The univariate approaches used to analyze heart rate variability have recently been extended by several bivariate approaches with respect to cardiorespiratory coordination. Some approaches are explicitly based on mathematical models which investigate the synchronization between weakly coupled complex systems. Others use an heuristic approach, i.e. characteristic features of both time series, to develop appropriate bivariate methods.In this study six different methods used to analyze cardiorespiratory coordination have been quantitatively compared with respect to their performance . Five of these approaches have been suggested in the recent literature whereas one method originates from older studies.The methods were applied to the simultaneous recordings of an electrocardiogram and a respiratory trace of 20 healthy subjects during night-time sleep from 0:00 to 6:00. The best temporal resolution and the highest number of coordinated heart beats were obtained with the analysis of 'Phase Recurrences'. Apart from the oldest method, all methods showed similar qualitative results although the quantities varied between the different approaches. In contrast, the oldest method detected considerably fewer coordinated heart beats since it only used part of the maximum amount of information available in each recording.The method of 'Phase Recurrences' should be the method of choice for the detection of cardiorespiratory coordination since it offers the best temporal resolution and the highest number of coordinated sequences and heart beats. Excluding the oldest method, the results of the heuristic approaches may also be interpreted in terms of the mathematical models. The time intervals between successive heartbeats  may be analyzed with different tools to obtain information about e.g. heart rate variability (HRV) , regularTo gain information that cannot be extracted from a single time series, the interaction between two (or more) physiological (sub-)systems has been investigated. This is often done on the basis of simultaneously recorded time series of each system. Hence, new bivariate techniques have been developed that aim to quantify the imprint of the physiological interaction between the two systems -17. EspeIn fact, physiologists had already investigated cardiorespiratory coordination in the human organism as early as the 1960ies . CalculaThe similarities or differences between the definitions of the different techniques and the advantages of each technique are not known yet. Unfortunately, a complete review and a  comparison of the recent literature is too extensive and beyond the scope of this paper. This study is restricted to the comparison of the performance of six different techniques in the detection of cardiorespiratory coordination. Although the comparison is limited, the insight that is drawn from it provides valuable information with regard to techniques that are not considered in this comparison. A second aim of this study is to investigate the cardiorespiratory interaction during night-time sleep since previous studies only dealt with relatively short recordings (no more than one hour).The electrocardiogram  and the uncalibrated nasal airflow (derived by a thermistor-technique) were recorded simultaneously in 20 healthy subjects  using an ambulatory device . In each subject the night-time sleeping period was recorded, in some subjects the complete evening before the sleep period was also recorded. Hence, the total recording time varied from 7–16 hours. The device's internal sampling rate of the ECG was 3000 Hz. Thus, the times of the automatically identified R-peaks had an accuracy <1 ms and were written into a file. To save memory, the ECG was recorded using a rate of 250 Hz. The data were transferred to a PC and analyzed using Matlab  and C routines. The times of the automatically identified R-peaks were visually controlled, i.e. the times of the R-waves were marked in the ECG, and edited if the times of the automatically identified R-peaks did not match the R-peak in the ECG . The times of the edited R-peaks had an accuracy of 4 ms because the recorded ECG had a lower sampling rate. Ectopies and artefacts were marked and excluded from the analysis.Ri  and the times of the inspiratory onsets Ij  were necessary and saved into a file. These data served as the basis for further calculations, see Figure The device's sampling rate of the nasal airflow was 100 Hz. The respiratory trace was saved into a file. Low frequency baseline trends were avoided using an internal filter with a 0.01 Hz cut-off frequency. The trace did not need any further pre-processing or filtering because it was smooth  is the calculation of temporal distances between the event markers of the two time series. Generally, two different useful possibilities have to be considered. (1) The temporal distance between inspiratory onsets and successive R-peaks, or, (2) the temporal distance between an inspiratory onset and the preceding R-peak.The first step towards the analysis of cardiorespiratory coordination with respect to different 1. Temporal distance between inspiratory onsets and successive R-peaks:ti = Ri - Ij     (1)absolute distance: 2. temporal distance between an inspiratory onset and the preceding R-peak:tj = Ij - Ri-1     (3)absolute distance: ti and n > 1 the appropriate settings of the indices i and j have to be considered. Furthermore, the relative distance φi corresponds to a definition of a phase angle of an oscillator on the interval . This phase (multiplied by 2π) is equivalent to the cyclic phase calculated via the Hilbert-transformation . This approach may easily be generalized for m:n-synchronization if the phases φ1 and φ2 are replaced by φ1/m and φ2/n. The distribution of φ2 may be quantified by the first Fourier mode.Along with the development of the Synchrogram a quantification on the basis of a mathematical model has been proposed ,47,48. Cφ2 at the times t the phase φ1 is φ1= θ :The formalization of this idea is as follows: Observe the phase ξi which is known as circular variance  they may be mapped onto the interval  by multiplication with 2π. In this study, the distribution of βj in a data window of length nF is quantified by the calculation of the first Fourier mode of the distribution (the circular variance):The quantification of the distribution of λ' defined above, the range of γ is 0 ≤ γ ≤ 1. If γ = 0 βj is equally distributed indicating the completely de-coordinated case. If γ = 1 the βj is constant in the data window indicating the coordinated case. Practically, the inspiratory onsets in a data window are marked as being coordinated if γ ≥ thresγ. Subsequently, the R-peaks in the respiratory cycle that follow a coordinated inspiratory onset are marked as coordinated. The window length nF is set to 10 and thresγ = 0.5. The data window is forwarded one inspiratory onset to achieve the maximal temporal resolution.Analogously to the 'Synchronization-λ', (2) 'Phase Recurrence' of relative distances φi, (3) 'Phase Recurrence' of absolute distances ti, (4) 'Quantification of Histograms' of relative distances φi, (5) 'Quantification of Histograms' of absolute distances ti, and (6) 'Quantification of Distribution of relative distances βj'. The detected R-peaks in horizontal structures, i.e. coordinated R-peaks, are marked with their corresponding number m of the m:n-ratio . This allowed the distinction of coordinated R-peaks with respect to their m:n-ratio. Next, to reduce the amount of information, the percentage of coordinated R-peaks with a certain m:n-ratio in a window of 500 consecutive R-peaks was calculated. To get an adequate temporal resolution, the window was forwarded by 100 R-peaks until the entire series of R-peaks is covered. The percentage of coordinated R-peaks was coded as a greyscale plot because the percentage was plotted versus time for all analyzed m:n-ratios. This plot is called 'coordination diagram'  'Synchronization-nces ti,  'Quantifr between the coordination diagrams of the six methods was obtained as follows. Each correlation is calculated comparing the percentages of coordinated R-peaks with a m:n-ratio of two different methods for each data window. Furthermore, to enhance the validity of this comparison, only those pairs of percentages are used in which at least one percentage is greater than zero. Otherwise the large number of pairs with , i.e. the white area that both coordination diagrams have in common, would lead to a strong correlation even if the other parts of the coordination diagrams show apparent divergent m:n-coordination ratios.The total number of coordinated sequences and the total number of coordinated R-peaks during night-time sleep, i.e. from 0:00 to 6:00 assuming that all subjects were continuously asleep, served as quantitative measures of the performance of each method. They were calculated for each recording. Furthermore, the matrix of Pearson's correlation coefficients φi. Since the heart rate decreases during night-time sleep whereas the respiratory rate remains almost constant the m:n-ratios of cardiorespiratory coordination also decrease during this period. Thus the sleeping period may be estimated from the 'coordination diagram' by looking at the low m:n-ratios. In the example, the subject slept from 23:30 to 6:30 and had a predominant 4:1-coordination during night-time sleep. At midnight the maximum of approximately 68% is reached, i.e., 68% of all R-peaks in a window of 500 R-peaks showed a 4:1-coordination with respect to respiration. A second local maximum of 4:1-coordination is observable at 1:30. The rest of the night shows sequences with 4:1-, 7:2- (shown in between 4:1- and 3:1-coordination) and also some 3:1-coordination. Although not shown, it has to be added that qualitatively similar results were obtained with the other techniques. Furthermore, the analysis of the other subjects showed that the predominant m:n-coordination ratio during night-time sleep of each subject varies inter-individually and is one of the following ratios: 3:1, 4:1, 5:1, 7:2 or 9:2.An example of a 'coordination diagram' is shown in Figure m:n-ratio. As indicated by the time course of the separate m:n-coordinations the total coordination is not constant or a simple function of time. Instead, the total coordination seems to oscillate. In the course of the night-time sleep the frequency of the oscillation increases whereas the amplitude decreases. Similar qualitative results were obtained using the other techniques.The additional line drawn in the coordination diagram represents the 'total coordination', i.e., the percentage of coordinated R-peaks in a window of 500 consecutive R-peaks regardless of the NR and the total number of coordinated sequences Nseq during night-time sleep contain important quantitative information about the performance of the different methods  to NR = 6158 R-peaks for the detection via 'Phase Recurrences' of phases φi (PhRec φi), i.e. 7.7% to 30.4% of the total number of R-peaks during night-time sleep are coordinated with respiration. The 'Phase Recurrences' of times ti and the 'Quantification of Histograms' of phases φi also detect a high number of coordinated R-peaks during night-time sleep . Notice that the number of coordinated sequences is highest for the 'Phase Recurrences' (Nseq = 416 for relative distances and Nseq = 398 for absolute distances) compared to other methods. Hence, the coordinated sequences detected by the 'Phase Recurrences' are shorter than the sequences detected by other methods. Although not shown, it has to be added that the high number of coordinated sequences and R-peaks for the 'Phase Recurrences' is due to the setting k ≥ m for equation (7). If this setting is changed to k ≥ m + 8, i.e. the requirement |φi - φi+m| <ω has to be fulfilled at least m + 8 times, the median number of coordinated epochs decreases to Nseq = 206 and the median number of coordinated R-peaks decreases to NR = 4273.The total number of coordinated R-peaks r ranges from r = 0.63 to r = 0.91 indicating a good agreement between the different coordination diagrams. The coordination diagrams for 'Synchronization-λ' and 'Quantification of Histograms' of relative distances φi correlate strongest (r = 0.91). And also the coordination diagrams for 'Phase Recurrence' of absolute distances ti and 'Phase Recurrence' of relative distances φi show a strong correlation (r = 0.90).An example of the correlation between the coordination diagrams of the different methods is shown in Figure λ' and the 'Quantification of Histograms' of relative distances φi correlate strongest (median correlation r = 0.89), followed by the correlation between the 'Phase Recurrences' and the 'Quantification of Histograms' of relative distances φi (r = 0.82) and the correlation between the 'Phase Recurrences' of relative distances φi and the 'Phase Recurrences' of absolute distances ti (r = 0.81). In all rows of the matrix except the last the correlation coefficients are r > 0.64 indicating a strong correlation between results of the different techniques. Since all correlation coefficients in the last row are r < 0.5 the correlation between the results of the 'Quantification of inspiratory onsets in RR-intervals' (Hist βj) and all other techniques is weaker.The matrix of correlation coefficients was calculated for each subject. Table In recent years a variety of different methods have been proposed to detect cardiorespiratory interaction on the basis of simultaneously recorded time series of heart beat and respiration. In this study, six different methods to identify cardiorespiratory coordination based on mathematical models or heuristic approaches have been quantitatively compared. The main issue of the different methods is the recognition of coordination in synchrograms and post event time series, i.e. the detection of structures with horizontal lines in these representations. Thus, the procedures permit the quantification of qualitative information that is contained in these representations and may provide new information that is not extractable from the isolated univariate time series.