The success of coronary artery bypass grafting (CABG) is limited by poor long-term graft patency. Saphenous vein is used in the vast majority of CABG operations, although 15% are occluded at one year with as many as 50% occluded at 10 years due to progressive graft atherosclerosis. Intravascular ultrasound (IVUS) has greatly increased our understanding of this process. IVUS studies have shown that early wall thickening and adaptive remodeling of vein grafts occurs within the first few weeks post implantation, with these changes stabilising in angiographically normal vein grafts after six months. Early changes predispose to later atherosclerosis with occlusive plaque detectable in vein grafts within the first year. Both expansive and constrictive remodelling is present in diseased vein grafts, where the latter contributes significantly to occlusive disease. These findings correlate closely with experimental and clinicopathological studies and help define the windows for prevention, intervention or plaque stabilisation strategies. IVUS is also the natural tool for evaluating the effectiveness of pharmacological and other treatments that may prevent or slow the progression of vein graft disease in clinical trials. The success of coronary artery bypass grafting (CABG), although the gold standard for the treatment of multivessel coronary artery disease, is limited by poor long-term vein graft patency . Early vAlthough the cellular and molecular mechanisms underlying vein graft disease have been systematically investigated, the time course and development of this process in patients after coronary bypass has only recently been defined as a consequence of the increasing use of intravascular ultrasound (IVUS). Quantitative coronary angiography, traditionally the predominant imaging modality used to assess the severity of vein graft disease underestimates the severity of vein graft remodeling and athermanous plaque development by measuring the vessel lumen in only two dimensions ,14. In cGlagovian remodeling was first described as a radial enlargement of the entire cross sectional area of a vessel in response to intramural atheroma . First iIVUS changes correlate closely with the findings of experimental and clinical studies. In porcine saphenous vein bypass grafts, in the first week after grafting, adventitial medial and neointimal thickening occurs as a consequence of increased shear stress, surgical preparative injury and the subsequent activation of multiple growth factor and cytokine cascades. This is associated with the infiltration of inflammatory cells, medial smooth muscle cell proliferation and migration to form a neointima . AdventiThe early changes seen in the vessel wall of vein grafts are similar to those seen during vessel remodeling in atherosclerotic coronary artery segments . In normEarly vein graft changes can be viewed as adaptive, however they also predispose the graft to later accelerated graft atherosclerosis . SeveralRisk factors for, and the microscopic appearance of vein graft atherosclerosis are largely similar to those in coronary arteries and it is reasonable to suggest that similar pathological mechanisms are at work, however these occur over a much more rapid time course in vein grafts ,4. AtherIVUS studies have clearly shown that early 'adaptive' or pathological changes occur within weeks of grafting and that occlusive atheroma, in susceptible individuals occurs within 1 year. IVUS studies have therefore defined the window in which strategies to inhibit vein graft disease might be effective. Furthermore, in addition to its advantages over coronary angiography, IVUS in vein grafts has been shown to be both accurate ,16 and rex vivo prior to implantation using pharmacological or other methods that may inhibit subsequent disease is a feature unique to vein graft disease. There are many examples of this being successfully achieved in experimental models. Pre-treatment with rapamycin [The ability to manipulate vein grafts apamycin , paclitaapamycin , have beapamycin  and endoapamycin  as has tapamycin . Targeteapamycin . StabiliIVUS has significantly contributed to our understanding of vein graft failure. It also serves as the natural tool for the development of clinical strategies that may lead to significant improvements in vein graft patency and more importantly for better long-term quality of life and longevity for patients with coronary artery disease. The introduction of clinical trials to address this Achille's heel of coronary bypass surgery are long overdue.None declared.G Murphy performed the literature review and prepared the manuscript. G Angelini conceived the idea for the manuscript and prepared the manuscript.


During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis.The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA.The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope). Granulopoiesis, the differentiation of peripheral blood granulocytes, involves dramatic nuclear and cytoplasmic structural changes . Committenvelope-limited chromatin sheets" or ELCS. The suspected role of LBR was confirmed in subsequent studies [Several established tissue culture cell lines have been investigated as model systems for understanding the events and mechanisms of granulopoiesis . Previou studies ,9, which studies  and the  studies . The mecbcl-2 cells [in vitro granulopoiesis. Prolonged exposure of HL-60-bcl-2 cells to CD or TX does lead to perturbations of nuclear shape, independently of RA induced nuclear differentiation.The position of the interphase nucleus within the cell appears to be regulated by microtubules and associated dynein  and acti-2 cells , we demoHL-60/S4 cells were cultivated for up to four days in medium with (or without) 1 μM RA and with (or without) 1 μM CD. Daily samples of these four cultivation conditions were cytospun on microscope slides and Wright-Giemsa stained for examination. The image data from day 3 are presented Figure . Controlbcl-2 cells, which are more refractory to undergoing rapid apoptosis.The previous publication  also exabcl-2 cells for up to three weeks following addition of 1 μM RA. For the sake of analysis, we distinguished four nuclear morphology categories: ovoid  1 μM RA for up to 7 days, cytospun onto microscope slides, methanol-fixed, air-dried and stained with Wright-Giemsa. The results were generally similar to those described above for HL-60/S4 cells. Several examples of Wright-Giemsa stained HL-60-bcl-2 cells treated with CD or with RA plus CD are shown in Figure -bcl-2 cells.HL-60-bcl-2 cells were exposed to 0, 0.1 or 1.0 μM NC, with 1 μM RA for up to 10 days. Samples were harvested for Wright-Giemsa staining at 1, 2, 3, 4, 7 and 10 days; for immunostaining at day 8. Figure bcl-2 cells at day 2 or day 4 after the addition of RA and nuclear morphology was observed on day 8. NC had a clear inhibitory effect upon RA induced nuclear lobulation, even when added 4 days after RA. Thus the inhibitory effect of NC is not an early event in granulocytic differentiation. As noted earlier, we have observed essentially 100% cell death of HL-60/S4 cells exposed to 0.1 μM NC by day 2. Viability of HL-60-bcl-2 cells was much better; but protection was not complete. By day 8 of 0.1 μM NC, viability had dropped to ~23%. This could also be observed as increased levels of cellular debris in the Wright-Giemsa stained slides. The cells shown in Figures -bcl-2 cells.HL-60myc [myc amplification [bcl-2 cells indicated that as early as day 2, addition of 0.1 or 1.0 μM TX resulted in the appearance of indented and lobulated nuclei in the absence of RA. Consequently, Wright-Giemsa staining analysis of TX treated cells (in the absence of RA) was performed for up to 10 days  of RA. Apparently the cytotoxicity of TX is potentiated in cells with elevated levels of c-myc . HL-60 cfication ,25. Prels Figure . There ws Figure . Clearlybcl-2 cells contrasts with earlier observations on RA treated "polarized" HL-60/S4 cells [bcl-2 cells, than previously observed for HL-60/S4 cells (data not shown). It is possible that the HL-60/S4 cells are more readily "activated", like normal neutrophils, than are HL-60-bcl-2 cells. They also differentiate to granulocytic form faster (~3 to 4 days), compared to HL-60-bcl-2 cells (~7 days).The position of the centrosomal region was visualized with monoclonal antibodies against γ-tubulin Figure . In all bcl-2 cells, compared to HL-60/S4 cells, allowing a window of time for determining the effects of these MT modifying chemicals on nuclear shape and nuclear differentiation.Prolonged exposure of HL-60 cells to various cytoskeleton-modifying chemicals (i.e., nocodazole and taxol) are very harmful to cell viability, inducing rapid apoptosis. The present study was conducted primarily on a Bcl-2 overexpressing subline of HL-60 cells, which is more refractory to apoptosis and exhibits the chemically induced differentiation properties of the parent cell line . Lethal bcl-2 and HL-60 cells observed in this study. The first difference: ~10% of the undifferentiated HL-60-bcl-2 cells exhibit micronuclei, compared to ~0.8% of HL-60 cells [-myc) and appear to form dynamically during S phase [bcl-2 cells exhibited immunostaining of centromeres, heterochromatin and nucleolar antigens  with multilobed nuclei, which we almost never observed with HL-60/S4 cells. There may be some clinical significance to this latter observation. A small percent of granulocyte nuclei with 5 or more nuclear lobes in human blood smears is considered diagnostic for megaloblastic anemia (vitamin B12 or folic acid deficiency) [bcl-2 cells suggests that delayed or dysfunctional apoptosis might play a role in these human diseases. It is of interest that neutrophil nuclear multilobulation (hypersegmentation) has been described in two circumstances that delay apoptosis: 1) Glucocorticoid administration to patients induces hypersegmentation [in vitro glucocorticoid treatment of neutrophils prolongs their survival [in vitro treatment of neutrophils with G-CSF prolongs their survival [The second difference: RA treated HL-60-on up to % with muentation , and in survival -39. 