activated and an unactivated enzyme complex, both of which can bind nucleotide triphosphates (NTPs) from solution. The complex exists most often in the long-lived activated state, and only becomes unactivated when transcription is slowed. This model permits multiple levels of nucleotide discrimination in transcription, since the complex can be "kinetically trapped" in the unactivated state in the absence of the correct NTP or if the 3' terminal residue is incorrectly matched. The transcription cleavage factor GreA (or an activity enhanced by GreA) increased the fidelity of transcription by preferential cleavage of transcripts containing misincorporated residues in the unactivated state of the elongation complex. This cleavage mechanism by GreA may prevent the formation of "dead-end" transcription complexes in vivo.

Base Sequence↗
PubMed1993

Direct observation of UV-crosslinked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry.

Interactions between proteins and nucleic acids are important in the fundamental cellular processes that drive replication, recombination, dynamic alteration and repair of DNA, transcription and processing of RNA, synthesis of proteins, and regulation of enzyme activities. As part of an effort to develop a general, sensitive mass spectrometric strategy for the characterization of protein-nucleic acid interactions, we have used matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to analyze protein-nucleic acid complexes that have been covalently crosslinked by ultraviolet (UV) light. In general, the application of MALDI mass spectrometric techniques to studies of UV-induced crosslinking of nucleoprotein complexes is demonstrated to be feasible. Specifically, MALDI mass analysis was used to determine the molecular weights of the phage T4 gene 32 protein (gp32) crosslinked to the oligonucleotide (dT)20, and the Escherichia coli transcription termination factor rho, photoaffinity labeled with 4-thio-uridine-diphosphate (4sUDP). The covalent gp32:(dT)20 complex is readily detected at a concentration of 1-2 microM in 1 microL of an unpurified solution of reactants that has been exposed to a single, 266 nm UV laser pulse. Mass spectrometric molecular weight determinations of the covalent rho:4sUDP complex add directness and specificity to the ATPase inactivation assay normally used to monitor the formation of 4sUDP photoaffinity labeled rho. It is found that successful MALDI mass spectrometry of protein-nucleic acid complexes is as critically dependent on the choice of solvents and additives as it is on the primary matrix compound.

Mass Spectrometry↗
PubMed1992

Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex.

A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA. Formation of these structures, which we term "hash-marks" for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 62), ATP and appropriate DNA cofactors. ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures. If ATP hydrolysis is stopped by chelation of Mg2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime. The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible. Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis. Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation. Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement. These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes. Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures. The three-dimensional size of the hash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44/62 protein subcomplex. Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis. The hash-mark structures often appear in clusters, even in DNA that has a single nick. We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.

Adenosine Triphosphatases↗
PubMed1991

Loss of therapeutic control in congenital adrenal hyperplasia due to interaction between dexamethasone and primidone.

A 14-year-old girl with congenital adrenal hyperplasia showed rapid conversion from undertreatment to overtreatment when primidone, used to treat coexistent epilepsy, was withdrawn while on a constant steroid dose. In addition to signs of hypercortisolism and a decrease in random measurements of plasma testosterone and 170H-progesterone concentrations, the deterioration in control was also illustrated by changes in the pattern of blood spot steroid profiles. Adequate control was achieved only after a 3-fold reduction in steroid dose. The decrease in dose requirement was probably the result of a reversal of liver enzyme induction consequent upon the withdrawal of primidone.

Adolescent↗
PubMed1990

Dexamethasone treatment for congenital adrenal hyperplasia.

Ten patients with congenital adrenal hyperplasia (three males, seven females; aged 12-29 years) had their usual glucocorticoid treatment changed to dexamethasone in three crossover dosage regimens. A starting dose of 5 micrograms/kg/day is suggested but as no one dose regimen resulted in adequate control the timing of the dose must be decided for each patient.

Adolescent↗
PubMed1990

Response to treatment of congenital adrenal hyperplasia in infancy.