φi. Since the coordinated R-peaks are distributed in the largest number of coordinated sequences this method has the best temporal resolution compared to all other methods used in this study. Furthermore, it is able to detect even very short and intermittent cardiorespiratory coordination because the implementation used in this study only required at least 2m R-peaks to detect a m:n-coordination . But, if the minimal number of required coordinated R-peaks is increased to e.g. 2(m + 8), this method shows approximately the same number of coordinated epochs and R-peaks as e.g. the 'Quantification of Histograms'. Hence, if the 'window length' of this method is increased, the results are similar to those of the methods with a window length of 20 R-peaks. Since the 'Phase Recurrences' of absolute distances ti also showed a large number of coordinated R-peaks and coordinated sequences, respectively, and the correlation to the results of the 'Phase Recurrences' of phases φi is strong, these procedures showed approximately the same characteristic features. All other methods were less sensitive in the detection of cardiorespiratory coordination because the identification of cardiorespiratory coordination required a minimal window length of 20 R-peaks. The method of 'Quantification of inspiratory onsets in RR-intervals' had an even longer window length, provided that the window length of 10 inspiratory onsets is equivalent to approximately 40 R-peaks since the average ratio of heart rate and respiratory rate, irregardless of any cardiorespiratory coordination, is 4 [m:n- ratio) was not constant but oscillated during night-time sleep. Since this oscillation correlates with heart rate variability (HRV) [The quantitative comparison of the six different methods to detect cardiorespiratory coordination showed that the group's median total number of coordinated R-peaks and the median total number of coordinated sequences varied depending on the detection method used. The largest number of coordinated R-peaks  and coordinated sequences was detected by the 'Phase Recurrences' of phases on, is 4 . Thus, tty (HRV)  and diffty (HRV) ,55, thisλ' and the 'Quantification of Histograms' of relative distances φi. Furthermore, the 'Phase Recurrences' of relative distances φi and of absolute distances ti correlated strongly. These results indicate that the amount of information contained in the relative distances φi, i.e. the relative timings of the R-peaks in a full respiratory cycle, and in the absolute distances ti, i.e. the timings of R-peaks with regard to the preceding inspiratory onset, was approximately the same. Furthermore, the results of the 'Synchronization-λ' showed strong correlations to the 'Phase Recurrences' and the 'Quantification of Histograms'. Thus, the latter methods, which have heuristic origins, may also be explained mathematically by the mathematical models used to develop the method of 'Synchronization-λ'. However, the correlation coefficients concerning the 'Quantification of Histograms' of relative distances βj, i.e. the relative timing of an inspiratory onset in the respective RR-interval, were lower. Thus, these distances seem to contain different information. This difference is attributed to the fact that the calculation of the relative distances βj is not based on the full amount of information available in the recording.Another quantitative comparison was carried out by intra-individually correlating the 'coordination diagrams' of each method. The correlation coefficients served as a measure of similarity between the different diagrams. The strongest correlation was observed between the coordination diagrams derived from the 'Synchronization-Taken these findings together, it is apparent that the different methods may lead to similar results if the maximum amount of information is used. It is important to keep in mind that all quantifications are based on the 'Post Event Time Series' or the 'Synchrogram'. Thus, although some methods have heuristic origins and some have a mathematical model as its origin and although the different approaches differ considerably with respect to their calculations, the relevant information of the 'Post Event Time Series' and the 'Synchrogram' is captured by the different methods.m:n-ratios. Further methods and models have to be developed to explore these features of the cardiorespiratory system.A debate exits whether the cardiorespiratory coordination is due to physiological interaction between the involved systems or other mechanisms might be responsible, like e.g. a reduced variability in at least one of the involved systems ,56. Surrβj contains different quantitative information since only parts of the full amount of information available is used. Hence, this method is not recommended because a comparison with the other methods is limited. Generally, using appropriate methods, cardiorespiratory coordination is detectable in approximately 20–25% of all heart beats during night-time sleep. Future work should focus on the prerequisites of the appearance of cardiorespiratory coordination during night-time sleep and whether this appearance is linked to sleep stages. Furthermore, a preliminary study has shown the loss of cardiorespiratory coordination in patients after acute myocardial infarction [In conclusion, the different methods to detect cardiorespiratory coordination show similar qualitative results. The method 'Phase Recurrences' has the ability to detect most sequences of heart beats coordinated with respiration. It is able to maximize the temporal resolution because even very short sequences may be detected. Thus, this method should be preferred. The method of 'Quantification of Histograms' of relative distances farction ,62. HencDC and HB designed the study, recruited the subjects and collected the data. DC carried out the analysis and drafted the manuscript. SL, DG and PvL were involved in the interpretation of the data and participated in the final revision.


Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers  were organized into linkage groups using Map Manager software.The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of Plasmodium falciparum614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.The  Plasmodium chabaudi chabaudi is a malaria parasite of murine rodents. It has been widely used as a model to study various aspects of parasite biology and disease which are difficult to investigate using human malaria parasites. For instance, P. c. chabaudi is being used to study the genetic basis of drug resistance  ATP. PCR products were run on acrylamide gels and AFLP bands visualised on autoradiography films. Polymorphic bands between the two parental strains were used as markers for the genetic linkage map.The AFLP method was carried out according to the original protocol  with sliet al . BrieflyFor every marker, the parental alleles identified in each of the progeny clones were entered in an Excel spreadsheet. The absence of a band in one parent was treated as the presence of the other parental allele at that locus. Data were then prepared for analysis with the Map Manager QTX software  accordinPrior to linkage analysis, markers were tested for random assortment using a chi-square test, to exclude markers segregating in a non-random fashion from the initial analysis with Map Manager, and thus to avoid spurious linkage inferences between such markers. Because of the large number of tests for non -random assortment performed (n = 672), some markers showing apparent non-random assortment may have been falsely excluded from our analysis; i.e. some valid markers may indeed show non-random assortment and should be included. A Bonferroni correction was therefore applied to the chi-square test to decrease the stringency of the statistical test. The value of the Bonferroni correction represents the number of 'independent' comparisons and here was arbitrarily set at 24. This value was chosen as representing the likely number of chromosomal fragments at meiosis, and is supported by data presented in this paper. Markers within these fragments are not independently inherited. The chosen value (24) is a compromise between 672  and 14 . Markers not following random assortment in the initial test were thus divided into two groups, i.e. those segregating in a non-random fashion before and after Bonferroni correction, and those segregating in a non-random fashion before but not after the Bonferroni correction. The markers in the latter group were added separately after linkage groups had been determined (see below).Linkage groups using AFLP and RFLP markers were formed with an initial p-value of 0.0001 using the "Make Linkage Groups" command in Map Manager. p-values in Map Manager indicate the probability of a Type 1 error; that is, the probability of a false positive linkage. Following formation of linkage groups, the p-value was raised to 0.001. Linkage at p < 0.001 is considered significant. Using the command "Distribute", linkage groups were brought together. Then, other previously unlinked markers were allocated to these new linkage groups, again using the "Distribute" command. Markers with non-random assortment after statistical analysis without Bonferroni correction were added next, and those still segregating in a non-random fashion after Bonferroni correction were added last. The "Ripple" function was then used to position markers in an order which maximizes the total LOD (logarithmic odds) score for linkage. The software also estimated the optimum order and genetic distance between markers in centiMorgans (cM) by using the "Kosambi" function in the software. It was then possible to calculate a map unit size .P. c. chabaudi chromosomes [The presence of 42 previously characterised RFLP markers which had been physically mapped onto omosomes  served aP. c. chabaudi AJ and either clone AS (3CQ) or AS (30CQ). The majority of the AFLP markers showed independent assortment in the 28 progeny clones, as illustrated previously [The inheritance of 672 AFLP markers was determined in 28 progeny clones derived from two crosses between eviously . Howevereviously . EstimatP. c. chabaudi chromosomes 1 and 5–13, by virtue of their linkage to RFLP markers previously assigned to specific chromosomes by physical mapping [The 672 AFLP markers formed a total of 22 linkage groups with a final p-value of 0.001. 