2) Gsurvival , and in survival ,41.bcl-2 cells by simultaneous exposure to NC  to 0.01–0.1 μM TX for up 20 hours, followed by incubation in drug-free media for up to 72 hours, led to nuclear envelope "unraveling" and clustering of nuclear pores . The autr Figure . The bunAlthough far from a complete understanding of all the molecular forces involved in shaping the granulocytic nucleus, a working model that incorporates existing data and emerging concepts provides a useful perspective for future experimentation Figure . The probcl-2 demonstrates that nuclear lobulation correlates with low levels of lamins A/C and B1 coupled with a rise in LBR levels  has been correlated with nuclear envelope folds or invaginations . We haveThe proposed model for granulocytic nuclear lobulation yields several testable predictions: 1) Expression of lamins A/C and B1 in HL-60 cells should strengthen the nuclear envelope, minimizing nuclear lobulation following exposure of the cells to RA; 2) "Knock-down" or expression of a "dominant negative" form of LBR in HL-60 cells would be expected to suppress granulocytic lobulation; 3) Overexpression of dynamitin in HL-60 cells should inhibit dynein activity , preventEmploying Bcl-2 overexpressing HL-60 cells, which are more refractory to induced apoptosis than the parent cell line, we demonstrated that disruption of the MTs by nocodazole prevented nuclear lobulation in response to RA treatment. These results implicate the necessity of an intact MT system for granulocytic nuclear shape. Cytochalasin D, on the other hand, did not suppress RA induced nuclear lobulation. Combined with the decreasing levels of intermediate filaments (vimentin) during differentiating granulocytic forms of HL-60 cells, the role of the MT system appears to be quite central to the nuclear shape change. Recent models on the role of a MT bound motor (dynein) in facilitating mitotic nuclear envelope breakdown suggest that similar tension forces on the differentiating granulocyte nucleus, combined with continued influx of nuclear envelope components, could explain nuclear invaginations and lobulation.bcl-2 [Two HL-60 cell sublines were employed in this study: HL-60/S4 , which abcl-2 , which abcl-2 ,4.all-trans retinoic acid (RA), nocodazole (NC), taxol (TX), cytochalasin D (CD). Stock solutions of these chemicals were stored at -20°C as described earlier [The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO):  earlier ,4.Guinea pig antisera were the generous gifts of two Ph. D. students in the laboratory of H. Herrmann (German Cancer Research Center, Heidelberg): anti-LBR and anti-emerin, from C. Dreger; anti-lamin A and anti-lamin B1, from J. Schumacher. Goat anti-lamin B was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit anti-4 × dimethyl H3K9 (lysine 9 on histone 3) was generously provided by T. Jenuwein (Biocenter, Vienna). Human auto-antisera anti-centromere (CREST) and anti-nucleolus (antigens unknown) were purchased from Antibodies Inc. (Davis, CA). Rabbit anti-calreticulin was from Calbiochem (San Diego, CA). Mouse monoclonal antibodies against α-tubulin, γ-tubulin and Golgi p58 were all purchased from Sigma-Aldrich. Mouse monoclonal anti-vimentin (3B4) was a gift of H. Herrmann and has been described before . FITC-, For Wright-Giemsa staining, cells were cytospun onto ethanol-cleaned microscope slides, fixed in room temperature methanol for 15 min, air-dried and stained as described earlier . For anaIn the majority of immunostaining experiments the procedure followed that described previously , with sobcl-2 cells over a three-week period (simultaneously with preparation of slides for Wright-Giemsa staining), were analyzed by immunoblotting as previously described [SDS total cell extracts, obtained from undifferentiated and RA treated HL-60-escribed . No atteRA, retinoic acid; MT, microtubules; NC, nocodazole; TX, taxol; CD, cytochalasin D; ELCS, nuclear envelope-limited chromatin sheetsALO performed the microscopy and prepared the figures. DEO performed the tissue culture, immunostaining and immunoblotting. Both authors were involved in the conception of the study and have read and approved the final manuscript.


Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides.A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3' D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition.The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences. The variable domain of the immunoglobulin heavy chain (IGH) is encoded by the IGHV (variable), the IGHD (diversity) and the IGHJ (joining) genes. In developing B cells these genes are brought together via a process of recombination involving the selection of one of each gene type from sets of genes present within the genome . The briThe addition of N nucleotides is performed by the enzyme terminal dideoxynucleotidyl transferase (TdT), and in the IGH locus this addition can occur at both the D to J and the V to D-J joins . The regP nucleotides are derived from the asymmetric opening of hairpin loops that form at gene ends as part of the rearrangement process . The opeThe mechanism of immunoglobulin gene rearrangement was first proposed by Tonegawa in the late 1970's [The very few reports of exonuclease removal in the literature mainly describe analysis of murine sequences -14. ThesR, also offers standardised nomenclature [Recent advances in data standardisation in immunogenetics has allowed for improved statistical analysis. The standardisation emanates from IMGT-ONTOLOGY ,17 upon nclature  and stannclature . Despitenclature ,22.The development of a statistically based algorithm for the partitioning of immunoglobulin sequences  as an alThe collection of human IgM sequences from public databases resulted in a dataset of approximately 1500 sequences. The exclusion of fetal, moderately and highly mutated (>5 mutations) and disease associated sequences reduced the dataset to 306 sequences. Further exclusions were made of those sequences that showed signs of IGHV gene replacement or the utilization of multiple D genes. Five sequences utilized two D gene segments, as identified using strict criteria as previously described  . Average removals from IGHV region ends were significantly lower than removals from IGHD and IGHJ region ends (p < 0.001, Dunn's Multiple Comparison Test). On average, only 1.2 nucleotides were lost from IGHV region ends while average removals of 4.7 nucleotides were evident from each of the IGHD region ends as well as from the IGHJ region ends.The average number of nucleotides removed from each gene subgroup within the three genes was calculated to identify differences in processing of sequences at the gene subgroup level Figure . It was Removals from D gene subgroups were examined at each of the region ends. The removals from the 5' D end did not reveal significant differences, but significant differences were seen between the subgroups at the 3' end (p < 0.0001, one-way ANOVA) Figure . More exComparison of average nucleotide loss for each of the six IGHJ subgroups revealed significant differences between the average removals (p < 0.0001, one-way ANOVA). The low level of removals from IGHJ3 was notable. On average just 1.3 nucleotides were removed from IGHJ3 sequences, while an average of 6.4 nucleotides were removed from IGHJ6 sequences (p < 0.002, Tukey's Multiple Comparison Test) Figure .A more detailed examination of exonuclease removals from the IGHJ genes was undertaken, to investigate the influence of W and S motifs. The presence of these motifs in the first 15 5' nucleotides of the IGHJ ends was considered. IGHJ ends containing 5' S motifs showed significantly lower average removals than those lacking a 5' S motif . The IGHJ genes whose sequences did not possess an S motif within the first 15 5' nucleotides had, on average, three more nucleotides removed Figure .Putative P nucleotides were identified among those gene ends that remained untrimmed by exonuclease activity during the process of IGH V-D-J rearrangement. Examples of nucleotides that satisfied the P nucleotide criteria were observed at 3' V, 5' D, 3' D and 5' J gene ends. The identified nucleotides ranged in length from 1 to 4 nucleotides. The observed P nucleotides fell into twenty-three sets based upon unique sequences, and the gene end at which they were observed (Table A single case of significance was observed among the putative P nucleotide sequences. This was for a sequence of 3 nucleotides (GTT) which was associated with the 3' end of the IGHD region. The occurrence of 3 'GTT' sequences in the dataset remains the only significant putative P nucleotides even if the alpha value is increased to 0.01. Using a 0.05 significance level, 6 sets out of 23 appear significant, however this conclusion carries a 69% chance of being incorrect and that the results occurred by chance.Although putative P nucleotide sequences are present in 10% of sequences most of these are likely to have arisen as the result of N nucleotide addition. P nucleotides can only be confidently attributed to less than 1% of sequences with three sequences from the IgM dataset contained statistically significant P nucleotides out of the 245 IGH V-D-J rearrangements examined. The overall contribution of P nucleotides to junctional nucleotides was 9 nucleotides out of 2899 junctional nucleotides, or 0.3% of junctional nucleotides, within the IgM dataset. The probability of 'GTT' occurring within an N region is 0.007875, therefore, the sequence could be expected to occur twice at the observed position in the 245 junctions examined, by chance alone. Of the three identified P nucleotides it is therefore possible that only one is a true P nucleotide. The contribution of P nucleotides to junctional nucleotides could therefore be as low as 0.1%, with P nucleotide inclusions occurring in less than 0.5% of sequences.H gene replacement [Investigation of the role played by nucleotide loss and addition in the generation of immunoglobulin diversity has been limited by the ability of researchers to accurately determine gene ends. The development of a statistically based partitioning method has allowed this study to gain insights into the nature of nucleotide loss and addition in the expressed human immunoglobulin repertoire. Analysis of 294 human IgM sequences revealed significant differences between average nucleotide losses from different heavy chain genes segments. IGHV genes suffer less removal in comparison to other genes, suggesting that a process or processes may act to prevent the removal of critical components or to select against sequences in which such removals have occurred. Critical components may include the conserved TGT that defines the start of the CDR3  and the lacement -25.The extension of the analysis to the gene subgroup level showed significant differences among removals from the IGHV gene subgroups, among the IGHD subgroups at the 3' end of the D gene and between the six IGHJ gene subgroups. The most striking contrast was observed for the IGHJ gene subgroups, specifically for IGHJ3 and IGHJ6. Removals from IGHJ3 averaged only a single nucleotide, while IGHJ6 on average lost in excess of 6 nucleotides. The differences observed as part of this study suggest that the loss of nucleotides during the creation of human heavy chain immunoglobulin sequences is not random.Unique 'patterns' of exonuclease removal between gene subgroups have previously been reported in murine immunoglobulins ,28, howeThe nucleotide composition of gene has previously been