Nine infants with congenital adrenal hyperplasia were started on replacement doses of hydrocortisone (20.6-32.6 mg/m2/day) without receiving a high dose for an initial period first. Plasma adrenal steroid concentrations fell to acceptable levels by 3 months of age. Adequate biochemical control was maintained and satisfactory growth achieved even though the mean dose of hydrocortisone had been reduced to 15 mg/m2/day by the age of 3 years. Inadvertent overtreatment and growth suppression in infants with congenital adrenal hyperplasia may be avoided by using replacement doses from the start, and by permitting the relative dose of hydrocortisone to fall as the body surface area increases during the first year of life.

17-alpha-Hydroxyprogesterone↗
PubMed1989

The pharmacokinetics of low-dose dexamethasone in congenital adrenal hyperplasia.

The pharmacokinetics of dexamethasone, given at low dose, were studied in 13 patients with congenital adrenal hyperplasia (CAH) to ascertain whether kinetics differed in this inherited disorder of cortisol metabolism from those seen in healthy individuals. Changes in plasma dexamethasone concentration after intravenous bolus, measured using a simple novel radioimmunoassay, were well described by a two-compartment open model with first-order kinetics. Values for lambda 2: 0.206 h-1, t1/2: 3.53 h, Vc: 24.41 and f: 0.64 were similar to those previously reported for normal subjects. There were considerable interindividual differences in parameter values and Cmaxp.o. (range 22-67 nmol/l). As suppression of the hypothalamo-pituitary-adrenal axis correlates with plasma dexamethasone levels, this variability may partly explain the differing dose and dose schedule requirements necessary to achieve adequate therapeutic control in the clinical management of CAH.

Administration, Oral↗
PubMed1989

Growth and body proportions in congenital adrenal hyperplasia.

Total height, sitting height, and subischial leg length were measured in 27 patients (19 girls and eight boys aged 4.3-21.1 years) with congenital adrenal hyperplasia to determine the influence of chronic hyperandrogenaemia on body proportions. Proportions were normal in 24 patients with classical congenital adrenal hyperplasia who had received steroid treatment since birth, but one of three patients with non-classical (late onset) congenital adrenal hyperplasia had a disproportionately large trunk. Eleven patients with classical congenital adrenal hyperplasia had completed growth, of whom seven had height standard deviation (SD) scores for chronological age less than zero, and one had extremely short stature (SD score -3.25). In 13 patients who were still growing, nine had height SD scores for chronological age of less than zero despite having mean (SD) advances in bone age over chronological age of 1.64 (1.68) years. Height SD scores for bone age were less than 0 in all 13 patients, indicating a loss of height despite advanced skeletal maturation. Doses of glucocorticoid that permit mild chronic or intermittent hyperandrogenaemia also seem to be associated with mild growth retardation. An adult height below average may be an inevitable consequence for many patients with congenital adrenal hyperplasia receiving conventional glucocorticoid treatment.

Adolescent↗
PubMed1989

Relationship between fetal adrenal morphology and anterior pituitary function.

A comparative study of adrenal morphology between normal fetuses and those with anencephaly or congenital adrenal hyperplasia (CAH) was performed in order to examine the hypothesis that fetal adrenal mass and structure are adrenocorticotrophin (ACTH)-dependent throughout gestation. Combined adrenal weight in 102 normal fetuses was used to establish a reference range for the gestational ages of 15-27 weeks. During this period, mean adrenal weight showed a 6-fold linear increase. In 38 anencephalic fetuses of similar gestation age, adrenal weight was below the normal range and did not show a rise. Three fetuses with CAH (18, 22 and 30 weeks gestation) had adrenal weights considerably above the normal range. Adrenal cortical thickness was significantly increased in CAH fetuses, largely as a consequence of cell hypertrophy, whereas decreased cortical thickness in the anencephalic group represented cellular hypoplasia. Conspicuous secretory granules in the cytoplasm was the electron-micrographic feature of the adrenal gland in the 22-week fetus with CAH. These observations are consistent with close dependency of fetal adrenal growth and development upon fetal pituitary function from an early age, mediated primarily through ACTH.

Adrenal Cortex↗
PubMed1989

17OH-progesterone response to acute dexamethasone administration in congenital adrenal hyperplasia.