58 AFLP markers, 21 of which segregated in a non-random fashion, could not be allocated to any linkage groups. Physical mapping of these markers would be required to assign them to specific chromosomes. Alternatively, unassigned linkage groups or unallocated markers might map to the small mitochondrial or apicoplast genomes, although these combined represent only 0.2% of the genome. Several RFLP markers could not be allocated to linkage groups by the Map Manager software, probably due to the small numbers of clones analysed for these markers. These markers were added to assigned linkage groups according to their chromosomal assignment, as previously determined by physical mapping [assigned markers because of factors such as a high rate of recombination between two linkage groups (one linked to the RFLP anchor) or because of an intervening region with a low density of AFLP markers. Both factors, or a combination of the two, may prevent two physically linked groups from being identified as genetically linked. Conversely an apparent unusually high frequency of AFLP markers (as in chromosome 7) may arise from strong linkage disequilibrium between loci on two different chromosomes. Some markers located on one chromosome may thus appear to be genetically linked to markers on another. This could arise where one locus exerts a strong constraint on another unlinked locus. For instance, the AJ allele of an enzyme in a metabolic pathway may only function with the presence of the product of the AJ allele encoding another enzyme in the same pathway. This constraint might be structural or functional. The same may be true of AS alleles of the same enzymes. In this case, the genes encoding these enzymes, and markers strongly linked to them, may appear in the same genetic linkage group.With the exception of chromosomes 2, 3, 4 and 14 (discussed above), chromosomes 9 and 10 showed the lowest density per Mb of AFLP markers. Chromosome 7 showed the highest density. This may simply reflect natural variation in the frequency of AFLP polymorphisms on particular chromosomes. However, for chromosomes with a low apparent density of AFLP markers such as chromosomes 9 and 10, it is likely that some of the markers in unassigned linkage groups would physically map to these chromosomes. These unassigned groups may not show genetic linkage with (groups of) AFLP markers were initially ordered on the linkage groups as described in Materials and Methods. After inspection of the predicted marker order, occasional manual adjustments were made to correct markers which appeared to be inappropriately positioned.Because Map Manager failed to allocate some RFLP markers to an assigned linkage group, these were positioned manually. The final distribution of the markers on the 10 linkage groups assigned to chromosomes is shown in Fig. If markers and recombination events were both uniformly distributed across the genome, then we would expect the number of predicted recombination events  in each chromosome to correlate with its physical size. The predicted total number of recombination events occuring in each linkage group is shown in the P. c. chabaudi genome consists of about 20 Mb [Plasmodium falciparum [Assuming that the ut 20 Mb  the valulciparum .P. falciparum, and were interpreted as being due to non-reciprocal conversion events [A significant number of double crossover events around a single AFLP marker were observed in many linkage groups. Any such occurrences were re-evaluated on the original X-ray film to detect any possible errors or ambiguous bands. Many distinct double crossover events were observed. The same phenomenon was also commonly observed in n events .The apparent genetic length of each linkage group in cM was calculated by Map Manager based on the number of recombination events . When adran, 40 cM from its nearest AFLP marker. At its other end, an RFLP marker, Ag 3027, lies about 35 cM from its nearest AFLP marker. Chromosome 10 has RFLP marker cDNA121 about 70 cM from its nearest AFLP marker and RFLP marker, 5S rRNA, a further 70 cM distant. These large gaps may be artefacts which arise for two reasons. Firstly, some unreliability in the typing of clones using RFLPs was previously noticed [ran, Ag3027, cDNA121 and 5S rRNA were typed for only 9, 17, 9 and 16 clones, respectively. The characterisation of inheritance of RFLP markers in all of the 28 progeny clones, and the correction of possible mistakes may reduce the estimated genetic length. This would lead to an increase in map unit size. Nevertheless, it is notable that the value reported above (13.7 kb/cM) is close to estimates made for P. falciparum , althou30 kb/cM .Of the 400 AFLP markers placed on chromosomes, 37 AS-AJ pairs  shared the same selective bases at both primer ends and showed complementary segregation in the cross-progeny clones. Most of these markers also differed in size by only a few base pairs. They are likely to be alleles at the same loci. This was confirmed by sequencing two such pairs, namely ASTA01AC and AJTA01AC (chromosome 5), and ASTT02CA and AJTT02CA (chromosome 13) (sequence data not shown). This suggests that a significant proportion of the polymorphisms observed between AS and AJ may be due to small insertions or deletions. In fact, it was observed that small indels tend to occur in introns or intergenic regions (data not shown).A few AFLP markers which were originally identified between clones AJ and AS  were notOngoing work on linkage between chloroquine resistance and markers on chromosome 11  suggesteP. falciparum [P. c. chabaudi [Eimeria tenella [Toxoplasma gondii [Theileria parva [Cryptosporidium parvum [P. falciparum exceeds its resolutionGenetic or physical linkage maps have been determined and reported for a number of apicomplexan parasites, including lciparum , P. c. cchabaudi , Eimeria tenella , Toxoplaa gondii , Theileria parva  and Crypm parvum . The mapOf the AFLP markers analysed, most were assigned to 10 of the 14 chromosomes, while some were placed on 12 unassigned linkage groups, which probably include groups located on chromosomes 2, 3, 4 and 14. The remaining AFLP markers could not be allocated to any linkage group. Unallocated AFLP markers and unassigned linkage groups may arise in a number of possible ways, discussed in the Results section above, including mistakes or considerable gaps in the recorded inheritance pattern of RFLP markers and, to a lesser extent, AFLP markers. Other factors include variations in the density of AFLP markers or polymorphism in general, areas of the genome where the rate of recombination is particularly high, linkage disequilibrium between loci on different chromosomes and non-random representation of clones in our sample. The numbers of markers allocated, the approximate genetic lengths and the number of recombination events were estimated for each of the linkage groups. The genetic length of the entire genome was estimated to be 1684 cM and the overall size of map unit 13.7 kb/cM. The genetic length and number of recombination events were expected to increase with the size of chromosomes. This was generally found to be the case, although a number of factors may influence these data. These factors include the failure to assign some linkage groups, incomplete or incorrect inheritance data, particularly for RFLPs, variation in the frequency of AFLP markers across the genome, variation in the recombination rate across the genome, and incorrect assignation of some linkage groups due to linkage disequilibrium.The presence and frequency of small indel mutations was confirmed. These markers could prove suitable for rapid typing of clones by size polymorphism and quantitative analysis by Real Time Quantitative PCR.P. chabaudi was originally conceived as an essential step towards the identification of loci linked to genes encoding important phenotypes, such as drug resistance. Indeed, the identification of a locus underlying chloroquine resistance in P. chabaudi within approximately 250 kb of chromosome 11 [P. c. chabaudi  [P. falciparum (complete) [P. falciparum genome. Because of the extensive gene synteny between P. chabaudi  and P. falciparum [P. falciparum are likely to be closely linked in P. chabaudi too.The generation of a complete AFLP genetic linkage map for osome 11  relied uosome 11  which moosome 11 . A genetpartial)  and P. fomplete) , and seqlciparum ,9,10, maP. falciparum genome in the studies discussed above [P. c. chabaudi, Plsmodium yoelii and P. falciparum genomes [P. falciparum genome , will allow us to assign unallocated AFLP markers and unassigned linkage groups to particular chromosomes on the assumption that gene synteny is conserved.The correspondence between the genetic linkage map reported here and the mapping of AFLP markers to the ed above ,5,26 has genomes ,10. The AM characterized the AFLP markers in the cross progeny, generated the genetic linkage map and drafted the article, PH helped in the generation of the linkage map, analysed genetic data from it and drafted the article, RF helped in the characterization of the AFLP markers in the cross progeny, PC and DW provided the recombinant clones and revised the article, RC designed and coordinated the study, revised the article and gave final approval. All authors read and approved the final manuscript.The file (.XLS) contains the following data for markers assigned to chromosomes and, where possible, for markers in unassigned linkage groups and the overall genome: – physical size of each chromosome (Mb), the number of markers , the number of AFLP markers per Mb, the number of recombination events predicted for the 28 clones, the frequency of recombination events per Mb and per AFLP marker, the predicted genetic length of all linkage groups, and the estimated size of map unit (kb/cM).Click here for fileThis file is the original PPT files from which figure 1 was derived.Figures 1-3 contain the linkage map for the chromosomes 1 and 5-13.Click here for fileThis file is the original PPT files from which figure 2 was derived.Figures 1-3 contain the linkage map for the chromosomes 1 and 5-13.Click here for fileThis file is the original PPT files from which figure 3 was derived.Figures 1-3 contain the linkage map for the chromosomes 1 and 5-13.Click here for fileThis file is the original PPT files from which figure 4 was derived. Figure 4contains various unassigned linkage groups.Click here for file


Retrovirology commemorates these two land mark findings by publishing separate personal recollections by Takatsuki and Gallo respectively on the discovery of ATL and HTLV.In 1977, Takatsuki and co-workers described in Japan a human malignant disease termed adult T-cell leukemia (ATL). Three years later, in 1980, Gallo and colleagues reported the identification of the first human retrovirus, human T-cell leukemia virus type I (HTLV-I), in a patient with cutaneous T-cell lymphoma. This month,  Retrovirology as a medical study first emerged in the early 1900s. In 1908, Ellermann and Bang reported on the transmissibility of avian leucosis by cell-free filtrates, suggesting the involvement of a virus [ a virus . Today, v-onc) encoded by many retroviruses were captured originally from cellular sequences (i.e. c-onc). To date, three general models of retroviral transformation are accepted: a) over-expression of v-onc; b) cis-oncogenic effect from promoter insertion; and c) cis-oncogenic effect from enhancer insertion (Fig. Understanding how retroviruses cause cancer took a major step forward with the development of the cellular oncogene hypothesis in 1976. Thus Varmus, Bishop and colleagues  demonstrion Fig. .c-onc nor by cis-insertion effects on the cellular genome. Pioneering molecular biology studies by Mitsuaki Yoshida and colleagues led to the delineation of the HTLV-I transforming gene, Tax [trans to disrupt cellular checkpoints and destabilize genome integrity [Retrovirology.Although not yet fully understood, HTLV-I is believed to transform human T-cells neither through the acquisition of a ene, Tax . Tax hasntegrity  leading Two articles in this month's Retrovirology describe respectively the discovery of adult T-cell leukemia  and HTLV