stated to influence exonuclease processing in the murine system . The preConsideration of the W and S motif composition of the 3' D gene segments showed the IGHD2 and IGHD3 gene subgroups to be rich in W motifs and to lack S motifs (data not shown). These two subgroups showed higher average removals compared to other IGHD subgroups. The relationship between nucleotide composition and exonuclease activity could therefore explain the significant differences observed at the 3' end of the IGHD gene subgroups.IGHD2 and IGHD3 are both long D genes. A relationship between D gene length and exonuclease activity may have therefore been acting to influence exonuclease processing. Examination of exonuclease activity of D genes grouped by length did reveal significant differences (data not shown), however, these differences were only evident at the 3' end of the D genes and as the analysis was confined to IGHD2 and IGHD3 sequences, it is difficult to conclude whether sequence length has a role.The influence of nucleotide composition on exonuclease removals from heavy chain gene is easily examined in the IGHJ genes, due to the small number of alleles and the clear division of sequences based on the presence or absence of S and W motifs. Significant differences in the V genes were not further examined as the larger number of alleles made sample groups too small to allow for meaningful statistical analysis. The absence of significant differences at the 5' end of the IGHD genes may result from the lack of distinct differences in the nucleotide composition of these sequences. This would make any variations in exonuclease processing more subtle, and thus a larger sample size would be necessary to observe any differences.P nucleotide addition has been reported to contribute to diversity in between 10% ,8,29 andIt should be noted that even those P nucleotides accepted on the basis of statistical analysis carry a degree of uncertainty. Accounting for the possible misidentification of TdT additions among P nucleotides suggests that the contribution of P nucleotides to junctional nucleotides may be even lower than 0.3%. The identification of P nucleotides during partitioning of immunoglobulin sequences introduces a greater margin of error than would result from their exclusion from partitioning processes. For example, the rare nature of P nucleotide inclusion in rearranged immunoglobulins means that in a dataset of 1000 sequences less than 10 sequences may possess P nucleotides. Arbitrary identification of putative P nucleotides in the same dataset could however lead to 100 of the sequences being identified as having P addition. The error is therefore smaller if P nucleotides are not allocated as part of the partitioning process.The statistical demonstration of the phenomenon of P nucleotides by Meier and Lewis utilized altered recombination substrates, where the IGHV, IGHD and IGHJ gene regions were replaced by restriction sites on a plasmid vector . The recin vitro evidence in support of the formation of hairpin loops as part of the immunoglobulin rearrangement mechanism [Substantial echanism ,30,31 anechanism ,33. The R, [Human IgM sequences were obtained from public nucleotide databases; IMGT/LIGM-DB available through the IMGT, The IMmunoGeneTics Information SystemR, , the EntR,  and the R, . The seqThe determination of genes and N regions of the sequences within the dataset was performed with the aid of a statistical analysis of point mutations . This meThe focus of the study upon exonuclease removal required careful definition of rules for the identification of nucleotide losses. Preliminary alignment of sequences were performed using IMGT/V-QUEST . Where iD gene determination was performed using previously described criteria , where tDifficulties with immunoglobulin partitioning are often experienced, especially in the determination of gene ends. In this study, runs of consecutive nucleotide differences between a IGH V-(D)-J rearrangement and a germline sequence at a gene end were always attributed to exonuclease removal, rather than mutation of the gene end. The portion of a IGH V-(D)-J rearrangement that contained such differences, with respect to the germline sequence, was allocated to the N region, and the gene was considered to have undergone exonuclease processing at the gene end.Situations where IGHV or IGHJ ends revealed a series of differences and similarities to the germline sequence required the consideration of each possible combination of exonuclease removal and point mutation that could have led to the creation of the observed region end. Probabilities were calculated for each 'path' to the observed segment end. Mutability scores for the region end were used to determine the likelihood of point mutations contributing to the region end. TdT addition probabilities were used to calculate the likelihood of N additions generating particular nucleotide sequences. The TdT probabilities used were p(G) = 0.35, p(C) = 0.35, p(A) = 0.15, p(T) = 0.15 [Sequences that lacked exonuclease removals from gene region end(s) were examined for the presence of P nucleotides . Self-coThe contribution of P nucleotides to immunoglobulin diversity was investigated through data generated by the investigations reported here. Unique putative P nucleotides were identified and their appearance was tallied within the dataset. The number of junctions that displayed a lack of exonuclease activity at one or more gene ends was also calculated. The cumulative binomial probability of observing a given number of P nucleotides or greater was then calculated for each putative P nucleotide sequence in an attempt to estimate the contribution of P nucleotides to immunoglobulin diversity.The effect of W and S motifs upon exonuclease activity was investigated within the IGHJ genes. The IGHJ genes were grouped based upon the relative location of such motifs. W motifs were defined as sequences of 3 or more consecutive A or T nucleotides. S motifs were defined as sequences of 2 or more consecutive G or C nucleotides. The presence of these motifs was considered within the first 15 5' nucleotides of the IGHJ genes. Three sets were established, based upon the observed configurations of the motifs in the IGHJ subgroups; S motif followed by W motif, S motif only and W motif only  with an alpha value of 0.05.An analysis of P nucleotides was performed by calculating the probability that putative P nucleotides may actually have resulted from N nucleotide addition by TdT. Probabilities were calculated as described by Meier and Lewis . The pron is the observed number of P nucleotides and N is the total number of sequences containing junctional inserts equal to or greater than the length of the P nucleotide(s) being examined, and p is the expected frequency of the observed P nucleotide sequence, which was calculated using reported TdT frequencies [where, quencies . ProbabiKJ carried out the dataset creation and partitioning, performed the statistical analysis and drafted the paper. BG aided in the design of the study and the development of the statistical analysis. WS advised on study design and development. AC devised the study, aided in study design and co-ordination and revised the manuscript. All authors read and approved the final manuscript.294 Partitioned human IgM Sequences 294 human IgM sequences were partitioned using a statistically based model and used in the examination of exonuclease activity and P nucleotide addition in the expressed human repertoire. The file is in Microsoft Excel format.Click here for file


The cause-and-effect relationship between innate immune activation and neurodegeneration has been difficult to prove in complex animal models and patients. Here we review findings from a model of direct innate immune activation via CD14 stimulation using intracerebroventricular injection of lipopolysaccharide. These data show that CD14-dependent innate immune activation in cerebrum leads to the closely linked outcomes of neuronal membrane oxidative damage and dendritic degeneration. Both forms of neuronal damage could be blocked by ibuprofen and alpha-tocopherol, but not naproxen or gamma-tocopherol, at pharmacologically relevant concentrations. This model provides a convenient method to determine effective agents and their appropriate dose ranges for protecting neurons from CD14-activated innate immunity-mediated damage, and can guide drug development for diseases, such as Alzheimer disease, that are thought to derive in part from CD14-activated innate immune response. Activated innate immunity is associated with several degenerative and destructive brain diseases including Alzheimer disease (AD), HIV-associated dementia (HAD), ischemia, head trauma, stroke, cerebral palsy, and axonal degeneration in multiple sclerosis . In thisLPS specifically activates innate immunity in peripheral organs through a well-described Toll-like receptor (TLR)-dependent signaling pathway ,3. There2, respectively [2 has relatively low receptor binding affinity but is rapidly and efficiently converted to multiple PGs or thromboxane A2, each of which are potent activators of a large family of G protein-coupled receptors [) fibrils have been shown to activate the microglial innate immune response through CD14-dependent mechanisms [in vitro and cell culture experiments raise the possibility that CD14-dependent signaling may be a common process shared in the pathogenesis of neurodegenerative diseases, especially AD.TLR-4 is the receptor for LPS in peripheral organs ,3. Howeveceptors . The comeceptors , and poleceptors . With rechanisms . Relevanchanisms  also actin vivo. It is important to stress that several laboratories have shown that glia, predominantly microglia, are activated by LPS but that neurons do not respond to LPS because they lack the appropriate receptors [4-neuroprostanes (F4-NeuroPs) [Here we present our results from studies that have identified the molecular and pharmacologic determinants of ICV LPS-initiated cerebral neuronal damage eceptors ,11. We mNeuroPs) . In addiNeuroPs) .Despite the expectation that LPS would produce a febrile response with widespread damage to brain and an acute encephalitis, we observe that ICV LPS does not yield any of these outcomes Figure  14]. In. In14]. in vitro, important limitations arise in living systems where extensive, highly active enzymatic pathways have evolved to metabolize many of the commonly measured products, such as 4-hydroxynonenal [in vivo is measuring F2-isoprostanes (F2-IsoPs) [in situ  , product-NeuroPs . Since D in vivo .4-NeuroP accumulation in cerebrum of wt mice exposed to ICV LPS and observed a delayed, transient elevation that peaks at approximately 24 hr after exposure and then returns to baseline by 72 hr post exposure [We first determined the time course of Fexposure . It