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I Gilbert

Publications and source records attributed to I Gilbert.

13 recordsLinked to original sources
PubMed2003

A force-based protein biochip.

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Base Sequence↗
PubMed1998

Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase alpha-primase.

The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). Fluorescence titrations revealed noncompetitive inhibition of TNP-ADP binding by origin DNA, and noncompetitive inhibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able to bind the TNP nucleotide. From these titrations, we have measured a binding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral origin DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier. The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a preferred ligand over free T antigen in the double hexamer assembly reaction, preformed T antigen hexamers were incapable of forming double hexamers on the DNA. The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.

Antigens, Polyomavirus Transforming↗
PubMed1994

Effects of follicular phase exercise on luteinizing hormone pulse characteristics in sedentary eumenorrhoeic women.

OBJECTIVE: Current studies reveal little regarding the inception of exercise-induced LH changes during physical training. This study aimed to assess the susceptibility of the hypothalamic-pituitary axis to the acute physical stress of exercise in untrained, physically inactive women. The acute effects of submaximal endurance exercise upon the pulsatile LH secretion in the follicular phase were compared with those accompanying leisurely strolling for a similar time period. SUBJECTS: All subjects were eumenorrhoeic, as determined by biphasic temperature patterns, detection of the urinary LH surge, and mid-luteal serum progesterone levels. Subjects were not physically active and had little history of strenuous exercise (VO2max = 38.0 +/- 1.8) (mean +/- SEM) ml/kg/min). DESIGN: All women completed a 13.5-hour pulsatility test which included three consecutive 20-minute runs on a treadmill at 50, 60 and 70% of the subjects' maximum oxygen uptake (n = 16). Six of these same subjects completed a separate test on another occasion in which one hour of leisurely strolling was substituted for exercise. Blood was sampled every 10 minutes via an indwelling cannula for 4.5 hours before and 8 hours after one hour of exercise and or strolling. MEASUREMENTS: A pulse algorithm (Pulsar) was used to quantify LH pulse characteristics. RESULTS: Exercise produced no significant effects upon LH pulse frequency or mean serum LH concentration. However, exercise of moderate intensity caused a significant increase in LH pulse amplitude (P < 0.05). Strolling produced no significant changes in LH secretion. CONCLUSION: Acute exercise of moderate intensity in the follicular phase of untrained women is an insufficient stimulus to inhibit the GnRH pulse generator in the post-exercise period, yet may produce a slight stimulatory effect on the amount of LH released per pulse.

Adult↗
PubMed1993

Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.

Alanine↗
PubMed1992

Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system.

The single-stranded DNA binding protein RP-A is required in SV40 DNA in vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replication in vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase alpha-primase complex.

Ammonium Sulfate↗
PubMed1990

The effects of submaximal endurance exercise upon LH pulsatility.

The acute effects of submaximal endurance exercise (three consecutive 20-min runs on a treadmill at 50, 60 and 70% of the subjects' maximum oxygen uptake) upon the pulsatile release of LH were compared with those accompanying leisurely strolling for a similar period in seven normally menstruating young women. All trials were conducted during the early to mid-luteal phase, as determined by body temperature patterns, ultrasonic scans of the ovaries, detection of the LH surge in first morning urine specimens, and serial measurements of plasma progesterone. Blood was sampled every 10 min via an indwelling cannula for 8 h before and 12 h after exercise and serum LH measured by radioimmunoassay. LH pulsations were analysed by a time series method. Following cannulation, mean LH levels declined but then rose to reach a maximum 2 h before the beginning of the exercise bout. LH concentrations remained virtually unchanged during exercise itself, and exhibited a declining trend throughout the post-exercise period. The findings in the two groups were similar in all respects, except that in the control study the rate of LH pulsatility was significantly diminished (P less than 0.05) during the first 2 h of sampling as compared with the subsequent 2-h period. The approximate half-life of LH varied from 27 to 57 min, with a mean of 41 min.

Adult↗
PubMed1989

The detection of peaks in luteinizing hormone secretion.

Fluctations in luteinizing hormone are believed to consist of irregularly spaced sharp increases separated by periods of exponential decay. A simple method is presented for analysing such fluctuations when the data consist of uniformly sampled observations of hormone. Specific allowance for the exponential decay in the absence of pulses is made via a time series model before assessing the number and extent of pulses. All calculations are done using MINITAB regression programs. The results have been compared with those obtained by three established models and are in general agreement.