During excessive pressure or volume overload, cardiac cells are subjected to increased mechanical stress (MS). We set out to investigate how the stress response of cardiac cells to MS can be compared to genotoxic stresses induced by DNA damaging agents. We chose for this purpose to use ionising radiation (IR), which during mediastinal radiotherapy can result in cardiac tissue remodelling and diminished heart function, and ultraviolet radiation (UV) that in contrast to IR induces high concentrations of DNA replication- and transcription-blocking lesions.Cultures enriched for neonatal rat cardiac myocytes (CM) or fibroblasts were subjected to any one of the three stressors. Affymetrix microarrays, analysed with Linear Modelling on Probe Level, were used to determine gene expression patterns at 24 hours after (the start of) treatment. The numbers of differentially expressed genes after UV were considerably higher than after IR or MS. Remarkably, after all three stressors the predominant gene expression response in CM-enriched fractions was up-regulation, while in fibroblasts genes were more frequently down-regulated. To investigate the activation or repression of specific cellular pathways, genes present on the array were assigned to 25 groups, based on their biological function. As an example, in the group of cholesterol biosynthesis a significant proportion of genes was up-regulated in CM-enriched fractions after MS, but down-regulated after IR or UV.Gene expression responses after the types of cellular stress investigated  have a high stressor and cell type specificity. The mammalian myocardium contains several cell types, of which the cardiac myocytes (CM) make up most of the heart's mass. Although a small proportion of CM in the adult myocardium remains mitotic, most CM lose the capacity to undergo cell division shortly after birth . In the In cardiac cells, MS is increased during excessive pressure or volume load of the heart, as seen in hypertensive and valvular heart disease. This results in an adaptive growth response leading to structural and functional cardiac changes, including CM hypertrophy and hyperplasia of fibroblasts, to compensate for the increased workload . In vitrRadiation induced heart disease (RIHD) has been recognised as a late adverse effect of thoracic radiotherapy if the heart was situated in the radiation field . IR induThe density of DNA damaging events after UVC, which include helix-distorting photolesions, is three orders of magnitude higher than the density of DNA damage that occurs after IR  and suffThe aim of the study was to identify and compare differentially expressed genes (up-regulated or down-regulated when compared with untreated controls) in cardiac cells in response to MS, IR or UV. To this purpose, cultures enriched for ventricular CM or fibroblasts were exposed to one of the three stressors. Differentially expressed genes were identified using Affymetrix GeneChips. Several statistical methods have been developed to analyse Affymetrix gene expression microarrays. We used a method based on Linear Modelling on Probe Level  to descr2) between the signals calculated by the model and the actual PM signals obtained. Figure 2 values for fibroblasts after UV. The majority of probe-sets have a correlation coefficient ≥ 0.90, indicating that the model used fitted the data accurately.As an example for the accuracy of the linear model that was used to describe the PM probe signals, Figure A Series entry (accession number GSE2032) at Gene Expression Omnibus (GEO), a public gene expression database of NCBI , gives aBased on information available at NettAffx™ , extendeFigure Probe-sets that showed an up-regulation or down-regulation after more than one stressor are listed in table 2 see . The oveGene expression changes detected using microarrays were validated by semi-quantitative PCR, using RNA from CM-enriched cultures at 24 hours after MS. The increased gene expression of Tenascin C and biglycan were confirmed with PCR in two independent experiments. In these two experiments, Tenascin C gene expression increased 1.59 and 1.64 times, respectively. Biglycan gene expression increased 1.42 and 1.25 times, respectively. Figure In this study, a linear model was used to describe GeneChip PM probe signals and to determine effects of three types of stressors on gene expression in two neonatal rat heart cell populations, consisting of CM that are known to be terminally differentiated cells and fibroblasts that still have the capacity to undergo mitosis. Several studies have shown a good correlation between Affymetrix GeneChip data and RT-PCR  or NorthHere, differences between the two cell populations are observed in the predominant type of gene expression response, i.e. up-regulation versus down-regulation. After all three stressors, differentially expressed genes were mostly up-regulated (q < 0.005) in CM-enriched fractions, while in cultures of fibroblasts the majority of changed genes were down-regulated. After MS, these differences were most pronounced. Paracrine signalling is involved in the response of CM and cardiac fibroblasts in co-cultures subjected to MS . In the Of the three stressors examined in this study, both IR and UV are known to induce oxidative stress. Accordingly, both stressors induced high numbers of up-regulated genes involved in anti-oxidative processes. The high percentages of up-regulated genes encoding heat shock proteins in both cell populations after IR and UV might indicate oxidative stress induced protein damage in these cells. In comparison, after MS only few genes encoding heat shock proteins were up-regulated in either cell population.Heinloth et al. (2003) proposed a model for the regulation of several gene expression patterns after IR and UV in human dermal fibroblasts, based on microarray analysis. The general view that p53 plays a central role in signal transduction after IR and UV was confirmed in their study. Moreover, IR down-regulated the expression of genes involved in mitosis. UV, on the other hand, induced the expression of genes involved in protein degradation and the MAPK pathway . The latThe high numbers of up-regulated genes involved in ubiquitination and protein degradation in both cell populations after UV are also in accordance with the study of Heinloth et al. 2003) and with other studies on cultured cells after UV 03 and wi.In vivo hypertrophy of the heart is also associated with elevated myocardial cholesterol contents [In CM-enriched cultures, MS led to a relatively high proportion of up-regulated genes that are involved in cholesterol biosynthesis. Among these genes, expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, which forms the starting enzyme of the mevalonate pathway and is considered to be the key enzyme in cholesterol biosynthesis, was up-regulated. This suggests that in neonatal rat cardiac myocytes cholesterol biosynthesis is stimulated after MS. Neonatal rat heart myocytes that undergo hypertrophy in culture show an increased biosynthesis and intracellular accumulation of cholesterol . Moreovecontents . An incrcontents -32. Morecontents . The rolHigher numbers of differentially expressed genes involved in ECM formation, including genes encoding collagens, fibronectin and laminin, were determined in CM-enriched cultures after MS than in cultures of fibroblasts. As mentioned before, paracrine signalling between CM and remaining fibroblasts might play a role in cellular responses in these CM-enriched cultures. Alterations in expression of genes involved in ECM formation might originate from remaining fibroblasts after stimulation by CM. Interestingly, MS does not affect the expression of these genes in cultures of fibroblasts, which suggests that paracrine signalling from CM is necessary for alterations in expression profiles of genes involved in ECM formation in fibroblasts.The down-regulated genes encoding cytoskeletal components in cultures of fibroblasts after IR and UV, including myosin and troponin, are mostly CM-specific. Therefore, these genes are likely to originate from remaining CM in the fibroblast cultures, although their numbers were low (3–5%). The extremely low numbers of differentially expressed genes encoding for ion channels and exchangers might be explained by a low number of cardiac cell type-specific genes within this functional group.MS, IR and UV mainly induce stress-specific and cell-type specific gene expression profiles in neonatal rat CM-enriched cultures and cultures of neonatal rat heart fibroblasts. Functional groups that show significant percentages of differentially expressed genes suggest that certain cellular pathways are activated after one or more stresses.The experiments were performed with permission of the local committee on animal experiments, installed by the University of Leiden according to the Dutch law. Cardiac cells were isolated from ventricles of neonatal rat hearts, and cultured as described before ,34. By aAfter 2 days of culturing with 5% horse serum (HS), culture media of CM-enriched cultures were replaced by media containing 2.5% HS. One day later, these cultures were subjected to UV, IR or MS.Three days after isolation, cultures of fibroblasts were subcultured for 6 days in medium containing 10% foetal bovine serum (FBS). Then, culture medium was replaced by medium containing 2.5% FBS. One day later, confluent cell cultures were subjected to UV, IR or MS.-1. During irradiation, sham-irradiated control cells were kept at room temperature. After irradiation, cell cultures were maintained at 37°C for 24 h.Cells were subjected to a single dose of 8.5 Gy of X-rays at room temperature, using a 6 MV accelerator (SL 75-5 Philips), operated at a dose rate of 8 Gy min2 UVC at room temperature at a dose rate of 0.2 J/m2 per s. Before irradiation, medium was collected and cells were rinsed with PBS. Following irradiation, growth medium was returned to the cultures and the cells were incubated for an additional 24 h. Control cells received a similar treatment except for UV irradiation.In another experiment cells were irradiated with 10 J/m® (Flexcell) in which the frequency and magnitude of stretch were regulated by a computer-controlled vacuum pump. The apparatus applied an equiaxial cyclic stretch of 20% elongation to the wells at a frequency of 60 cycles/min (1 Hz). Stretch was applied for 24 h. Control cells were grown in identical culture plates and incubated in the same incubator as the stretched cultures, but were not mounted in the Flexercell Strain Unit.To subject cells to cyclic stretch, CM-enriched cells (54 ± 5% CM) and fibroblasts (95 ± 2% non-myocytes) were cultured in 6-well Flex I culture plates , coated with collagen I. Medium was changed to medium containing 2.5% serum and after 24 h plates were placed in the Flexercell Strain Unit FX-2000Twenty-four hours after IR or UV, non-irradiated and irradiated CM-enriched cultures were trypsinised and subjected to centrifugal elutriation . The proAfter undergoing MS or control treatment for 24 h, cells were collected from CM-enriched cultures and from cultures of fibroblasts by trypsinisation. The proportion of CM in the cell cultures was determined by flow cytometric analysis of myosin expression as described before . CM-enri® kit , according to the manufacturer's instructions. Ten to 14 μg of total RNA from CM-enriched fractions and 7.5 to 14 μg of total RNA from fibroblast cultures was used for labelling. Per experiment, the quantities of total RNA from irradiated and control cells were equal. cDNA was synthesised using the Gibco BRL Superscript system . Briefly, single stranded cDNA was synthesised using Superscript II reverse transcriptase and T7-oligo(dT)24 primers at 42°C for 1 h. Double stranded cDNA was obtained by using DNA ligase, DNA polymerase I and RNAse H at 16°C for 2 h, followed by T4 DNA polymerase at 16°C for 5 min. After clean up with a phase lock gel (Qiagen), ds-cDNA was used for in vitro transcription (IVT). cDNA was transcribed using the BioArray HighYield® RNA transcript labelling kit  in the presence of biotin-labelled ribonucleotides, or using the MEGAScript T7® kit , in the presence of biotin-labelled CTP and UTP. In each experiment, cDNAs from control cells and stressed cells were labelled simultaneously using the same labelling kit. After clean up with a RNeasy kit (Qiagen), the biotin-labelled IVT-RNA was fragmented in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium-acetate and 30 mM magnesium-acetate, at 94°C for 35 min.RNA was isolated applying an RNeasyRG-U34A arrays  were used, representing ~7000 transcripts, of which 6399 had a unique LocusLink identification number (ID) at the time of data analysis. Biotin-labelled IVT-RNA was hybridised to the arrays at 45°C for 16 h according to the manufacturer's instructions. After hybridisation, the arrays were washed in a GeneChip Fluidics Station 400 with a non-stringent wash buffer at 25°C followed by a stringent wash buffer at 50°C. After washing, the arrays were stained with a streptavidin-phycoerythrin complex. After staining, intensities were determined with a GeneChip scanner, controlled by GeneChip software (Affymetrix). The intensities were background corrected using gcrma  and normFor each stressor, three independent experiments were performed. To describe the signal of every PM probe, the following