is i4-NeuroPs (Table 2 receptor subtype 2 (EP2), one of four prostaglandin E2 (PGE2) receptors expressed in brain and one of the two PGE2 receptors expressed by microglia, have no neuronal oxidative damage in response to ICV LPS [in vivo [in vivo is restricted to microglia among parencymal cells in brain [in vivo is dependent on CD14, MyD88, p50 of NF-κB, iNOS, and EP2.We next used a series of mice, all on the C57Bl/6 genetic background, lacking specific genes to establish the determinants of neuronal oxidative damage in this model. Our results showed that genetic ablation of one co-receptor CD14), the required adaptor (MyD88), or one arm of the initial signal cascade (the p50 subunit of NF-κB) each completely blocks an LPS-induced increase in cerebral F4, the re ICV LPS . There a ICV LPS ,11,22-24[in vivo ,26. Seco[in vivo -32, it din brain ,26. Thusin vivo to the extent that is observed in AD brain without any neurodegeneration?These data left us with an apparent conflict. We have clearly demonstrated neuronal oxidative damage to mouse cerebrum following ICV LPS that is of a magnitude comparable to diseased regions of AD brain . HoweverTo address this question, we decided to examine directly the dendritic compartment of neurons, which is largely transparent to the standard histological techniques used so far to investigate ICV LPS-induced damage. Using Golgi impregnation and Neurolucida-assisted morphometry of hippocampal CA1 pyramidal neurons , we firsWe next pursued the molecular determinants of ICV LPS-induced dendritic degeneration using the same genetically altered mice that we used above Table . We obsein vivo neuroprotective doses of nonsteroidal anti-inflammatory drugs and anti-oxidants that are being evaluated as potenital protectants from AD. Indeed, a major criticism leveled against nonsteroidal anti-inflammatory drugs (NSAIDs) is that the concentrations that appear to be neuroprotective in epidemiologic studies are lower than those that classically considered anti-inflammatory doses. Moreover, there is some data suggesting that some NSAIDs, such as ibuprofen and naproxen, that may differ in their effectiveness as AD protectants despite being equivalent anti-inflammatory agents in peripheral assays of inflammation suggesting alternative mechanisms of action in AD [4-NeuroPs. For ibuprofen, the EC50 for suppressing ICV LPS-induced F4-NeuroPs is between 0.1 and 0.5 μg/ml and the maximal effect is reached by 1.4 μg/ml, considerably lower than the classic anti-inflammatory dose. In contrast, naproxen is without effect up to 1.4 μg/ml and thus an EC50 cannot be calculated from these data. As with F4-NeuroPs, ibuprofen completely protects both dendrite length and spine density . AT partially suppresses ICV LPS-induced F4-NeuroPs at 10 mg/kg and completely suppresses F4-NeuroP formation and both reduction in dendrite length and reduction in spine density at 100 mg/kg (Figure in vitro and lacks a specific transporter in vivo, does not, as expected, protect from neuronal oxidative damage or dendritic degeneration at the same dose.Next, we extended our studies to tocopherols, natural antioxidant products with a number of proposed actions  including Figure . GT, an Our data show that CD14-dependent activation of cerebral innate immunity leads to an acute, transient increase in oxidative damage to neuronal membranes that coincides with reversible dendritic degeneration. Although we did not directly test TLR-4 deficient mice in our studies, given what is know about LPS receptor activation and the fact that TLR-2-/- mice were not protected from neuronal damage caused by ICV LPS, these data argue strongly for CD14/TLR-4-dependent neuronal damage in our model. Moreover, using a wide array of genetically altered mice, we observed complete concordance between dendritic degeneration and neuronal membrane oxidative damage. In combination, these data suggest that these two events are mechanistically related, perhaps with neuronal membrane oxidative damage being a proximate contributor to dendritic degeneration in the context of innate immune activation.One obvious, commonly voiced criticism of the model described here is that it produces an acute stress that does not correspond to chronic neurodegenerative diseases. However, it has yet to be shown whether the stress to individual neurons in these protracted diseases truly is chronic or instead the integration of innumerable microscopic acute stresses over many years. Finally, to the extent that CD14-dependent innate immunity activation contributes to neurodegenerative diseases, such as AD and HAD, the model described here provides a convenient means to screen experimental therapeutics and rapidly optimize dosing and timing parameters before moving to more complex animal models or clinical trials.2 receptor subtype 2; F2-IsoPs: F2-isoprostanes; F4-NeuroPs: F4-neuroprostanes; GT: γ-tocopherol; HAD: HIV-associated dementia; ICV: intracerbroventricular; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; MPO: myeloperoxidase; NSAIDs: nonsteroidal anti-inflammatory drugs; PG: prostaglandin; PGE2: prostaglandin E2; TLR: Toll-like receptor; wt: wild type.AA: arachidonic acid; AD: Alzheimer disease; AT: α-tocopherol; Aβ: amyloid beta; COX-2: cyclooxygenase 2; DHA: docosohexaenoic acid; EP2: prostaglandin EThe authors declare that they have no competing interests.


Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid. Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(λDE3) reproducibly restored high level protein production.Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase. Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate. Escherichia coli using a gene expression system that allows selective overexpression of a cloned gene [Often the first step in protein purification is production of the protein of interest in ned gene . In the lacUV5 promoter [One commonly used system for achieving high levels of target protein production depends on the extremely selective nature of the bacteriophage T7 RNA polymerase for specific promoters -4. In thpromoter ,3. The tE. coli [The T7 RNA polymerase system is extremely effective, resulting in very high levels of target protein production, and the controllable promoters allow even the cloning and expression of genes encoding highly toxic proteins in E. coli ,3. HowevAlthough poor protein synthesis, or complete lack of protein production, appears to be a common problem, strategies to cope with it are varied. Some protocols suggest "pre-induction" tests to determine specific colonies that will result in overproduction . We founWe initially examined the conditions required for optimal target protein production from pET plasmids because we observed inconsistent results upon induction of plasmid-borne genes. This inconsistency was most apparent with the plasmid that encoded SecB, pJW25. Following the protocol provided [While overproduction of SecB was unreliable from frozen stocks, we found that BL21(λDE3) that had been newly transformed with pJW25 overproduced SecB to a much higher level and that every colony examined synthesized SecB to a similar extent. Continued subculturing, however, resulted in a decrease or loss of overproduction Figure . These rE. coli proteins in BL21(λDE3); the plasmid pJGGV encodes the secretory protein, proOmpA, pT7SecA produces SecA, the translocation ATPase [To verify that our observations were not unique to SecB, we examined a variety of other plasmids for overproduction of n ATPase , and pETn ATPase . Similar600 = 0.4), a sample was removed and tested for the ability to overproduce plasmid-encoded protein. The amount of SecB synthesized by pJW25 decreased gradually with each subculturing, so that by the eighth subculture SecB protein production was minimal .To more accurately determine the effect of subculturing on target protein production, protein levels were monitored as the strains were allowed to grow. Newly transformed BL21(λDE3) were grown as overnight cultures, then repeatedly subcultured as described in Methods. After each subculturing, when cultures reached early log phase  the plasmid was not stably maintained in all bacteria, even with the constant presence of selective agent, 2) the plasmid was accumulating mutations that decreased expression levels, or 3) the bacterial strain accumulated mutations that inhibited production of functional T7 RNA polymerase, resulting in decreased or no expression from the plasmid. We examined each of these possibilities.Previous protocols highly recommend that strains be examined for loss of plasmid prior to induction, and suggest that this is the major contributor to decreased expression levels ,3. HowevTo address possible plasmid loss more directly, we compared newly transformed strains and ones that had been subcultured repeatedly for their ability to form individual colonies on LB agar or LB agar containing ampicillin. As β-lactamase is a periplasmic enzyme, it is possible that sufficient leakage of the enzyme occurred to inactivate the ampicillin in liquid culture and allow growth of bacteria that no longer retained plasmid DNA despite the high dilution (1:1000) upon subculturing. If that were the case, we would expect the strain that had been subcultured many times to contain fewer plasmid-containing cells and therefore to form fewer colonies on LB+ampicillin. If no significant plasmid loss were occurring, we would expect the number of colonies to be equal whether or not ampicillin was present. We found that the pJW25-containing strain formed equivalent numbers of colonies on LB and on LB-ampicillin, even after subculturing eight times to result in a culture that did not overproduce SecB (data not shown). Thus, plasmid loss is not sufficient to account for the observed decrease in protein overproduction in these strains.The second hypothesis was that the plasmids had suffered mutations that rendered them unable to overexpress target genes, for example mutations to the T7 RNA polymerase binding site or to the Shine Dalgarno region. To observe such a great decrease in protein production, a mutated plasmid must confer a growth advantage that results in cells containing the mutated plasmid to overtake the culture. Therefore, strains that no longer overproduce significant amounts of target protein should contain mutated plasmid. To test this possibility, we isolated plasmid DNA from strains that no longer synthesized significant amounts of target protein and retransformed BL21(λDE3) with the plasmid DNAs. If plasmid mutations were occurring, we would expect a large number of the transformants to be poor expressors. On the other hand, if plasmid mutations were not responsible for decreased expression, then we would predict that the new transformants would produce high levels of protein. Indeed, this was the result we