Activity Cycles↗
PubMed1989

Role of gamma globulin.

The introduction of preparations of immune serum globulin that are safe for intravenous use (IVIG) has made possible safe and effective prophylactic treatment for patients with a variety of humoral immunodeficiencies. These include not only primary agammaglobulinemia and common variable hypogammaglobulinemia but also the antibody deficiencies that accompany chronic lymphocytic leukemia (CLL) and multiple myeloma, as well as the hypogamma-globulinemia found in very low birth weight newborns who have not received adequate transplacental IgG from their mothers. In contrast, trials to date have not shown efficacy of IVIG in preventing sepsis in burn patients. The ease of administration and efficacy of IVIG in preventing respiratory symptoms in hypogammaglobulinemic patients has suggested that many other patients presenting with sinusitis and asthma, recurrent bronchitis, and other chronic chest symptoms might also benefit from IVIG and that they should be worked up for IgG subclass or specific antibody deficiencies. Side effects of IVIG administration are generally minor and may be prevented by slow administration and/or pretreatment with aspirin or Benadryl. The only contraindication to IVIG treatment is anaphylactic sensitivity to IgA, which is extremely rare. IVIG is thus an effective and safe form of prophylaxis that can reduce the incidence of pneumonia and other respiratory infections in patients with antibody deficiency as a predisposing factor.

Adult↗
PubMed1988

Ultra rapid blood sampling for the determination of short-term variations in the circulating concentration of oestradiol in man.

Blood was taken from three healthy female and three healthy male volunteers every 20 s for 15 min. The serum level of oestradiol was measured and the pattern of variation assessed by a cusum plot of the sequential data, by autocorrelation of the detrended serial data, and by looking for pulses. In two cases the variation in oestradiol values was greater than that which could be attributed to variation in the assay. Both these subjects showed a significant overall change in values during the sampling period (an increase and a decrease). There was no trend in the remaining four subjects. In two of the six subjects there was significant autocorrelation of detrended sequential levels. Defining a 'pulse' as three times the assay coefficient of variation no more pulses were identified than was expected from random fluctuations. By frequency analysis the two subjects with significant autocorrelation showed periodic fluctuations of approximately 70/h and 9/h respectively. It is apparent that both the rate of sampling and the method of analysis greatly influence the evaluation of pulsatile release of oestradiol.

Adult↗
PubMed1985

Adenocarcinoma of the lung presenting with pericardial tamponade: report of a case and review of the literature.

In a patient with cancer, a diagnosis of cardiac tamponade should be considered when there is dyspnea, cough, thready pulse or pulsus paradoxus, low systolic blood pressure, engorged neck veins, an enlarged cardiac silhouette, and total or ventricular electrical alternans. Immediate pericardiocentesis is indicated in such patients to avoid the risk of sudden death. A pericardial window should be created for more prolonged palliation of cardiac tamponade. Cytologic examination of the pericardial fluid often reveals malignant or highly suspect cells. Metastatic carcinomas from the lung and breast are the most common tumors that involve the heart when they spread in a retrograde fashion through the cardiac lymphatic system. Total pericardiectomy for the treatment of cardiac tamponade that is due to cancer is not generally advisable. Radiation therapy in the cardiac area with or without systemic chemotherapy is effective in decreasing the amount and the recurrence of neoplastic pericardial effusion.

Adenocarcinoma↗
PubMed1984

Particle-counting immunoassay of human somatotropin.

Human somatotropin was assayed by a novel automated nonradioisotopic technique, "particle-counting immunoassay," that requires a 45-min incubation and only 60 microL of 10-fold diluted sample. The principle of the assay is agglutination of antibody-coated latex particle by somatotropin, the reaction being measured by the (instrumented) counting of residual non-agglutinated particles. The dynamic range in serum extends from 0.2 to 40 micrograms/L. The between-assay CV was 12% for a concentration of 3.3 micrograms/L and 8.5% for 31.9 micrograms/L. The coefficient of correlation (r) with radioimmunoassay was 0.97. Curves for various dilutions of the macromolecular and monomeric forms of the hormone were not parallel.

Agglutination Tests↗