Biomedical subjects
Publications and source records attributed to K McEntee.
We have developed a simple PCR based approach for synthesizing high-molecular-weight DNA multimers of short oligonucleotide elements in yields sufficient for preparing DNA affinity columns or for using as probes to detect sequence-specific DNA binding proteins. The technique is rapid, efficient and produces multimers consisting of 30-100 repeating units.
The acute polymyopathy in a seven-year-old German shepherd dog was attributed to the muscular hypertonia, tremors and seizures which developed during the acute phase of carbamate poisoning. After two days of generalised muscular rigidity, the dog adopted a characteristic fetal position which could be explained by the imbalance between the injuries to the extensor and flexor muscles. The polymyopathy resolved gradually over the course of a week.
DNA polymerase II of Escherichia coli, an alpha-like or group B polymerase, has been crystallized. The crystals are orthorhombic, space group P2(1)2(1)2, with cell dimensions a = 94.4 A, b = 118.2 A, c = 84.2 A and diffract to at least 3.0 A resolution. This is the first example of a group B polymerase to be crystallized.
This study was designed to test the applicability and the reproducibility of the thermodilution method in the measurement of right ventricular ejection fraction (EF), end-diastolic volume (EDV) and end-systolic volume (ESV) in unsedated cattle. In the 16 healthy calves studied, it was possible, using a fast response thermodilution catheter, to obtain characteristic exponential steplike washout curves, allowing the calculation of right ventricular EF, EDV and ESV. Five to 10 successive thermodilution curves were recorded within a few minutes and two to five similar sets of measurements were performed throughout the same day to test the reproducibility of the technique. The same protocol was repeated the following day. Right ventricular EF, EDV and ESV were reproducible successively, throughout a given day and on following days. The mean intraset, intraday and interdays coefficients of variation ranged from a mean of 4.0 (SD 4.1) per cent to a mean of 18.2 (SD 7.9) per cent. Values of right ventricular volumes and EF agreed with those previously reported in various animal species. Thermodilution was, therefore, shown to be a useful method for measuring with a satisfactory reproducibility, right ventricular EF, EDV and ESV in the unsedated healthy calf.
Bovine muscle biopsies were analysed for fiber type and capillary density by the acid ATP-ase and amylase-PAS methods respectively. Samples obtained from Belgian blue double muscled (DM) calves were compared for their fibre type, size and vascularization with samples from Friesian (F) calves. The animals were sampled both at 2 and 7 months of age. Changes in histochemical composition occurring with ageing, independently of the breed, and differences related to the muscle sampled by comparing biopsies obtained from the longissimus dorsi (LD) and gluteobiceps (GB) muscles in animals of both breed were also investigated. Independently of age and muscle, fibre type I percentage and area were significantly higher in F than in DM muscles. Type IIa area was significantly larger in F than in DM, while fibre type IIb percentage and area were higher in DM. The number of capillaries/mm2 was significantly higher in F than in DM. Fibre type IIa and IIb percentage as well as the diameter of the three fibre types was significantly higher in the oldest animals. Finally, independently of age and breed, fibre type I and IIb percentages were significantly higher in LD but lower in GB. The results mainly demonstrated that Belgian blue DM muscles present a higher proportion of IIb fibres and a lower capillary supply than F muscles.
DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.
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During growth, central venous, right ventricular, pulmonary arterial, pulmonary capillary wedge, and systemic arterial pressures, heart rate, and cardiac output were repeatedly measured in 41 Friesian calves, considered as having conventional muscular conformation, and in 19 Belgian White and Blue double-muscled calves. A total of 123 and 70 recordings were collected in conventional and double-muscled calves, respectively. These circulatory indices were calculated: stroke volume, cardiac and stroke indices, pulmonary and systemic pulse pressures, pulmonary and systemic vascular resistance indices, and right and left ventricular work indices. Results indicated that systemic arterial and pulse pressures, as well as cardiac output, stroke volume, cardiac and stroke indices, and right and left ventricular work indices were significantly (P < or = 0.05 to 0.001) lower but, in contrast, pulmonary and systemic vascular resistance indices were significantly (P < or = 0.001) higher in double-muscled than in conventional calves. Right-sided vascular pressures and heart rate were similar in the 2 groups. These results indicated that global cardiac performance may be considerably poorer in double-muscled calves. Diminished cardiac performance of double-muscled calves appears to be related neither to relative bradycardia nor to reduced ventricular preload. The potential role of increased ventricular afterload or of reduced myocardial contractility in double-muscled cattle should be determined by direct measurements.