observed. Every colony examined, from every plasmid tested, synthesized target protein efficiently after retransformation. Results with pJW25 and pJGGV are shown in Figure The third hypothesis was that mutation of the bacterial chromosome might lead to decreased production of active T7 RNA polymerase, in turn decreasing expression from the plasmid. We felt this explanation was probable as a single chromosomal mutation that diminished T7 RNA polymerase expression could be sufficient to abolish protein production from all resident plasmids and this mutant could quite likely overtake the culture in a fairly short period of time, even in the presence of antibiotic to maintain the plasmid. We examined this possibility by titering a mutant T7 phage, Δ4107, that requires host production of T7 RNA polymerase for growth . T7 Δ410The loss of T7 RNA polymerase activity could be due either to mutation of the polymerase gene or excision of the λ prophage. To distinguish between these possibilities, we performed PCR using primers that amplify an 1100 base pair fragment of the T7 RNA polymerase gene. As template DNA, we used either BL21(λDE3), BL21(λDE3) freshly transformed with pJW25, or BL21(λDE3) containing pJW25 that had been subcultured eight times and no longer produced significant amounts of SecB. In all cases, a DNA product of the correct size was observed, indicating that prophage excision was not the major cause for decreased T7 RNA polymerase activity (data not shown). Therefore, our results suggest that mutation to the prophage, resulting in decreased levels of functional T7 RNA polymerase, was the predominant contributory factor for decreased production of target proteins.Optimal overproduction is an important first step towards high quantity purification of target proteins. It is critical that one be certain that the culture used will synthesize large quantities of the protein of interest, particularly if it will be used for large scale production procedures, such as growth in a fermentor. When used correctly, the T7 RNA polymerase/promoter system is an excellent choice for such overproduction. However, care must be taken to achieve optimal protein production levels.It had been suggested previously that plasmid loss is the primary cause for decreased expression from target genes in the pET system ,3. Our rSurprisingly, there was no clear growth disadvantage to strains carrying pJW25 as assessed by monitoring growth over time (data not shown). We do not think that this finding negates our proposal that chromosomal mutation occurs, however. Rather, this may explain why we see loss of expression more rapidly with some plasmids than with others; for example, synthesis of proOmpA was drastically decreased after only three subculturings. Apparently, even in the uninduced state, sufficient expression of target protein occurred, so that a detrimental effect resulted. If that target protein is sufficiently detrimental, selection for polymerase mutations will occur. For this reason, the steps that are taken to ensure optimal expression from target genes must be directed at limiting opportunities for mutation to the T7 RNA polymerase and for those mutants to overtake the bacterial culture.Two methods have been suggested previously to ensure high levels of protein production; either screening individual colonies for protein synthesis  or testiWe found that the quickest and most reliable method for obtaining optimal protein production was to freshly transform BL21(λDE3) with the desired plasmid, and use a colony directly from the transformation plate for expression studies with only a single subculture of an overnight culture. Should decreased levels of target protein production be observed, the problem can be simply remedied by isolating plasmid DNA from poor expressors and retransforming BL21(λDE3). However, we acknowledge that this approach may not be realistic for very large scale production facilities. Nevertheless, understanding the molecular basis for decreased protein production will facilitate development of techniques that will minimize conditions that may lead to selection and outgrowth of mutant strains.A common difficulty with overproduction of proteins using the T7 RNA polymerase based system is decreased target protein synthesis or even lack of detectable protein production. It is clear that synthesis of proteins, even at low basal levels, leads to the loss of induction capability. Effective strategies have been presented previously to avoid loss of expression, all of which are based on preventing basal expression levels. In this report, we demonstrate that a significant underlying mechanism leading to loss of expression is a decrease in functional T7 RNA polymerase, and selection of mutants unable to express the recombinant gene.Escherichia coli B strain BL21(λDE3) was used for overproduction of proteins from plasmids containing T7 promoters and was obtained from Stratagene. All plasmids are derivatives of pET11 (Stratagene). Plasmids encoding SecB (pJW25) [ompA gene from E. coli K12 strain MC4100 [atgaaaaagacagctatcgc) and ompA-2 (ggtcatcccgggtgatcattaagcctgcggctgagttac, underlining indicates regions of homology to the ompA gene). The PCR product was digested with restriction enzymes NdeI and BclI and cloned into pET11c that had been digested with NdeI and BamHI. Standard protocols were used for PCR, restriction digests, ligations, and transformations [SecB pJW2 [5], Sec (pJW25) , and Tus (pJW25)  were genrmations . Plasmid600 of 0.4 by addition of IPTG to a final concentration of 1 mM. Growth was allowed to continue for 2 hours after addition of IPTG. Uninduced controls were grown the same except no IPTG was added. Cells were lysed by boiling in SDS [All strains were grown in LB medium . When plg in SDS , and prog in SDS .600 of 0.4 for induction. Thus, the bacteria were subcultured only once. For continuous subculturing experiments, samples were removed before addition of IPTG and used to inoculate fresh LB plus ampicillin media at a dilution of 1:1000.For experiments using newly transformed BL21(λDE3), colonies were picked directly from the transformation plate and inoculated into 5 ml LB containing ampicillin for overnight growth. The overnight culture was diluted 1:1000 into fresh LB with ampicillin and grown to an ABacteriophage T7 mutant Δ4107 , which wGenomic DNA was isolated using the Qiagen DNeasy kit. Oligonucleotides used as primers (t7pol1 – gattaacatcgctaagaacg and t7pol2 – gattcatgtcgatgtcttcc) were obtained from Midland Certified Reagents, Midland, TX. PCR was performed using the FailSafe PCR kit (Epicentre Technologies, Madison, WI) following manufacturer's recommendations. PCR products were visualized by agarose gel electrophoresis.IPTG – isopropyl-β-D-galactosidePCR – polymerase chain reactionLB – Luria-BertaniSDS – sodium dodecyl sulfateJGGV was responsible for the original observations of inconsistent expression levels and the data for Figure


In 2002, the World Health Organization published a health system performance ranking for 191 member countries. The ranking was based on five indicators, with fixed weights common to all countries.We investigate the feasibility and desirability of using mathematical programming techniques that allow weights to vary across countries to reflect their varying circumstances and objectives.By global distributional measures, scores and ranks are found to be not very sensitive to changes in weights, although differences can be large for individual countries.Building the flexibility of variable weights into calculation of the performance index is a useful way to respond to the debates and criticisms appearing since publication of the ranking. The World Health Organization recently published a performance ranking of the health systems of its 191 member countries, and intends to update it at regular intervals . It was Publication of the analytical framework and the resulting ranking provoked considerable comment, and a variety of issues concerning the methodology and country positions in the ranking have been raised. A central component of the methodology for measuring overall attainment was the use of fixed weights, common to all countries, to aggregate the five indicators. This feature has been controversial, with some arguing that people in different cultural and social settings value individual health system goals in different ways -13. The In this paper we examine the sensitivity of the attainment scores to alternative weighting schemes that allow weights to vary across countries. These country-specific weights may reveal varying objectives of policy makers or constraints under which they operate. Melyn and Moesen  have refWHO used fixed weights (0.25, 0.25, 0.125, 0.125, 0.25) to aggregate five health system outputs  into a scalar health system attainment index. Overall attainment ranged from 35.7 (Sierra Leone) to 93.4 (Japan) on a [0–100] scale.We propose here an analytical framework that reduces to the WHO fixed-weight methodology as a special case, but that allows varying degrees of freedom for weights to be defined that – in the sense of Melyn and Moesen – implicitly take into account individual country circumstances. For shorthand, we say countries "choose" such weights, which in reality are determined as solution values of a linear program. If the linear program is a fair representation of the objective function of and constraints faced by decision makers, the weights are indeed "chosen", but even if this condition is not necessarily met, the resulting weights may still be of interest.The extent of freedom to choose weights in a linear program can be set by the analyst. The analyst can enforce fixed weights common to all countries, allow countries complete freedom to choose their own weights, or adopt a middle ground in which countries are granted limited freedom to choose weights within bounds thought to be sensible by experts.A generic statement of the performance evaluation problem in primal-dual linear programming format is:In these programs y is a country's output vector, x is its input vector, Y is the sample output matrix and X is the sample input matrix. In the present context y is (5 × 1), Y is (5 × 191), x is (n × 1) and X is (n × 191), with n to be specified below.