linear model was used: signal ~P + T + E + TE, based on a method described before . In our To determine significant proportions of differentially expressed genes within functional groups, the hypergeometric probability P was calculated. P < 0.005 was considered significant.2 was calculated for every probe-set, using the following standard formula: R2 = 1-ΣRi2/Σ2, where Ri = observed-fitted value for signali and mean signal = the mean of observed signali.To determine the accuracy of the linear model that was used to describe PM signals, RTo compare data on differentially expressed genes with microarray data in literature on species other than rat, RESOURCERER of the Institute of Genomic Research  was used® kit (Qiagen). Following DNAse  treatment, cDNA was synthesised using M-MLV reverse transcriptase  and oligo(dT) primers (Amersham). Semi-quantitative PCR was performed, using the following primers and annealing temperatures: Tenascin C sense: CGA CAG TTT TGT TAT CAG GAT CAG, Tenascin C antisense: GGC ACA TAA GTA ATC CGG AAA T, 60°C; Biglycan sense: CAA CAA CCC TGT GCC CTA CT, Biglycan antisense: GGT GT GCT TCT TTG CTT CC, 65°C. A competitive PCR was performed on GAPDH to correct for differences in cDNA concentrations. After agarose gel electrophoresis, intensities of PCR product bands were determined by Scion image analysis software .Total RNA isolation, cDNA synthesis and semi-quantitative PCR were performed as described before . In shorMB performed experiments, developed and analyzed functional groups of genes and prepared the manuscript. CGCW performed experiments, developed and analyzed functional groups of genes and participated in discussions. HV and AL participated in discussions and gave textual advice. PS performed microarray normalization and data analysis. JW and LHFM participated in discussions. AAZ participated in discussions, suggested the assignment of genes to functional groups and gave textual advice. All authors read and approved the final manuscript.q-values of genes within functional groups Table 1. Excel-file containing the 25 functional groups of genes used in this study. Of every gene, Affymetrix probe-set IDs and q-values after the three types of stress are listed. Down-regulated genes are represented by a minus-sign in front of their q-value. q-values < 0.005 are marked red (in case of up-regulation) and green (in case of down-regulation).Click here for fileGeneral stress response genes Table 2. Excel file containing Affymetrix probe-set IDs, LocusLink ID and gene description of all probe-sets that showed an up-regulation or down-regulation after two or more stresses.Click here for file


Although soy protein may have many health benefits derived from its associated antioxidants, many male exercisers avoid soy protein. This is due partly to a popular, but untested notion that in males, soy is inferior to whey in promoting muscle weight gain. This study provided a direct comparison between a soy product and a whey product.Lean body mass gain was examined in males from a university weight training class given daily servings of micronutrient-fortified protein bars containing soy or whey protein . Training used workouts with fairly low repetition numbers per set. A control group from the class (N = 9) did the training, but did not consume either type protein bar.Both the soy and whey treatment groups showed a gain in lean body mass, but the training-only group did not. The whey and training only groups, but not the soy group, showed a potentially deleterious post-training effect on two antioxidant-related related parameters.Soy and whey protein bar products both promoted exercise training-induced lean body mass gain, but the soy had the added benefit of preserving two aspects of antioxidant function. Many male exercisers avoid soy protein because there is a perception that it is inferior to proteins like whey for supporting lean boss mass gain. This perception persists even though there are no studies comparing whey and soy for effects on lean body mass gain. Soy may actually help promote lean body mass gain by the antioxidants associated with soy protein. Antioxidants are agents, either consumed in the diet or made by the body, which work against molecular damage due to oxidant reactions caused by free radicals, which are reactive molecules with an unpaired electron . Soy proIf soy protein can promote lean body mass gain at least as well as whey, there may be one advantage to consuming soy protein. Soy protein contains antioxidants which may not only help with lean body mass gain, but which can also promote other aspects of health. Antioxidant actions are thought to work against the onset and severity of many diseases and health problems . This maThe present study compared a soy protein product to a whey protein product in subjects undergoing a 9 week weight training program. Subjects were evaluated for lean body mass gain and changes in antioxidant status. The latter was done using one measurement of a component of antioxidant capacity and one for a component of oxidant stress. The former was based on an assay called plasma antioxidant status which assesses the ability to scavenge certain chemically generated radicals. The oxidant stress parameter was plasma myeloperoxidase, a measure of neutrophil activation, which is associated with increased secretion of superoxide radical .This study was approved by the Human Subjects Review Committee for Biomedical Sciences at The Ohio State University. All subjects signed an informed consent form. Male subjects, aged 19–25, were recruited from the Sport, Fitness and Health Program courses at The Ohio State University to participate in the present 9-week study. All subjects were considered experienced weightlifters with at least 1 year or more experience in strength training, which was confirmed by a questionnaire. Subjects were reported to be non-smokers, non-vegetarians, not currently taking supplements of any kind, and having no major health problems . All subjects had a body mass index (BMI) of less than 30.At the start of the study, each subject was put on a common strength training program to strictly follow for the duration of the 9 week study. Subjects were given either workout 1 or workout 2. The two workouts were identical with the exception of exercise order and were designed to prevent subjects in the strength training classes from having to perform the same exercises at the same time. Midway through the program, subjects with workout 1 were given workout 2 and vice versa in order to maintain consistency.The strength training protocol was 3 sets of 4–6 repetitions for 14 exercises so that strength was the variable being maximized. The following exercises were performed to work all major muscle groups: 1) chest press; 2) chest fly; 3) incline press; 4) lat pull-down; 5) seated row; 6) military press; 7) lateral raise; 8) preacher curl; 9) bicep curl; 10) supine tricep extension; 11) seated tricep extension; 12) leg press; 13) calf raise; and 14) abdominal crunches.® Bars, which contained 11 grams of protein and an assortment of micronutrients. The whey bars were made using the same recipe as the DrSoy® Bars except that whey protein was substituted for soy protein. The products were supplied to study personnel in plain wrappers with different colors for each product. The color code was unknown to the subjects and study personnel who were in contact with the subjects. Each subject was instructed to consume 3 bars per day for the 9-week training period. This was in addition to the subjects' self-selected diet. Subjects were instructed not to change eating patterns during the course of the study. The time of the day when the bars were consumed was recorded daily in the subject's fitness log so that compliance could be monitored.Subjects were randomly assigned in a double-blind manner to either a soy, whey, or control group. The controls did the exercise program but did not consume a protein product (n = 9/each group). The soy protein product was DrSoyLean body mass was analyzed by hydrostatic weighing. Each subject performed at least 3 efforts and an average reading was taken. Blood was drawn into heparin tubes before and after the 9 week treatment period on a day when the subjects did not exercise. Blood was spun at 3000 × g and the plasma was stored at -70°C until analysis. Unfortunately, a problem during blood processing made some plasma samples unavailable for analysis. Plasma was analyzed for free radical scavenging capacity using the Total Antioxidant Status Assay Kit from Calbiochem-Novachem Corp. . Plasma myeloperoxidase was analyzed using an ELISA kit from Calbiochem-Novachem.Statistical analysis was done by the Jump 3.1 program , with significance at p < 0.05. For each parameter and treatment group, values prior to the 9 week treatment were compared to values after treatment by paired, 2-tailed Student's t-test. In addition, for lean body mass, the changes in values for soy treatment were compared to the change in values for the other two groups by Tukey test.Baseline subject characteristics are given in Table In this study, soy and whey were both effective at increasing lean body mass with exercise training, but the soy had the added advantage of inhibiting two negative effects of training on antioxidant status. The percent change in the radical scavenging capacity  seen with training alone and training plus whey was substantial compared to the differences typically seen for these types of measurements-13.The lean body mass data seen here contradicts the common, but unconfirmed notion that soy is inferior to whey for promoting lean body mass gain. It should be noted, however, that the general trend for this study may or may not be duplicated for other study designs. For example, the time frame used here, 9 weeks, is not overly long for seeing lean body mass gain, which may explain why the training alone did not produce an effect on lean body mass gain. Thus, the effects of soy or whey on lean body mass gain versus training alone may be more pronounced than in longer studies. It should also be noted that the training program used here emphasized low exercise repetitions in subjects not used to this type of training. In addition, this study included only subjects that were still relatively early in their training experience, and placed no restriction on Calorie intake. These design considerations were geared toward gaining bulk and power. The effects of whey or soy on lean body mass might be different in a design that emphasizes higher repetitions or Calorie restriction in other types of subjects. In addition, it can be noted that the current study diet intervention used bars which included added micronutrients. Thus, this study did not determine if the effects of the soy or whey protein required co-administration of micronutrients.It is not known whether the negative effects of training seen here for antioxidant status in the whey plus training alone groups would continue upon longer training. The current state of knowledge concerning exercise training effects on antioxidant defenses does not present a clear pattern i.e. ,5,8,9], ,8,9, 5,8Another unresolved issue is whether the effects on lean body mass seen here for the two proteins were due to increased total protein intake or other factors. In regard to the former, the data regarding the amount and type of protein intake necessary to produce optimal strength training gains is conflicting. While a diet meeting the current RDA for protein intake (0.8 g/kg body mass) may be sufficient for the sedentary individual, recent studies suggest dietary protein exceeding that of the RDA is needed for muscle hypertrophy ,15. One If soy and whey promotion of lean body mass gain was not due to increased total protein intake, which remains uncertain, then other factors were responsible. In the case of soy protein, there are associated antioxidants . As presIn summary, soy and whey protein bars both supported lean body mass gain in conjunction with a short term power-based weight training program, but only the soy bar prevented a training-induced drop in antioxidant capacities.Author AB owns the company that produces the soy bars used in the study.ECB planned and carried out specifics of the intervention. RAD conceived the general aims of the study and chose the blood measurements. AB invented the protein bars and planned specifics of the nutrition intervention. STD planned the general aspects of the exercise intervention.


