Quantitative analysis of the time courses of enzyme-catalyzed reactions

The catalytic properties of enzymes are usually evaluated by measuring and analyzing reaction rates. However, analyzing the complete time course can be advantageous because it contains additional information about the properties of the enzyme. Moreover, for systems that are not at steady state, the analysis of time courses is the preferred method. One of the major barriers to the wide application of time courses is that it may be computationally more difficult to extract information from these experiments. Here the basic approach to analyzing time courses is described, together with some examples of the essential computer code to implement these analyses. A general method that can be applied to both steady state and non-steady-state systems is recommended. (C) 2001 academic Press.

On the heritability of inspection time and its covariance with IQ: A twin study

Using the classical twin design, this study investigates the influence of genetic factors on the large phenotypic variance in inspection time (IT), and whether the well established IT-IQ association can be explained by a common genetic factor. Three hundred ninety pairs of twins (184 monozygotic, MZ; 206 dizygotic, DZ) with a mean age of 16 years participated, and 49 pairs returned approximately 3 months, later for retesting. As in many IT studies, the pi figure stimulus was used and IT was estimated from the cumulative normal ogive. IT ranged from 39.4 to 774.1 ms (159 +/- 110.1 ms) with faster ITs (by an average of 26.9 ms) found in the retest session from which a reliability of .69 was estimated. Full-scale IQ (FIQ) was assessed by the Multidimensional Aptitude Battery (MAB) and ranged from 79 to 145 (111 +/- 13). The phenotypic association between IT and FIQ was confirmed (- .35) and bivariate results showed that a common genetic factor accounted for 36% of the variance in IT and 32% of the variance in FIQ. The maximum likelihood estimate of the genetic correlation was - .63. When performance and verbal IQ (PIQ & VIQ) were analysed with IT, a stronger phenotypic and genetic relationship was found between PIQ and IT than with VIQ. A large part of the IT variance (64%) was accounted for by a unique environmental factor. Further genetic factors were needed to explain the remaining variance in IQ with a small component of unique environmental variance present. The separability of a shared genetic factor influencing IT and IQ from the total genetic variance in IQ suggests that IT affects a specific subcomponent of intelligence rather than a generalised efficiency. (C) 2001 Elsevier Science Inc. All rights reserved.

Effect of probe stimulus intensity on the dissociation between autonomic orienting and secondary probe reaction time

Information processing accounts propose that autonomic orienting reflects the amount of resources allocated to process a stimulus. However, secondary task reaction time (RT), a supposed measure of processing resources, has shown a dissociation from autonomic orienting. The present study tested the hypothesis that secondary task RT reflects a serial processing mechanism. Participants (N = 24) were presented with circle and ellipse shapes and asked to count the number of longer-than-usual presentations of one shape (task-relevant) and to ignore presentations of a second shape (task-irrelevant). Concurrent with the counting task, participants performed a secondary RT task to an auditory probe presented at either a high or low intensity and at two different probe positions following shape onset (50 and 300 ms). Electrodermal orienting was larger during task-relevant shapes than during task-irrelevant shapes, but secondary task RT to the high-intensity probe was slower during the latter. In addition, an underadditive interaction between probe stimulus intensity and probe position was found in secondary RT. The findings are consistent with a serial processing model of secondary RT and suggest that the notion of processing stages should be incorporated into current information-processing models of autonomic orienting.

Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Accuracy and cost- and time-effectiveness of digital clip versus videotape interpretation of echocardiograms in patients with valvular disease

Background. Although digital and videotaped images are known to be comparable for the evaluation of left ventricular function, their relative accuracy for assessment of more complex anatomy is unclear. We sought to compare reading time, storage costs, and concordance of video and digital interpretations across multiple observers and sites. Methods. One hundred one patients with valvular (90 mitral, 48 aortic, 80 tricuspid) disease were selected prospectively, and studies were stored according to video and standardized digital protocols. The same reviewer interpreted video and digital images independently and at different times with the use of a standard report form to evaluate 40 items (eg, severity of stenosis or regurgitation, leaflet thickening, and calcification) as normal or mildly, moderately, or severely abnormal Concordance between modalities was expressed at kappa Major discordance (difference of >1 level of severity) was ascribed to the modality that gave the lesser severity. CD-ROM was used to store digital data (20:1 lossy compression), and super-VHS video-tape was used to store video data The reading time and storage costs for each modality were compared Results. Measured parameters were highly concordant (ejection fraction was 52% +/- 13% by both). Major discordance was rare, and lesser values were reported with digital rather than video interpretation in the categories of aortic and mitral valve thicken ing (1% to 2%) and severity of mitral regurgitation (2%). Digital reading time was 6.8 +/- 2.4 minutes, 38% shorter than with video (11.0 +/- 3.0, range 8 to 22 minutes, P < .001). Compressed digital studies had an average size of 60 <plus/minus> 14 megabytes (range 26 to 96 megabytes). Storage cost for video was A$0.62 per patient (18 studies per tape, total cost A$11.20), compared with A$0.31 per patient for digital storage (8 studies per CD-ROM, total cost A$2.50). Conclusion. Digital and video interpretation were highly concordant; in the few cases of major discordance, the digital scores were lower, perhaps reflecting undersampling. Use of additional views and longer clips may be indicated to minimize discordance with video in patients with complex problems. Digital interpretation offers a significant reduction in reading times and the cost of archiving.