-1 ≤ 1. Best practice countries have φ-1 = 1, other countries have φ-1 < 1, so φ provides a basis for acomplete ranking of countries on their relative ability to deliver five health system outputs. The dual program seeks a set of nonnegative weights μ,υ attached to a country's outputs and inputs that maximize its attainment. Each country's output weights are normalized by μy = 1, but each is free to select its own set of nonnegative weights.The primal program seeks the maximum radial expansion of a country's outputs, provided that it not exceed the standards established by a convex combination (λ ≥ 0, ∑λ = 1) of best-practice countries in the sample. The optimal value of φ provides a distance measure (i.e. an indication of how far a country has to go) to match best practice as observed in the sample. Since φ ≥ 1, the attainment of a country is evaluated as φIn constructing the overall attainment index, WHO identified five output indicators and no inputs, preferring to treat each country's health system as a "health output management unit". Consequently each country's input vector is represented as a scalar with unit value. Under these circumstances the performance evaluation problem simplifies to:0. The modified primal program seeks the maximum feasible radial expansion of a country's outputs consistent with best practice observed in the sample. The modified dual program seeks a set of nonnegative weights μ for a country's outputs that put it in the best light. A country can be expected to assign relatively large weights to those outputs at which it excels relative to best practice, and relatively low, possibly zero, weights to those outputs at which it lags behind best practice, subject to the normalization μy = 1. where ω = υ + υm(Ymλ - φym) = 0, m = 1, ..., M, so slack in any element of a country's projected output vector φy implies that the country assigns a zero weight to that output. Since it is unreasonable to allow a country to assign a zero weight to any output deemed sufficiently important to have been included in the WHO performance evaluation exercise, it is desirable to restrict weights in some way. This can be accomplished most easily by appending constraints to the dual side of (2) of the form:By complementary slackness, μm ≥ μmym/μy ≥ βm, m = 1, ..., M.     (3)γm) in total output.Restrictions (3) place lower and upper bounds on the relative importance of each output , with lower and upper bounds in (3) set by a modified AHP procedure.The fixed weights used by WHO))  to aggre.In the modified procedure, "expert opinion" was taken to mean the average values of weights arising from population-representative country surveys, each of which included a module on health system goals . Respondm = (0.19, 0.17, 0.12, 0.11, 0.22) and the upper bounds γm = (0.29, 0.25, 0.18, 0.17, 0.30). This specification allows limited freedom to choose. We refer to the four indexes as WHO, LP1, LP2 and LP3, respectively.For each output, the lower bound for calculation of the third index was taken as the minimum of the country average weights, and the upper bound the maximum . CountrySummary statistics of the four attainment indexes appear in Table Rank correlations between pairs of attainment rankings appear in Table Figure Results based on LP1 are unattractive. Over one-third of countries (70 of 191) assign a zero weight to four of the five indicators, and the vast majority of countries assign a weight in excess of 0.9 to either responsiveness distribution or fairness in financial contributions. This means they assign a low or zero weight to population health, the defining goal of the health system, which does not have face validity. In LP1, only Japan, Luxembourg and the United States assign positive weights to all five indicators. Consequently the attainment index is tightly distributed about a very high mean value. The ability to discriminate is sacrificed to freedom to choose, with 75% of countries receiving attainment indexes of 99 or above.Results from LP2 are somewhat more attractive. Nevertheless, when weights are bounded below by 0.1, over three-quarters (147 of 191) of countries assign the minimum weight to four of the five indicators and a 0.6 = 1 - (4 × 0.1) weight to either responsiveness distribution or fairness in financial contributions. The attainment scores are again compressed about a high mean value, and the ability to discriminate is not much improved, with 75% of countries receiving attainment scores of 93 or above. However despite this dramatic compression, the LP2 ranking is globally very similar to the WHO ranking. Eighteen of the countries ranked in the top 20 by WHO appear in the LP2 top 20, and 14 of the countries ranked in the bottom 20 by WHO appear in the LP2 bottom 20.Not surprisingly, the distribution of the attainment scores for LP3 looks even more similar to the distribution of the WHO scores, and has a similar mean and standard deviation. Rank correlation is very high, with only one country falling out of the WHO top 20 and only five countries rising out of the WHO bottom 20. Nonetheless, limited freedom to choose has an important impact on individual countries. The USA, given an ability to emphasize the importance of responsiveness level and responsiveness inequality, and to de-emphasize the importance of health level and inequality in the distribution of health, improves its ranking from #15 to #3. Australia improves from #12 to #7 for similar reasons. Italy, on the other hand, falls from #11 to #18, primarily as a result of the movement of other countries. In LP3, the largest positive changes in ranking are for Mauritius (+25) and Saint Vincent (+22), while the largest negative changes in ranking are for Kazakhstan (-39) and Albania (-36).We began by questioning the appropriateness of the fixed weight approach to aggregating indicators adopted by WHO, on the grounds that fixed weights deny countries at varying stages of development the freedom to choose. We then proposed a sequence of linear programming models that allowed countries varying freedom to choose the weights assigned to their indicators. LP1 allows complete freedom to choose, and generates weights we consider unacceptable, particularly because so many countries give a zero weight to improving health. LP2 allows considerable freedom to choose, but generates many country-specific weights falling outside the range of the within-country means used as bounds in LP3. Clearly, the validity of a procedure that routinely assigns weights out of the range of representative cross-population preferences should be questioned, even without a sophisticated theory of empirical ethics or democratic choice.LP3 applies the AHP procedure to set plausible bounds on weights, and allows limited freedom to choose. It generates a very similar distribution of the attainment index, and a very high linear rank correlation with the WHO ranking. Despite these similarities, we find the LP3 approach intuitively appealing, and are encouraged by its global concordance with the WHO index. However it is fair to ask: if LP3 and WHO generate such similar rankings, why bother? What value does LP3 add? Indeed, individual countries may come to diametrically opposed conclusions about the benefits of LP3 or WHO on the basis of their implied rank changes (e.g. Mauritius vs. Kazakhstan).Howbeit, our first response to the question "why bother" focuses on the distribution of the LP3 weights in comparison to the WHO weights. The WHO weight on responsiveness inequality was 0.125. But the LP3 upper bound of 0.17 is binding on 182 countries, which implicitly want a higher weight on this indicator. The WHO weight on fairness in financial contributions was 0.25. But the LP3 upper bound of 0.30 is binding on 167 countries that want a higher weight. At the other end, the WHO weight on health level is 0.25. But the LP3 lower bound of 0.19 is binding on 170 countries that want a lower weight. The WHO weight on responsiveness level is 0.125. But the LP3 lower bound of 0.12 is binding on 103 countries that want a lower weight. It appears that a majority of countries at all stages of development may implicitly assign greater importance to indicators of health distribution, and less importance to indicators of health level, than the experts whose judgments formed the basis of the original WHO weights.Our second response is more general. By allowing countries limited freedom to choose their weights, LP3 takes a small but nevertheless significant step toward respecting their varying circumstances. While the narrower the bounds on weights, the smaller the step, even the limited freedom embodied in LP3 makes an important difference to some countries.Building in the flexibility of varying weights might be a useful way for WHO to respond to the debates and criticisms appearing since publication of the ranking. We conclude by speculating that a variant of LP3 incorporating information regarding which weights are binding, and in which direction, might yield even greater benefits in terms of respecting individual circumstances.JAL, CJLM and DBE were part of the team at WHO that developed the methods for the world health system performance rankings published by the Organization in 2000.AHP DEA LP (Linear program)LP1 (Linear Program 1)LP2 (Linear Program 2)LP3 (Linear Program 3)WHO JAL developed methods inspired by the benefit-of-the-doubt concept and drafted an earlier version of the manuscript. CAKL adapted and applied the DEA methods described here to health system performance and drafted the initial version of the current manuscript. CJLM conceived the methods for measuring health system performance. DBE coordinated the research at WHO. All authors revised and approved the manuscript.The pre-publication history for this paper can be accessed here:Annex.Graphical interpretation of DEA and conceptual links with value theory.docClick here for file