Mre11 (which causes an ataxia-telangiectasia-like disease [ATLD]) and mutations in the ataxia-telangiectasia-mutated (ATM) gene lead to defects in handling damaged DNA and to similar clinical and cellular phenotypes. Using Xenopus egg extracts, we have designed a simple assay to define the biochemistry of Mre11. MRN is required for efficient activation of the DNA damage response induced by DSBs. We isolated a high molecular weight DNA damage signaling complex that includes MRN, damaged DNA molecules, and activated ATM. Complex formation is partially dependent upon Zn2+ and requires an intact Mre11 C-terminal domain that is deleted in some ATLD patients. The ATLD truncation can still perform the role of Mre11 during replication. Our work demonstrates the role of Mre11 in assembling DNA damage signaling centers that are reminiscent of irradiation-induced foci. It also provides a molecular explanation for the similarities between ataxia-telangiectasia (A-T) and ATLD.Mre11/Rad50/Nbs1 complex (MRN) is essential to suppress the generation of double-strand breaks (DSBs) during DNA replication. MRN also plays a role in the response to DSBs created by DNA damage. Hypomorphic mutations in  A defective cellular response to double-strand breaks in DNA is associated with several cancer-predisposition diseases. Specific defects affect the formation of a large protein complex that involves several key proteins Cellular response to DNA damage requires the coordinated activation of cell cycle checkpoints with DNA repair . FailurePhosphorylation of Nbs1 by ATM is critical for S-phase checkpoint . Nbs1 foATM function is defective in patients carrying the recessive genetic disorder ataxia-telangiectasia (A-T). A-T is characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability, and cancer predisposition .Mre11 and Nbs1 give rise, respectively, to an A-T-like disease (ATLD) and Nijmegen breakage syndrome (NBS) (Hypomorphic mutations in me (NBS) . The clime (NBS) . All thrme (NBS) . These sMre11 binds DNA and is both a 3′-to-5′ exonuclease and an endonuclease that cleaves hairpin DNA structures . Rad50 bRad50 dimer binds to two Mre11 molecules to form a stable tetrameric complex with enhanced nuclease activities . hRad50/Mre11 or Nbs1 form IRIF inefficiently. In ATLD cells, which carry a defective Mre11, ATM activation is inhibited. Furthermore, ATM fails to localize to sites of DSBs in cells lacking functional MRN . IRIF are believed to originate by chromatin modification, such as H2AX phosphorylation, at the site of the DSB, followed by the recruitment of signaling and repair factors. MRN localizes to DSBs, independently of H2AX phosphorylation, and is critical for the formation of IRIF and the consequent response to DNA damage . Thus, conal MRN . Taken tXenopus eggs recapitulate signaling pathways triggered by DNA damage and have been instrumental in unraveling the functions of ATM and Mre11  as a reporter substrate to monitor the response to DSBs. This peptide contains two putative SQ phosphorylation sites for ATM or ATR: serines 134 and 139. To test the specificity of the kinase(s) activated by DSBs, we synthesized four peptides: wild-type and alanine substitutions at serine 134 (S134A), serine 139 (S139A), and serines 134 and 139 (S134A/S139A).Addition of fragmented DNA to  pathway . We prev pathway . We now  pathway . We usedIncubation of interphase extracts for 30 min with fragmented DNA dramatically enhanced phosphorylation of H2AX peptide A. PhosphWe next monitored phosphorylation of H2AX peptide in extracts in which specific DNA damage response signaling pathways were inhibited. X-ATM- and X-ATR-neutralizing antibodies were used to abrogate ATM- and ATR-dependent signaling, respectively. We previously demonstrated that these antibodies completely inhibit ATM- and ATR-dependent checkpoints in extracts , 2003. HNbs1 or Mre11 , H2AX peptide phosphorylation became partly independent of ATM and Mre11. This phosphorylation was sensitive to vanillin, a specific inhibitor of DNA-PK , and to In contrast to its inactivity in the DSB checkpoint reaction, MRN-ATLD1/2 can fulfill the essential function of MRN in preventing the accumulation of breaks during DNA replication. These results establish that MRN is required to activate the DSB signal pathway and that the C-terminal region of Mre11 plays a critical role in this activation.32P-labeled, 1 kb linear double-strand DNA molecules and applied to a BioGel A15m column. This large-pore gel filtration resin includes most proteins and small DNA fragments, but excludes protein–DNA complexes larger than 1.5 × 107 kDa . When radio-labeled DNA at the concentration of 50 ng/μl  was applied to the column in the absence of extract . Fraction 10 from the excluded volume and fraction 25 from the included volume were precipitated with PEG, and biotinylated DNA molecules were affinity-purified using streptavidin-magnetic beads. The results of this assay show clearly that 32P-DNA was associated with biotinylated DNA in fraction 10, but not in fraction 25 . ATR activation might be triggered by processing of DSBs into single-strand DNA (ssDNA) . We prevates ATR . Since Mates ATR . AlternaWe propose that MRN interacts with linear DNA to form DNA–protein complexes that induce the phosphorylation cascade responsible for the G1–S checkpoint. MRN assembles with linear DNA molecules in vitro . We haveWe believe that the formation of these complexes is a critical step in the kinase cascade that leads to the G1–S checkpoint. Several lines of evidence support this idea: (1) Mre11-depleted extracts do not form complexes and fail to activate ATM in response to DSBs. (2) Mre11 is concentrated 18-fold in the DNA–protein complexes and is heavily phosphorylated. We previously established that phosphorylation of Mre11 correlates with increased nuclease activity . (3) ATMATM, and possibly ATR, participates in the assembly of the complexes. Pretreatment of extracts with caffeine, an inhibitor of ATM and ATR, significantly reduces the yield of complex.Some H2AX kinase activity is not associated with the DNA–protein complex. This activity is principally accounted for by DNA-PK.Both MRN components Mre11 and Nbs1 are phosphorylated in response to DSBs. Nbs1 phosphorylation is ATM-dependent . Once re10 breaks/μl), the ATM-dependent checkpoint does not require Mre11 function  intact Mre11 protein and, presumably, binding of Mre11 to DNA, and (2) that IRIF form independently of , but areAs shown in 2.CSF-arrested extracts were freshly prepared according to To prepare DNA fragments containing DSBs, we used pBR322 plasmid digested with restriction endonucleases to yield different types of ends . These DNA fragments behaved equivalently in our assay (data not shown). For the experiments shown in 32P-labeled fragment was obtained by addition of α-32P-dATP (10 mCi/μl) to the PCR. The biotinylated 1 kb fragment was obtained by PCR on M13 ssDNA template using a 22 nt primer complementary to position 5,570 and a 22 nt primer complementary to position 6,584, biotinylated on three thymidine residues .The 1 kb DNA fragment used for size fractionation experiments was obtained by PCR on M13 ssDNA template using 22 nt primers complementary to positions 5,570 and 6,584 . The 32P2, 1 mM DTT, 1 mM NaF, 1 mM Na3VO4, and 10 mM MnCl2) supplemented with 0.5 mg/ml histone H2AX peptide (Sigma-Genosys), 50 μM ATP, and 1 μl of γ-32P-ATP, 10 mCi/μl . Samples were incubated at 30°C for 20 min, and reactions were stopped by 20 μl of 50% acetic acid and spotted on p81 phosphocellulose filter paper . Filters were air-dried and washed three times in 10% acetic acid. Radioactivity was quantified in a scintillation counter.Interphase egg extracts were incubated with DNA fragments, DNA fragments and ATM-neutralizing Ab, ATR-neutralizing antibodies or 5 mM caffeine for 30 min at 22°C. Extract (2 μl) was mixed with 20 μl of EB kinase buffer  previously equilibrated with buffer A at 4°C. Extracts were mock-depleted, Mre11-depleted, or Mre11-depleted supplemented with 500 nM MRN or with 500 nM MRN-ATLD1/2. Control extracts were treated with 500 nM MRN and 100 μM TPEN or 5 mM caffeine or 1 mg/ml proteinase K at 37°C. After the samples were loaded, 15 ml of buffer A  were gently applied to the column. We collected 31 fractions of approximately 300 μl, and radioactivity was measured in a scintillation counter. For the elution profile of the circular plasmid in control extracts, a 1.8 kb plasmid derived form pUC19 with the SspI–SapI region deleted  were incubated with or without 50 ng/μl of  deleted  was used32P-labeled 1 kb DNA fragments. Samples were applied to the BioGel A15m sizing column and fractions were collected. Void volume peak fraction 10 and included volume peak fraction 25 were incubated with 50 μl of specific polyclonal antibodies against Mre11 prebound to protein A–Sepharose beads or beads alone overnight at 4°C . Beads were washed with buffer A, and radioactivity was counted in a scintillation counter.We incubated 200 μl of control, Mre11-depleted, or Mre11-depleted extract supplemented with Mre11 precipitated from the extract with 50 ng/μl of 32P-labeled 1 kb DNA fragments and 50 ng/μl biotinylated 1 kb fragments for 2 h at 22°C. Samples were applied to the BioGel A15m sizing column, and fractions were collected. Void volume peak fraction 10 and included volume peak fraction 25 were incubated with kilobase-BINDER dynabeads  or mock protein A dynabeads , and DNA fragments were isolated according to the kit protocol. Biotinylated DNA fragments bound to beads were washed with buffer A, and radioactivity was counted in a scintillation counter.We incubated 200 μl of control, Mre11-depleted, or Mre11-depleted extract supplemented with 500 nM MRN with 50 ng/μl Human MRN and MRN-ATLD1/2 were purified from baculovirus-infected cells according to published protocols . The recFor X-Mre11 complex depletion, 50 μl of interphase extract was incubated with 25 μl of protein A–Sepharose beads coupled with 50 μl of preimmune serum or with 50 μl of X-Mre11 antiserum for 60 min at 4°C. For Ku70/80 depletion, 50 μl of interphase extract was incubated with 25 μl of protein A–Sepharose beads coupled to 50 μl of Ku antiserum  for 60 min at 4°C.TUNEL assay was performed according to Xenopus ATM, Xenopus ATR, Xenopus Mre11, phosphoserine 1,981 of human ATM , and phosphorylated ATM/ATR SQ substrates .We incubated 2 μl samples of interphase egg extracts for 30 min at 22°C with 50 ng/μl DNA fragments, DNA fragments and 5 mM caffeine, or with 50 ng/μl circular plasmid, and 2 μl samples were recovered from the BioGel A15m column and precipitated with 9% PEG see A were di