Two randomly distributed groups of thoroughbred horses were compared during a 12-week period for their cardio-respiratory and metabolic adjustment to strenuous exercise, training and detraining. The horses were trained following the same standardized schedule and were regularly investigated using standardized treadmill exercise tests (SET) of increasing speed. After the first SET and during the whole experimental period, a group of 6 horses received a probiotic (Bioracing) once a day while a group of 5 horses received a placebo. All other conditions were similar for both groups. During each SET, the oxygen uptake, carbon dioxide output, tidal volume (inspired volume), respiratory rate and expired minute volume were obtained using 2 ultrasonic pneumotachographs and a mass spectrometer. All the parameters were the mean of the values calculated during the last 20 s of the SET. Heart rate was continuously measured with a polar horse tester. Venous blood was sampled before and after the test and analyzed for various biochemical parameters. In both groups, training induced significant modification in most of the cardio-respiratory parameters, ie peak oxygen uptake, peak carbon dioxide output, respiratory exchange ratio, ventilation/min to oxygen-uptake ratio and oxygen-uptake to heart-rate ratio. After the 3-week detraining period, most of the values were again similar to the pre-training values in both groups. However, the training-induced modifications of most of the cardio-respiratory parameters occurred earlier and were proportionally greater in the probiotic-treated group than in the control. The respiratory coefficient decreased in the control but not in the treated group. All other parameters changed similarly in both groups. This suggests that Bioracing could modify the physiological effects of training by improving some aerobic metabolic capacities for carbohydrate utilization, but that this effect occurs only during training and not during periods of physical inactivity.
This study investigated the pattern of growth-induced haemodynamic changes in normal calves during their first year of life. The central venous pressure (CVP), the right ventricular pressure (RVP), the pulmonary arterial pressure (PAP), the pulmonary capillary wedge pressure (PW), the systemic arterial pressure (SAP) and the cardiac output (CO) were measured in 41 healthy Friesian calves. The heart rate (HR), the stroke volume (SV), the cardiac and stroke indices (CI and SI, respectively), the pulmonary and systemic vascular resistance (PVR and SVR, respectively), the right ventricular and left ventricular work (RVW and LVW, respectively) and their corresponding indices (PVRI, SVRI, RVWI and LVWI, respectively) were also measured or calculated. The cardiac output, SV, SAP, PVRI, SVRI, RVW and LVW increased significantly while the HR, CI, PVR, SVR, RVWI and LVWI decreased significantly with somatic growth. The right-sided vascular pressures did not change significantly. The significant increase in systemic arterial pressure may be due to the simultaneous increase in CO. The high CI observed in the first few weeks of life was attributed to a high metabolic rate and might induce a reduced cardiac pumping reserve in young calves. In consequence, a therapeutic inotropic intervention may have little potential benefit at this age.
The stress-responsive DDR2 gene (previously called DDRA2) of Saccharomyces cerevisiae is transcribed at elevated levels following stress caused by heat shock or DNA damage. Previously, we identified a 51-bp promoter fragment, oligo31/32, which conferred heat shock inducibility on the heterologous CYC1-lacZ reporter gene in S. cerevisiae (N. Kobayashi and K. McEntee, Proc. Natl. Acad. Sci. USA 87:6550-6554, 1990). Using a series of synthetic oligonucleotides, we have identified a pentanucleotide, CCCCT (C4T), as an essential component of this stress response sequence. This element is not a binding site for the well-characterized heat shock transcription factor which recognizes a distinct cis-acting heat shock element in the promoters of many heat shock genes. Here we demonstrate the ability of oligonucleotides containing the C4T sequence to confer heat shock inducibility on the reporter gene and show that the presence of two such elements produces more than additive effects on induction. Gel retardation experiments have been used to demonstrate specific complex formation between C4T-containing fragments and one or more yeast proteins. Formation of these complexes was not competed by fragments containing mutations in the C4T sequence nor by heat shock element-containing competitor DNAs. Fragments containing the C4T element bound to a single 140-kDa polypeptide, distinct from heat shock transcription factors in yeast crude extracts. These experiments identify key cis- and trans-acting components of a novel heat shock stress response pathway in S. cerevisiae.