Escherichia coli, such as productivity and protein activity, are affected by the growth rate. This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis. To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate.Important parameters during recombinant protein production in E. coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate. However, the levels of cardiolipin are very high compared to continuous cultivation. The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy. The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust. The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol. The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium. At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication. This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate.During fedbatch cultivation, -1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point.The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest. However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch. If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected. Induction at low growth rate (<0.2 h Esherichia coli there seems to be a strong dependence of the productivity and product quality on the limiting substrate feed rate at induction [For several recombinant proteins produced in nduction -8. DiffeOf particular importance for protein translocation processes are the anionic phospholipids but also unsaturated fatty acids. It was reported a great number of times that increased amounts of phosphatidylglycerol could facilitate protein translocation ,13-15. I-1 simultaneously to an optimum in β-lactamase leakage over the outer membrane [-1 unsaturated fatty acids where replaced by cyclic fatty acids [In continuous cultivation it was observed that conditions for establishment of an optimum in phosphatidylglycerol and unsaturated fatty acids could be achieved. An optimum in the accumulation of these compounds occurred at a growth rate of 0.3 hmembrane . It was ty acids  which waty acids ,27. If tThere is thus a strong incentive to select and operate processes which allow the establishment of controlled growth conditions and to understand the specific growth characterises established with respect to the membrane at particular feed rates. Two main techniques are at present available: fedbatch and continuous cultivation. We have previously established data from continuous cultivation  which we-1 and above, the traditional fedbatch concept established early in the former century based on bakers yeast and later on for antibiotics production, is generally used. The feed profile, characterised by this general concept, is initially exponential until the oxygen or heat transfer capacity of the reactor is reached and thereafter constant. This implies that the substrate concentration and the specific growth rate decline with cultivation time after the constant feed profile is adopted. This constitutes the advantage: the declining growth rate allows the cell mass productivity to increase at constant oxygen consumption/heat production. A further advantage of this cultivation mode, in comparison to a standard batch process with substrate overflow and to fedbatch with high exponential feed, is the possibility to avoid inhibitory by-products such as acetate. At a preset time during the feeding process, induction is performed. The drawback is that when the cell mass is very high the substrate consumption is very low and this point might not represent the optimal condition for high product formation, achievement of high product quality or facilitated transport of the protein.The fedbatch technique is used when a high cell density is the primary goal. A variety of feeding strategies can be applied for different research purposes where exponential feed can be used to create conditions that resemble a steady state in all points except for the continuous cell mass accumulation. This technique can however only be used for a few number of generations since the oxygen transfer rate rapidly becomes limiting. In order to reach a high cell density, i.e. cell densities of 50 g, lEscherichia coli.In continuous cultivation the feed/dilution rate is also used to control the growth rate where there is a limit of the chemostat to cultivation at the maximum or near maximum rates when the pH-auxostat is the preferred method . ContinuThe goal of this work is to show how growth rate controls the performance in fedbatch cultivation with respect to the structure of the membrane and to compare these changes to the function of some selected process variables and methods. These data will, in turn, be compared to earlier data derived from continuous cultivation . From thEscherichia coli W3110, a K-12 derived strain, was grown in fedbatch cultivation. The fedbatch concept was characterised by an initial exponential feed, started at time zero, of the growth limiting component, glucose, leading to the establishment of a specific growth rate of 0.6 h-1. This was followed by a constant feed phase where the growth rate declined asymptotically to a value of approximately 0.05 h-1. During this phase the cell mass increases linearly to a cell density of approximately 27 g, l-1, as expected , unsaturated (UFA: C16:1, C18:1), and cyclic (CFA: C17:cyc). From this figure is evident that the amount of SFA's is the most stable during the declination of growth. There is only a slight reduction of the amount at a growth rate of approximately 0.3 h-1, which is accompanied by a maximum in the amount of unsaturated fatty acids. The unsaturated fatty acids are markedly decreased as the growth rate goes below 0.3 h-1 to 56/70 % of their maximum values, respectively. These maximal values, which coincide with the above mentioned minimum in CL/maximum in PG, thus constitutes a switch-point in the fatty acid and phospholipid metabolism during fedbatch cultivation. At this point there is also a minimum of cyclic fatty acids. The levels of the cyclic fatty acids are then rapidly increasing the lower the growth rate and from its minimum the cyclic fatty acids are dramatically increased by almost 100 % in less than two hours. This occurs although there is a supply of nutrients at all times which is in contrast to earlier literature where it is stated that it is the lack of carbon and energy in the stationary phase which results in cyclic fatty acid formation. During the continuing declination of the growth rate, the cyclic fatty acids continues to increase to a final of three times their minimum value. It should be noted that since the fatty acids of the lipopolysaccharide (LPS) were removed during the sample extraction, the results are only reflecting the inner part of the outer membrane and the cytoplasmic membrane.The fatty acids are shown as a function of the time from constant feed start in Figure The three phospholipids were furthermore examined for their individual fatty acid content. The results are shown in Figure The effects of these membrane changes were investigated on basis of the function of some selected methods and process variables: the mechanical strength of the membrane to ultrasound, the effect of osmotic chock, the shredding of endotoxin to the medium, the leakage to the medium and the amount of cell lysis.β-lactamase, at different time of sonication using the same amount of cell and sonication effect. Figure -1 but from this point on there is continuously increased membrane stability. A comparison to the pattern of unsaturated fatty acid accumulation in Figure The mechanical strength of the membrane during different growth rates was quantified as the release of the periplasmic marker, vice versa. The figure shows that the sucrose addition and the late addition of water results in the major effect on permeabilisation and, for unknown reason, also the initial dilution by Tris-HCl. The addition of lyzosyme, in itself, has no effect which is a result of the fact that it can only penetrate the membrane by the addition of water [Osmotic shock is a frequently used method to release protein in the small scale. The methodology chosen as a model system gives a step-wise permeabilisation of the membrane, which is followed by breakage of the cell wall murein saccculus and formation of sphaeroplasts when lysozyme enters the cell . Severalof water . The effThe release of lipopolysaccharide, LPS, to the medium was measured by means of detection of the presence of a specific inner part of this structure, lipid A, which has properties of an endotoxin. This compound has to be removed in downstream processing of pharmaceutical proteins since it gives a toxic response in patients and must always be proven absent in the final product. It was considered likely that the changes in the underlying phospholipid and fatty acid structure might have large consequences for the release of this component to the medium which was to some extent confirmed by literature studies . In FiguIn Figure -1 where after it slowly declines from 0.50 to a final value of 0.42 g protein, g cells-1.A major concern of this work was the effect of membrane structural changes on protein leakage from the periplasm to the medium. Since it is clear that a periplasmic protein can be present in the medium both by cell lysis and by true leakage from the periplasm, cell lysis was registered as a function of cultivation time. This is shown in Figure β-lactamase, which is constitutively produced from its own promoter. The total β-lactamase production is shown as well as the specific production values in the medium per gram of cells. It is clear that during fedbatch cultivation the total amount of this specific protein follows the total protein accumulation pattern and is constant independent if calculated per cell mass or per total cell protein except for the values at very low growth rate. The β-lactamase value per gram of cells in the medium is thus slightly higher during the first part of the cultivation down to μ = 0.3 h-1 where after the value declines. At very low growth rate there is yet another period of increased specific accumulation. It is realised that this latter period of slowly increased accumulation in the medium coincides with an increased however small lysis in the end of the cultivation. Since the leakage is not increased during this period lysis seems most likely cause for the accumulation. The level of β-lactamase in the medium amounts to approximately 10% of the total β-lactamase production throughout the cultivation as seen from the quotient of β-lactamase in the medium in relation to the total value.In Figure -1 is formed in expense of cyclic fatty acids which are accumulating at low growth rate. Compared to literature data the amount of cardiolipin is approximately 70 % higher than from earlier observations [The conditions during fedbatch leads to a structure, which is approximately constant with respect to phospholipids and saturated fatty acids compared to growth rate. The changes in lipid components arrive from changes in unsaturated fatty acids where the increased values above the maximum at 0.3 hrvations .-1. The high content of cardiolipin is to some extent balanced by high level of unsaturated fatty acids, which compensates the need for flexibility, permeability and movement of compounds within the membrane which growth rate. The growth dependent accumulation and the generally high level of unsaturated fatty acids influence the results of sonication. The high levels above μ = 0.3 h-1 thus supports a weaker membrane, in this respect, while the substitution of unsaturated to cyclic fatty acids reduces the effect at low growth rate. The transport of protein over the membrane is however only slightly higher at higher amounts of unsaturated fatty acids which is implicated by the larger effect of enzyme/water penetration during osmotic chock and the leakage of the model product, β-lactamase, at high growth rate. In total, β-lactamase is however always present in the medium at a level of approximately 10 % of the total production due to that the membrane flexibility caused by unsaturated fatty acid is balanced by the high and constant amount of cardiolipin.The resulting effects on the membrane function are several but are generally and overall characterised by their stability as a function of growth rate. Cell lysis is low at all times i.e. the membrane is not easily breaking-up and this is due to the high content of cardiolipin at all times which is further emphasised by high levels of cyclic fatty acids specifically at growth rates below 0.3 hThere is a very slight increase in cell lysis at the end of cultivation. The reduced capacity of this cell for total and specific protein production at low growth rate, which might be due to a reduced amount of ribosomes , low supContinuous cultivation gives theoretically always the highest productivity due to the reduced shut-down periods for cleaning and refilling. However this technique is rarely used in industrial production of recombinant protein production due to problems of separation of growth and product formation. The pharmaceutical industry will furthermore need a validation protocol, which must be derived for this technique and where the concept of batch reproducibility needs some clarification. There is also a tendency to use techniques which are since long established and to which the personnel are educated. However, sometimes reasons are not well developed and a problem is the lack of data for a scientific choice of production technique.