A number of studies have suggested that stress-echocardiography may be used for prognostic stratification in patients with idiopathic dilated cardiomyopathy. There is no consensus on which protocol or which measurements of left ventricular contractile reserve to use. The most frequently used protocol is low-dose dobutamine stress-echocardiography, and most commonly used measures of left ventricular systolic performance are ejection fraction, wall motion score index and cardiac power output.Stress-echocardiography has been shown to predict improvement in cardiac function in patients with recently diagnosed dilated cardiomyopathy, as well as to predict which patients will benefit from the treatment with beta-blockers. Most importantly, stress-echocardiography can identify patients with worse prognosis in terms of cardiac death and need for transplantation. Additionally, contractile reserve is closely correlated with maximal oxygen consumption and can even be used for further stratification in patients with maximal oxygen consumption between 10 and 14 ml/kg/min.Future studies are needed for head-to-head comparison of various protocols in an attempt to make standardization in the assessment of patients with dilated cardiomyopathy. Epidemiologic data from United States indicate that idiopathic dilated cardiomyopathy (DCM) is diagnosed in approximately 36/100.000 persons each year, and that it is responsible for more than 10.000 deaths per year , accuratThere is a widespread belief that maximal oxygen consumption, assessed by cardiopulmonary testing, is one of most, if not the most important prognostic variables in DCM patients . MaximalHowever, it appears that other factors have considerable influence on maximal oxygen consumption, since it has been shown that regular physical exercise can augment oxygen consumption with little or no impact on other parameters of cardiovascular function . AdditioA number of studies have shown that assessment of ventricular contractile reserve by means of stress-echocardiography may refine prognosis in patients with left ventricular systolic dysfunction -12. NeveThe aim of this review is to put stress-echocardiography in DCM patients in clinical context, to give practical tips how to perform it and what to measure, as well as to try to define its role in everyday clinical practice.Unlike protocols for stress-echocardiography for coronary artery disease, there is no consensus about the protocol to be used in patients with left ventricular systolic dysfunction.The majority of authors have used either low- or high-dose dobutamine echocardiography ,14. Low-Exercise testing has been used for the assessment of left ventricular contractile reserve but in conjuction with radionuclide angiography  and hemoDipyridamole stress-echocardiography has been proposed for stratification of patients with DCM. Standard high-dose dipyridamole protocol (0.84 mg over 10 minutes) has been used for the assessment of contractile reserve .All studies on stress-echocardiography in DCM measured contractile reserve of the left ventricle. Contractile reserve is defined as the difference between values of an index of left ventricular contractility during peak stress and its baseline values. There is no consensus on what index to use.This is the most frequently used index of left ventricular performance. However, it may not accurately reflect left ventricular contractility since it is heavily dependent on loading conditions  which isDespite all these potential drawbacks, the change in ejection fraction during stress has been shown to have crucial prognostic significance in patients with DCM. It is generally accepted that increase in ejection fraction by ≥ 5% or change from baseline ejection fraction by ≥ 20% during stress-echocardiography identifies patients with preserved left ventricular contractile reserve and better prognosis. Ejection fraction should be assessed by Simpson biplane formula.Wall motion score index has been traditionally used in stress-echocardiography for the detection of coronary artery disease . Only twThis index is not sensitive to changes in afterload, and after optimization for preload accurately reflects contractile properties of the myocardium . NoninvaCardiac power output =  × 2.22 × 10 -3where cardiac output is calculated by multiplying aortic velocity-time integral by aortic valve area, and mean arterial pressure is calculated in a standard manner.The major problem with this index is that its calculation is time consuming, requires skilled echocardiographer, and is subjected to numerous sources of error. Furthermore, very few cardiologist are familiar with this index which precludes its wider use. Suggested cut-off point between patients with respect to prognosis is dobutamine induced change in cardiac power output of ≥ 1 W.There is no doubt that change in left ventricular contractility during stress has considerable prognostic significance and may have profound effect on therapeutic strategy. We will review available data according to the means how contractile response was elicited.Most authors prefer low-dose dobutamine stress-echocardiography, probably because it is considered safe and is not time consuming. A report by Paelinck and coworkers has suggested that low-dose dobutamine can identify patients with atrial fibrillation induced dilated cardiomyopathy who will improve following restoration of sinus rhythm . These aIt has been shown, in a small number of patients, that changes in left ventricular wall motion score index and ejection fraction during low-dose dobutamine echocardiography are predictive of improvement of left ventricular systolic performance during medium term follow-up Figure  15]. Si. Si15]. These findings are further extended, so it has been shown that the presence of myocardial contractile reserve identifies patients who will respond favorably to beta-blocker therapy . FurtherContractile reserve has been shown to correlate well with peak oxygen consumption Figure  16]. At. At16]. Use of high-dose dobutamine is not associated with serious complications in DCM patients, and has an overall feasibility of 88.7%. The most common adverse event requiring discontinuation of dobutamine infusion is occurrence of complex ventricular arrhythmias, which was noted in 8% of patients  . AlthougVery intriguing finding is that early in the course of DCM, dobutamine induced change in left ventricular contractile response and geometry is able to predict late spontaneous recovery of left ventricular systolic performance . It is iThe largest study that studied prognostic significance of high-dose dobutamine included 186 DCM patients. The major findings of this study are that dobutamine induced change in wall motion score index is able to identify patients at greater risk for cardiac death during the follow-up Figure , and thaRecent data by our group demonstrate that contractile reserve indices assessed by high-dose dobutamine correlate with myocardial histomorphometric features, suggesting that contractile reserve is strongly related to the degree of hystological disruption in DCM patients. Myocyte diameter and interstitial fibrosis showed strongest correlation with change in wall motion score index , followed by change in ejection fraction  .It appears that dipyridamole may be used instead of dobutamine to evoke contractile response, since it is less arrythmogenic  better tAs previously said, there are no echocardiographic studies on exercise induced contractile response in DCM patients. However, Nagaoka and colleagues used radionuclide ventriculography to measure increase in ejection fraction during exercise in DCM patients with mild symptoms, and concluded that change in ejection fraction <4% identifies patients with worse prognosis . AdditioRight ventricular contribution to global cardiac performance is minor in subjects with normal or mildly depressed left ventricular systolic function, but may become more important in patients with advanced left heart failure . PreviouHowever, there are only limited reports of the prognostic value of right ventricular contractile reserve in patients with DCM. DiSalvo and colleagues have demonstrated that an increase in RVEF to >35% during exercise is the only independent predictor of event-free survival in patients with advanced heart failure . It has Data from our laboratory support prognostic significance of high-dose dobutamine induced change in right ventricular fractional area change . It appeDespite the wealth evidence that favor use of stress-echocardiography in patients with DCM, there is no clear-cut algorithm about its use in risk stratification and therapeutic strategy. The reasons for this are not clear, but probably reflect the lack of standardized protocol and measurements of left ventricular contractile reserve.We strongly believe that stress-echocardiography should be used as a standard procedure, at least in centers which do not have access to cardiopulmonary testing, since data obtained when patients are subjected to some form of stress have far greater prognostic significance than data obtained at rest. Furthermore, stress-echocardiography should be used in patients who are not able to exercise or fail to achieve expected work load. Stress-echocardiography may also play an important role for detailed risk stratification in patients with maximal oxygen consumption of 10–14 ml/kg/min. The choice of stress-protocol, at least for the time being, should be based upon local expertise and preferences of attending physician.There is an obvious lack of studies that will contribute to standardization of stress-echocardiographic protocol. Head-to-head comparison of stressors, including low- and high-dose dobutamine, dipyridamole, and exercise, has to be performed in order to rank their ability to predict prognosis. Similar comparisons have to be made for various indices of left ventricular contractility. Additionally, it is not clear should the patients be tested with or without beta-blocker therapy, and how this therapy may affect our choice of stressor. Last but not the least, novel echocardiographic techniques that can easily assess regional and global contractility, like tissue Doppler imaging and strain-rate imaging, have not yet been tested in a prospective manner.In conclusion, stress-echocardiography can be a valuable tool for the assessment of patients with DCM, but a lot of work has to be done before it becomes a part of a routine work-up.