Hereditary muscular hypertrophy is a character that has been selected in several animal species for industrial meat production. The selection of this character in cattle produces animals of exceptional commercial value but ones with a lower aerobic capacity than that of conventional cattle. The purpose of this work was to study the role of cardiac function as a potential limiting factor of aerobic capacity in double-muscled calves. Two groups of healthy calves were studied, one consisting of nine calves of conventional conformation and the other of nine double-muscled calves. Pulmonary arterial and capillary wedge, central venous, and systemic arterial pressures were measured by fluid-filled catheters and recorded together with the electrocardiogram. Cardiac output was measured by the thermodilution technique. From these measurements, the heart rate, the cardiac and the stroke indices, the pulmonary and the systemic vascular resistances, and the cardiac power output were calculated. The parameters were recorded under basal resting conditions and during incremental dobutamine challenge, which allowed determination of the resting cardiac power output, the cardiac pumping capability, and the cardiac reserve. Dobutamine challenge induced a significant rise in cardiac and stroke indices, heart rate, and cardiac power output, a significant decrease in pulmonary and systemic vascular resistances, and no change in systemic and pulmonary arterial pressures. The cardiac reserve obtained in the present study was low in comparison with those previously reported in humans, dogs, and horses. This may be related to the poor running capability of bovine species relative to that of the former species.(ABSTRACT TRUNCATED AT 250 WORDS)
An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.
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The purpose of this study was (1) to evaluate the technical and methodological problems associated with invasive haemodynamic measurements in unsedated cattle; (2) to assess the reproducibility of such measurements both within and between days; and (3) to compare the values with those previously reported. Twenty-one healthy calves, aged from 5.5 to 12 months, were studied. The central venous, the right ventricular, the pulmonary arterial, the pulmonary capillary wedge and the systemic arterial pressures were obtained by means of fluid-filled catheters, and the cardiac output was measured by the thermodilution technique. The heart rate, the stroke volume, the pulmonary and systemic vascular resistances and the pulmonary and systemic ventricular workloads were calculated. An adverse reaction, consisting of severe pulmonary hypertension, tachycardia, tachypnoea and transient weakness, occurred in 7 calves during the catheterization procedures. Such a reaction might be due to a local reflex induced by stimulation of mechano-receptors by the catheter tip. It should be avoided by reducing the manipulation of the catheter as much as possible and by inflating the tip of the balloon when moving it forwards. A comparison of the vascular pressures with those previously reported was difficult because of methodological or technical limitations, such as, for instance, a lack of standardization of the baseline. The reproducibility of the haemodynamic measurements obtained was satisfactory, in contrast to previous studies performed in conscious animals. This was attributed to our animals being better trained to the experimental conditions and emphasizes the importance of reducing mental stress in obtaining reliable haemodynamic measurements in unsedated and potentially uncooperative animals.
Sporadic cases of testicular hypoplasia were earlier found in bulls of the Swedish Red and White breed. An accumulation of cases have occurred since 1970 in sons of 2 outstanding progenytested bull sires, 2 F and 27 U, which had a common father, 545 B. The history and clinical examination of affected bulls varied. Some had azoospermia and very small testes at a young age, while others could be normal in all respect when they were young but had a short reproductive life and had to be culled at about 3 years of age. Most of the affected bulls were between these 2 extremes. The histologic examination showed principally different degrees of testicular degeneration. There were always some germ cells left in all affected seminiferous tubules indicating that there was not a lack of germ cells causing the hypoplasia. Germ cell weakness is obviously a hereditary condition. The sires 545 B, 2 F and 27 U had a relatively low fertility. In their pedigree were several bulls known to have had a low fertility. No sons of 2 F and only a few sons of 27 U were used for A.I. services and at present only few cases of testicular hypoplasia are seen.
The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.
The complete nucleotide sequences of the recA genes from Escherichia coli B/r, Shigella flexneri, Erwinia carotovora and Proteus vulgaris were determined. The DNA sequence of the coding region of the E. coli B/r gene contained a single nucleotide change compared with the E. coli K12 gene sequence whereas the S. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to the E. coli K12 RecA protein. The DNA sequences of the recA genes from E. carotovora and P. vulgaris were 80% and 74% homologous, respectively, to the E. coli K12 gene. The predicted amino acid sequences of the E. carotovora and P. vulgaris RecA proteins were 91% and 85% identical respectively, to that of E. coli K12. The RecA proteins from both P. vulgaris and E. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.