-1) or a low (0.05 h-1) growth rate. The table furthermore indicate if curves, with respect to growth rate, show a trend which reveals the existence of maximum/minimum values of the specific compound. Table In table One of the most significant structural differences between the cultivation techniques is the high content of CL in fedbatch cells in expense of both PG and PE. The formation of large amounts of CL is an obvious cell strategy to save energy and carbon by the release of glycerol to further metabolisation. Since the only difference between the two cultivation techniques is the constant conditions in continuous cultivation and the dynamically changing ones in fedbatch the increased need for carbon/energy must lie in the rapidly changing growth environment.β-lactamase due to the growth dependent changes of this fatty acid.In fedbatch there are also more unsaturated fatty acids accumulated, which develop in expense of saturated fatty acids, which are higher in continuous cultivation. The fatty acid level is also more or less constant in CL, which promotes stability. The conclusion is that cells in fedbatch cultivation interpret the environmental signals in a way that leads to a membrane structure with several features that generally only develop during nutrient exhaust in stationary phase. This is not the case for cells growth in continuous cultivation which develop distinct characteristics at each stable growth rate. This leads to a fedbatch membrane that is rigid compared to cells in continuous cultivation. The overall result is fewer changes in the membrane function due to growth rate. This is represented by constant leakage, constant lysis and an almost constant effect of permeabilisation. The fedbatch membrane shreds also less endotoxin although levels are low for both cultivation techniques. However, the fedbatch membrane is sensitive to sonication, specifically at high growth rate due to the comparatively high amount of unsaturated fatty acids and thus releases more -1. However, this is not allowed to develop in fedbatch to the extent seen in continuous cultivation where a specific growth rate is kept much longer of the process time. The result is that no optimum in the leakage to the medium is clearly established in fedbatch cultivation and the fedbatch process cannot be designed for this since a constant growth rate of 0.3 h-1 cannot be kept while at the same time leading to a high cell accumulation.From the comparative data of table Protein transport is generally considered to benefit from a high content of PG and unsaturated fatty acids. This accumulation leads to high leakage which is verified by the continuous cultivation leakage optimum  and whicThe overall conclusion is thus that if leakage is undesired, fedbatch should not be used but the process could well be run in continuous cultivation at low growth rate. If an increased leakage is preferred this can be optimised in the continuous operation mode principally by change of growth rate but probably also by mutants accumulating a combination of phosphatidylglycerol and unsaturated fatty acids if these are stable enough for prolonged cultivation.The stability of the fedbatch membrane, which is not growth dependent, is the strength of this cultivation technique. The time of cell harvest is thus not critical since the membrane strength and function does not rapidly change which allows for flexibility in operation. The stability of the fedbatch derived cell membrane might thus also by the same reasoning be an advantage in further downstream processing however depending on the chosen unit operations.The merits of the cell characteristics in continuous cultivation are due to the possibility to run the cultivation at a feed rate which is optimal for membrane leakage independent if this is to be small or maximised.-1 and induction should thus take place well above this value. This is a considerable drawback in fedbatch cultivation, which relies on high cell density accumulation leading to low growth rates before induction but easily controlled in continuous cultivation. The coupling of the productivity and even product quality to a specific growth rate further strengthens the implication of using continuous cultivation.Total and specific protein production is lowered at low growth rate and the data points to that recombinant protein production should not be performed below a growth rate of 0.2 hThe conclusion is that the drawbacks of industrial operation of continuous cultivation in the biopharmaceutical industry earlier mentioned should be preferably be solved to explore the benefits that are obviously present with this technique.Escherichia coli W3110 (F-, λ-, IN (rrnD-rrnE)) [-1, was used. The plasmid pBR322 (Pharmacia Biotech, gene Bank Accession number V0119) was transferred to the host cell. This plasmid carries the genes bla and tet where β-lactamase (TEM-1) was used as the model product. The maximum levels were approximately 1 % of the total protein. The medium for the bioreactor and the feed was a mineral salts medium composed of (g, l-1): (NH4)2SO2, 7.0; KH2PO4, 1.6; Na2HPO4 *2H2O, 6.6; (NH4)2-H-citrate, 0.5. Glucose was autoclaved separately and added to bioreactor together with a sterile filtered trace element solution and 1.0 M MgSO4 (1 ml, l-1, respectively). A further addition of magnesium, of the same amount, was done at eight hours of feeding. The glucose concentration in the feed was 300 g, l-1. The trace elements solution was composed of (per litre): CaCl2 *2H2O, 0.5g; FeCl3 *6H2O, 16.7 g; ZnSO4 *7H2O, 0.18 g; CuSO4 *5H2O, 0.16g; MnSO4 *4H2O, 0.15 g; CoCl2 *6H2O, 0.18 g; Na-EDTA, 20.0 g.D-rrnE)) , a K12 d600) of 2–3 was reached from which point the fedbatch cultivation was performed. The experiments were carried out in a 14-l bioreactor with a working volume of 8.0 l. The bioreactor was equipped with an air sparger and the air-flow was 0.5–1.0 vvm. By varying of the stirrer speed up to 1000 rpm the dissolved oxygen concentration did never go below (30 ± 2) % air saturation. A pH of 7.0 was kept by addition of 28% (w/w) ammonia solution. Temperature was controlled at 37°C. The cultivations were started as batch cultures with an initial glucose concentration of 2 g, l-1. The feed was started when glucose was almost exhausted. The feed profile consists of two different phases where an exponential feed was followed by a constant feed phase. The exponential feed profile was calculated from the following equation:Four independent fedbatch cultivations were performed in the following manner: inoculum was prepared from a shake flask culture grown overnight at 37°C at 150 rpm. This was transferred into the bioreactor when an optical density at 600 nm (OD0 * eμt-F(t) = Fμ the desired specific growth rate (1/h) and t is the process time (h). The calculation of the initial feed rate, F0, is based on a mass balance at the time of the feed i.e. after the batch growth assuming: pseudo steady state growth during the feed phase, a theoretical yield coefficient (Yx/s) of 0,5 g, g-1 and a negligable amount of the limiting substrate in the reactor.where F is the feed rate (L/h), 0 =(μ *V*X)/(Si*Yx/s)Fx/s is the cell yield on glucose (g/g), X is the cell concentration at F0 (g/L), V is the culture volume (L) and Si is the glucose concentration in the feed (g/L).Where YThe continuous cultivation conditions and data, compared to in the conclusions section, were reported elsewhere . These w-1) by 10 min centrifugation (4500 rpm, 2250 g) of 3*5 ml of cell suspension in preweighed test tubes and drying of the pellet overnight at 105°C before weighing. The supernatant from the CDW samples was sterile filtered and used for acetic acid and cyclic adenosine monophosphate, cAMP, analysis. Acetic acid was measured with the enzymatic method from Boehringer Mannheim GmbH (Cat. No 148 261) and cAMP was analysed by an enzyme immunoassay system from Amersham Pharmacia Biotech AB (code RPN 225).The cell growth was followed by optical density at 600 nm. Biomass concentration was determined as cell dry weight  at 55 bar. Data from French press was set as the 100% disruption level and all other values were taken as a percentage of this value.The mechanical strength was estimated by sonication  at constant frequency, cell dry mass, sample volume and acoustic power. Samples were placed in a water bath at +4°C during cell disruption to prevent overheating. The release of -1 cultures were harvested and suspended in 0.3 ml, 200 mM Tris-HCL (pH 8.0), (2) t = 1 min, 0.5 μl, 100 mM EDTA (pH 7.6) was added, (3) t = 2 min, 0.5 ml, 1 M sucrose (pH 8.0) was added thus giving excess of EDTA over Mg2+ ions, (4) t = 3.5 min, egg white lysozyme (EC 3.2.1.17, C. F. Boehringer und Soehne GmbH, Mannheim, Germany) was added to a final concentration of 60 μg/ml, (5) t = 4 min, the cell suspension was exposed to a mild osmotic shock by two-fold dilution in water to trigger lysozyme penetration of the outer membrane, and finally (6) t = 8 min, the cell suspension was diluted 11-fold in 10 mM EDTA (pH 7.6). Permeabilisation was recorded by optical density at 450 nm. The samples taken at different growth rate were diluted to the same cell concentration (0.5 g, l-1) and the same volume (0.2 ml) was used at the sampling point.Chemical strength was estimated by permeabilisation as described by Witholt et al . The folPhospholipids and fatty acids were determined by the method of Arneborg  where thAS made the fermentations, analyses and data treatments and evaluation. The project was conceived by GL who further participated in the project planning, design and data evaluation. All authors read and approved the final manuscript.