The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes.in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1.Here we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation These studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease. The generation and repair of blood vessels in adult life requires the regulation of endothelial cell survival, migration, proliferation and their differentiation from lineage-committed progenitors by the coordinated action of several classes of vaso-active agents including growth factors, cytokines, and the extracellular matrix (ECM) -4. Eluci5β1 through a mechanism involving the promotion of an integrin α5β1-VEGFR-2 signalling moiety generated as a consequence of receptor ligation by a VEGF-FN complex. These events promoted the sustained activity of Erk kinase, which was coupled to the migratory response. More recently, we presented data demonstrating that FN significantly enhanced VEGF-mediated migration of CD34+ cells and their differentiation into endothelial cells [in vitro studies have highlighted the importance of hepatocyte growth factor (HGF) as a pro-angiogenic mediator. HGF, also termed scatter factor, has a well-established role in tumourogenesis but may be an important mediator of neovascularization since studies show that HGF induces the expression of VEGF in endothelial cells in vitro and that HGF synergises with VEGF to promote capillary-tube assembly in collagen matrices [There is growing evidence pointing to a close collaboration between growth factors and the ECM in several biological processes including vasculogenesis and post-natal revascularization. Studies have shown that the response of cells to growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are potentiated by integrin ligation to specific ECM glycoproteins -8. In a al cells . In addimatrices ,12. In amatrices . The emematrices . Althougin vivo. Significantly, we show that HGF-FN and HGF-VN molecular complexes induce a unique and enhanced intracellular signal employing Ras, thereby highlighting an important mechanism of growth factor receptor tyrosine kinase and integrin cooperation in promoting pro-angiogenic responses.In the present work we show that HGF forms a specific physical complex with FN and VN and that these complexes are present in degranulated platelet suspensions implicating a putative role 125I-labelled HGF to a variety of ECM molecules immobilized on plastic wells. As shown in Fig. 125I-labelled HGF bound to both FN and VN specifically with residual binding observed to either collagen-1 or laminin. Further experiments were performed to locate the HGF binding site on the FN molecule using purified FN proteolytic fragments immobilised onto the polystyrene microtiter wells. In these experiments 125I-labelled HGF bound to the 70 kDa N-terminal fragment and the 40 kDa C-terminal fragment. No significant binding was observed to the 120 kDa fragment that harbours the internal cell binding domain .We recently identified binding domains on FN for VEGF, which played an important role in promoting the activity of VEGF . Since Hain Fig. . To furtin vivo we examined platelets, a rich source of growth factors, for the presence of these complexes. Washed human platelet suspensions were stimulated with thrombin (1 U/ml) to promote degranulation and the derived supernatants were immunoprecipitated with antibodies directed to FN or VN. The resulting immune complexes were analysed for co-precipitation of HGF  were incubated with an optimal concentration of HGF alone or in combination with fixed concentrations of FN, VN or collagen-1 Fig. . Signifi5β1 completely inhibited HGF-FN-induced endothelial migration  did inhibit endothelial cell migration to HGF-FN by 20% suggesting an ancillary role for this integrin in mediating HGF-FN responses. When endothelial cell migration was induced by HGF-VN complexes, the integrin dependence shifted as expected . This latter effect may be a consequence of integrin signal cross-talk (transdominant integrin regulation), as reported previously [To further characterize the degree and identity of integrin involvement in the observed migratory response, we investigated the consequences of blocking integrin receptors on HMVEC with specific integrin antibodies prior to HGF-ECM stimulation. Antibodies directed to the integrin αion Fig. . In contted Fig. . Under teviously ,15. Thes2β1, α5β1 and αvβ3 respectively, predominantly co-precipitated Met with the integrins α5β1 and αvβ3. In contrast, Met co-precipitation with the integrin α2β1 was minimal for lysates derived from cells stimulated with HGF and collagen-1. The level of Met expression in these samples was not altered by treatment of the cells with various combinations of HGF and ECM molecules  that were coupled to the migratory response, HMVECs were treated with specific inhibitors of MEK and PI-kinase. In cell migration assays, LY294002 but not U1026 inhibited endothelial cell migration induced by HGF-FN Fig.  and HGF-The effect of the co-administration of ECM molecules with HGF on endothelial cell proliferation was also investigated. In contrast to cell migration, HGF, in the absence of ECM molecules, induced a significant proliferative response Fig. . HoweverThe data shown above indicate that HGF-induced endothelial cell migration and proliferation were mediated by PI-3 kinase and Erk pathway respectively. We next investigated the role of Ras in regulating these two pathways induced by HGF-FN and HGF-VN complexes. Since Ras is a well-documented regulator of p85 PI-3 kinase and Erk and as well as a down stream effector of both the Met and integrin receptors, we assessed the activation status of Ras by measuring the comparative levels of GTP-loaded Ras after endothelial cells were stimulated with HGF in the presence and absence of ECM molecules Fig.  &6B. EndTo further characterise the role of Ras in regulating endothelial cell responses to HGF/ECM, the effect of the FPT-III inhibitor on the phosphorylation levels of Erk 1/2 and Akt was investigated. In cells stimulated with HGF alone, Erk 1/2 was activated and significantly inhibited by the FPT-III inhibitor and to a lesser extent by GGTI Fig. . A similThe major finding of the present report is that HGF-induced endothelial cell responses are significantly augmented through the formation of molecular complexes between this growth factor and the ECM glycoproteins FN and VN. The significance of this finding is highlighted by the observation that HGF-induced endothelial cell migration, a PI-3 kinase coupled response, did not occur in the absence of additional signals originating from the ECM. However, HGF-induced endothelial cell proliferation was evident in the absence of signals emanating from the ECM. These observations have led us to propose a model for the mechanisms of HGF-induced responses in endothelial cells Fig. . This mo5β1 derived from endothelial cell lysates stimulated with HGF-FN complexes but not with the integrin α2β1 derived from cells stimulated with HGF and collagen-1  in binding buffer (PBS containing 2% BSA) were added to the microtitre plates and incubated for 30 min at room temperature (RT) before washing and counting to determine bound radioactivity. SPR analysis was performed on the BIAcore X (Biacore Herts UK) as described previously [2, 2 mM CaCl2 and the sensograms recorded. The data was analysed by the ASSAY programme  in order to determine the EC50 value and Kd.ECM proteins and FN peptides were purchased from Sigma and Gibco and were further purified by gel filtration and ion exchange chromatography. The assay was performed as described previously . 125I-HGeviously . HGF  and then blocked by incubation in 3.5 mg/ml BSA in basal culture medium) for 1 hour at room temperature followed by extensive washes with basal culture medium. The remaining cells were measured using a fluorescence plate reader (measuring fluorescence in the upper transwell compartment). For proliferation experiments, cell division was measure by fluorescence labeling of DNA . HMVEC was plated on poly-D-lysine coated 48-well plates and cultured overnight in MCDB-131 medium containing 5% FBS. After washing plates with PBS, endothelial cells were then cultured in MCDB-131 medium + 0.1% FBS containing HGF (10 ng/ml) in the presence or absence of VN, FN or Collagen-1 (10 μg/ml). Cells incubated for 48 h and HGF/ECM was added every 24 hours. Cell proliferation was quantified using a fluorescence plate reader.Human dermal microvessel endothelial cells (HMVEC) were maintained in EBM-2 growth medium (Clonetics Corp). Migration studies were carried out essentially as described previously  using se202/Tyr204) and Akt (Ser473) respectively by Western blotting. These studies were performed with both cells in suspension and with adherent populations. Cells were grown to 80% confluence and serum starved for 2 hours prior to harvesting. Cells were resuspended in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA (resuspension buffer) at a concentration of 1–5 × 106 cells /ml. The cell suspensions were challenged 10 ng/ml HGF supplemented with 2 μg/ml collagen-1, or FN or VN for various durations ranging from 2 to 120 min at room temperature. Cells were harvested by centrifugation at 4°C and lysed in 10 mM Tris pH 7.4, 145 mM NaCl supplemented with 0.1% Triton X-100 and protease inhibitors. For inhibitor studies, serum-starved HMVEC suspensions were pre-treated with the inhibitor for 45 min prior to stimulation with HGF and ECM molecules for a further 60 min at room temperature. The cells were pelleted, washed in ice-cold resuspension buffer without BSA and lysed in a lysis buffer containing 1% (v/v) Triton X-100. Cell lysates were analysed by Western blotting using protocols specific for the phosphospecific antibodies according to the manufacturer's recommendations. Blots were cut along appropriate marker divides and probed with antibodies to phopho Erk 1/2 and Akt (Ser473)  simultaneously. For GTP-Ras pull down assays, serum-starved HMVEC were stimulated with HGF and ECM molecules for a desired time point and the cells were spun down and washed in ice-cold resuspension buffer without BSA. Cell pellets were lysed in MLB buffer  and 500 μg of cell lysate was mixed with a 10 μl suspension of RBD-Sepharose (Upstate Biotechnology) for each reaction at 4°C for 60 min. Sepharose beads were spun down and washed in MLB prior to solubilization and analysis by Western blotting probing for Ras using a monoclonal antibody (Upstate Biotechnology). For Ras inhibition studies cells were pre-incubated with FPT-III (100 μM) and GGTI (2 μM)  for 45 min at room temperature prior to cell stimulation for 60 min with HGF and ECM.HMVEC were assessed for the activation profiles of Erk1/2 and Akt using phosphospecific antibodies  to Erk (Thr2β1 (clone JBS2), α5β1 (clone JBS5) and αvβ3 clone (LM609). After analysis by SDS-PAGE and protein transfer, the blot was then probed with a monoclonal to Met  and developed by chemiluminescence. For Met tyrosine phosphorylation analysis, cells were stimulated with HGF alone or HGF-FN and HGF-VN complexes for various time points ranging from 15 mins to 2 hours at room temperature. Lysed samples were immunoprecipitated with a polyclonal anti-phosphoMet antibody  and the immune complexes analysed by SDS-PAGE and Western blotting using a monoclonal ant-Met antibody (Upstate). Met was visualised using chemiluminescence technology (Pierce).Human microvessel endothelial cells (HMVEC) in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA were plated on collagen, FN and VN coated petri dishes in the absence or presence of HGF (50 ng/ml) for 15 min to 1 hour at room temperature. Cells were then harvested as described previously  and inteSupernatants from non-stimulated and thrombin-stimulated washed platelet suspensions were prepared as previously described . SupernaHGF, hepatocyte growth factor, FN fibronectin, VN vitronectin, Col-1, collagen-1, HMVEC, human microvessel endothelial cells.SR, study design, HMVEC migration studies, SPR ligand binding analysis, HMVEC signalling studies, co-immunoprecipitation studies (integrin-Ras), manuscript preparation. ESW, study design, HMVEC proliferation studies, solid-phase ligand binding studies, co-imunoprecipitation (integrin-Met), manuscript preparation. YMP, study design, co-imunoprecipitation studies (HGF-FN/VN). JM, protein purification and preparation, KVP, GTP-Ras pull-down assays, RS, technical support. MS, study design.