Rearrangement of diflunisal acyl glucuronide into its beta-glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta -glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5 +/- 2.5, 28.8 +/- 8.3 and 11.0 +/- 1.6 mug DF/ml respectively, obtained at 3.8 +/- 0.3, 3.6 +/- 1.8 and 7.5 +/- 0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2 +/- 3.3, 19.8 +/- 0.8 and 42.9 +/- 10.1% of the doses respectively were recovered in feces, with less than or equal to 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine. whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degreesC. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 mug DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 mug DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta -glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon. (C) 2001 Elsevier Science Inc. All rights reserved.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

Smooth muscle cells of injured rat and rabbit arteries in culture: contractile and cytoskeletal proteins

The aim of this study is to determine whether subpopulations of smooth muscle cells (SMC). as distinguished by variations in contractile and cytoskeletal proteins, appear in the neointima at different times after vascular injury, and/or whether subpopulations develop during serial passaging of these cells. Rat aortae and rabbit carotid arteries were injured with a 2F Fogarty balloon catheter and cultures established from the resulting neointima and the media 2, 6, 12, 16 and 24 weeks later. Cultures were examined at passages 1-5 and subpopulations of SMC categorised by intensity of staining for each protein by immunohistochemistry. Two populations of SMC with different staining intensities ('+ +', '+') were observed for each of the following proteins: alpha -SM actin, SM-myosin, desmin and vimentin. Populations without these proteins were also found. Changes in the percentages of cells expressing these proteins were transitory, indicating that the populations were not limited to a particular tissue (neointima or media), time after injury or passage number. One exception was found in rabbit cultures where the number of desmin-expressing cells quickly decreased with both time after injury and time in culture. Subpopulations of SMC were found at all times after injury in the media and neointima of rat and rabbit arteries, and after multiple passage of these cells. There was no pattern of development of one population suggesting that either no subpopulation has a proliferative or migratory advantage over others, or that only one population exists: that is capable of diverse phenotypic changes. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Time-dependent master equation simulation of complex elementary reactions in combustion: Application to the reaction of (CH2)-C-1 with C2H2 from 300-2000 K

Computational simulations of the title reaction are presented, covering a temperature range from 300 to 2000 K. At lower temperatures we find that initial formation of the cyclopropene complex by addition of methylene to acetylene is irreversible, as is the stabilisation process via collisional energy transfer. Product branching between propargyl and the stable isomers is predicted at 300 K as a function of pressure for the first time. At intermediate temperatures (1200 K), complex temporal evolution involving multiple steady states begins to emerge. At high temperatures (2000 K) the timescale for subsequent unimolecular decay of thermalized intermediates begins to impinge on the timescale for reaction of methylene, such that the rate of formation of propargyl product does not admit a simple analysis in terms of a single time-independent rate constant until the methylene supply becomes depleted. Likewise, at the elevated temperatures the thermalized intermediates cannot be regarded as irreversible product channels. Our solution algorithm involves spectral propagation of a symmetrised version of the discretized master equation matrix, and is implemented in a high precision environment which makes hitherto unachievable low-temperature modelling a reality.

Time evolution in the unimolecular master equation at low temperatures: full spectral solution with scalable iterative methods and high precision

A new method is presented to determine an accurate eigendecomposition of difficult low temperature unimolecular master equation problems. Based on a generalisation of the Nesbet method, the new method is capable of achieving complete spectral resolution of the master equation matrix with relative accuracy in the eigenvectors. The method is applied to a test case of the decomposition of ethane at 300 K from a microcanonical initial population with energy transfer modelled by both Ergodic Collision Theory and the exponential-down model. The fact that quadruple precision (16-byte) arithmetic is required irrespective of the eigensolution method used is demonstrated. (C) 2001 Elsevier Science B.V. All rights reserved.

Flavour retention during high temperature short time extrusion cooking process: a review

Research on the stability of flavours during high temperature extrusion cooking is reviewed. The important factors that affect flavour and aroma retention during the process of extrusion are illustrated. A substantial number of flavour volatiles which are incorporated prior to extrusion are normally lost during expansion, this is because of steam distillation. Therefore, a general practice has been to introduce a flavour mix after the extrusion process. This extra operation requires a binding agent (normally oil), and may also result in a non-uniform distribution of the flavour and low oxidative stability of the flavours exposed on the surface. Therefore, the importance of encapsulated flavours, particularly the beta -cyclodextrin-flavour complex, is highlighted in this paper.