Glucocorticoid-related changes in 17OH-progesterone (17P) concentrations were studied in 13 patients receiving treatment for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH). Blood spot and saliva 17P levels were elevated, with or without loss of diurnal rhythm, within 24 h of stopping maintenance glucocorticoid therapy. A single dose of dexamethasone (0.01 mg/kg) given either intravenously or orally at 09.00 h resulted in rapid onset of complete pituitary-adrenal suppression characterised by a prompt and exponential fall in 17P levels (first-order elimination half-life, mean 2.87 h, range 1.98-3.98 h). Concentrations of 17P remained low throughout the day until the onset of an abrupt nocturnal rise, which occurred between 24.00 and 05.00 h. There were individual differences in both the rate of fall and the timing of the nocturnal rise in 17P levels which may partly explain the need to vary individual steroid requirements in the treatment of CAH.

17-alpha-Hydroxyprogesterone↗
PubMed1988

Increased in vitro bone resorption by monocytes in the hyper-immunoglobulin E syndrome.

Children with the hyper-immunoglobulinemia E syndrome are prone to bone fractures. We determined bone density in six patients with hyper-IgE syndrome using photon absorptiometry. All six patients had significantly reduced bone density compared with age- and sex-matched controls. The capacity of peripheral blood monocytes to degrade 45Ca-labeled bone in vitro was studied. The mean percent 45Ca release in four hyper-IgE patients (40.8 +/- 8.6) was significantly higher (p less than 0.01) than in ten age-matched healthy control subjects (7.1 +/- 2.6), seven age-matched patients with recurrent infections (7.9 +/- 1.6), or nine patients with severe atopic dermatitis and elevated serum IgE levels (5.8 +/- 1.3). Monocytes from four of four patients studied spontaneously released abnormally high levels of prostaglandin E2. Bone degradation by these monocytes was significantly reduced in the presence of 10(-6) M indomethacin in vitro. Administration of aspirin in vivo to two hyper-IgE patients reduced boned degradation by their monocytes to normal levels. These results suggest that monocytes from patients with hyper-IgE syndrome are activated to resorb bone via products of the prostaglandin synthase (cyclooxygenase) pathway. The activation of cells in the monocyte/macrophage family to resorb bone may contribute to the osteopenia observed in hyper-IgE syndrome.

Aspirin↗
PubMed1988

Congenital murine polycystic kidney disease. II. Pathogenesis of tubular cyst formation.

In the current study, the pathogenesis of proximal tubular cyst formation was studied in an animal model of polycystic kidney disease, the CPK mouse. The specific roles of (a) sodium-potassium adenosine triphosphatase (Na-K ATPase) activity, determined by an enzyme-linked kinetic microassay, (b) proximal tubular epithelial hyperplasia, determined by calculation of mitotic indices, and (c) altered proximal tubular basal lamina formation, determined by immunohistological localization of basal lamina glycoproteins, were investigated at progressive developmental stages of CPK proximal tubular cyst formation. Increases in renal Na-K ATPase were present at the earliest fetal stages of proximal tubular cyst formation, and subsequently paralleled the course of proximal tubular cyst progression. Proximal tubular epithelial hyperplasia, although not present at the earliest stages of cyst formation, was a consistent feature of progressive proximal tubular cystic enlargement. Abnormalities in basal lamina glycoprotein expression were not present at any stage of proximal tubular cyst development. We conclude that increased Na-K ATPase and tubular epithelial hyperplasia are significant features of proximal tubular cyst formation in the CPK mouse.

Age Factors↗
PubMed1988

Internal fixation of an unstable open fracture of a distal phalanx with a Herbert screw.

A 20-year-old patient was seen with an unstable, infected, open fracture of the distal phalanx of the long finger of the right dominant hand. The patient was treated by removal of the nail, debridement of the fracture site, and stabilization of the fracture with a Herbert screw. The Herbert screw compressed the fracture site and allowed early active motion of the distal inter-phalangeal (DIP) joint. The wound healed without incident, and the fracture was radiographically united 6 weeks after the procedure. The Herbert screw is useful in the treatment of unstable fractures of the distal phalanx, since the screw maintains reduction, compresses the fracture site, and allows early active motion of the DIP joint.

Adult↗