Saccharomyces cerevisiae is an important microorganism for both industrial processes and scientific research. Consequently, there have been extensive efforts to characterize its cellular processes. In order to fully understand the relationship between yeast's genome and its physiology, the stockpiles of diverse biological data sets that describe its cellular components and phenotypic behavior must be integrated at the genome-scale. Genome-scale metabolic networks have been reconstructed for several microorganisms, including S. cerevisiae, and the properties of these networks have been successfully analyzed using a variety of constraint-based methods. Phenotypic phase plane analysis is a constraint-based method which provides a global view of how optimal growth rates are affected by changes in two environmental variables such as a carbon and an oxygen uptake rate. Some applications of phenotypic phase plane analysis include the study of optimal growth rates and of network capacity and function.The yeast Saccharomyces cerevisiae genome-scale metabolic network was used to formulate a phenotypic phase plane that displays the maximum allowable growth rate and distinct patterns of metabolic pathway utilization for all combinations of glucose and oxygen uptake rates. In silico predictions of growth rate and secretion rates and in vivo data for three separate growth conditions  were concordant.In this study, the Taken together, this study examines the function and capacity of yeast's metabolic machinery and shows that the phenotypic phase plane can be used to accurately predict metabolic phenotypes and to interpret experimental data in the context of a genome-scale model. The development of numerous high-throughput experimental techniques such as DNA microarrays, genome sequencing, and protein chips has revolutionized the analysis of biological systems and generated a catalog of information about a cell's components -3. EfforSaccharomyces cerevisiae. Flux-balance models of yeast have appeared for small-scale network reconstructions. Most of these studies are specific for growth conditions, such as anaerobic, glucose-limited metabolism  (mM) is the substrate/product or DO concentration in the flask, q (mmole/g-dry weight/hr) is the substrate uptake rate or by-product formation rate or OURflask, and X(t) (g-dry weight/L) is the biomass concentration at time = t (hr). + is for the by-product formation and - is for the substrate consumption. The solution to this equation was fitted to the experimental data points using the nonlinear estimation routine in Statistica  or the solver in Microsoft Excel. All data, to be considered valid and included in the analysis, must have correlation coefficients of 0.95 or greater. The data that were generated in this way represented the "pseudo-steady-state" [flask.where y-state"  of the bS. cerevisiae metabolic network has been reconstructed [x, and reaction activities, v, is described by the dynamic mass balance equation [A genome-scale structed . The netequation ,44:S is an m × n matrix of stoichiometric coefficients, x is an m × 1 vector of metabolite concentrations, and v is and n × 1 vector of reaction activities. Thus, the rows of S correspond to the internal metabolites and the columns represent the reactions in the network. Under steady-state conditions, the dynamic mass balance equation simplifies to:where S • v = 0     (Eq. 6)S. cerevisiae for various growth conditions [Since the number of reactions is often greater than the number of metabolites, Eq. 6 is underdetermined and contains multiple solutions. One approach to solving Eq. 6 for microbial networks is to define a set of inputs and outputs that correspond to the growth conditions and use linear optimization to maximize the cell growth . This apnditions .S. cerevisiae PhPP displays optimal growth rates for all possible variations in two constraining environmental variables, such as the carbon substrate and oxygen uptake rates. In this study, the glucose uptake rate (x-axis) was allowed to vary from 0 to 20 mmol/gDCW/hr and the oxygen uptake rate (y-axis) ranged from 0.1 to 20 mmol/gDCW/hr. The oxygen uptake rate was not allowed to reach zero because anaerobic simulations required additional supplements to maintain cell growth (ergosterol and zymosterol). Linear programming was used to calculate the optimal growth rate for all points in the x-y plane. Growth rate values were then plotted as the z-axis to form the surface of a three-dimensional PhPP  to changes in the availability of each metabolite:i is the ith metabolite and γi is the ith shadow price. Shadow prices were calculated for each point in the x-y plane during the linear programming simulations. By definition, phases were identified as regions of the PhPP in which all of the points have the same shadow prices. Lines of optimality, which represent the optimal ratio of glucose and oxygen uptake rates for maximal biomass synthesis, were also identified using shadow price analysis [where banalysis .e.g. the value of the objective function increases if the metabolite's net production increases or its net consumption decreases. Similarly, a positive shadow price indicates that a metabolite is available in excess and a shadow price equal to zero indicates that a change in the availability of the metabolite does not affect the objective value.To obtain a physiological interpretation of the differences between the oxidative fermentative phases (phases 2–6), we analyzed how the shadow prices of key metabolites changed across the phase boundaries. Simulations were run at a fixed glucose uptake rate of 5 mmol/gDCW/hr and an oxygen uptake rate ranging from 1.5 to 15 mmol/gDCW/hr. The sign of the shadow price was used to identify whether a small change in the metabolite's availability would positively or negatively affect the objective value. According to the convention defined in , a negatin silico . Single-5 and 1.27 × 10-4 mmol/gDCW/hr, respectively, were used for the calculation at OUR = 0). The simulations were then re-run with a fixed glucose uptake rate, oxygen uptake rate, and growth rate to determine the maximum and minimum secretion rates of each metabolite with a shadow price equal to zero.The first step in generating the secretion profile was to calculate the optimal growth rate for a given glucose and oxygen uptake rate. For the simulations in Figures NCD calculated the phenotypic phase plane Fig. , carried


Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses. The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: [kcat/Km for DNA cleavage by a factor ≥106 [Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: ). The "otor ≥106 .D-Xn-(D/E)-X-K (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity : BfiI iStreptomyces fimbriatus. It recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3', where N denotes any base, and cleaves it as indicated by "^", leaving 3' extensions 3 nt long [SfiI is a REase isolated from  nt long . SfiI is nt long . However nt long -25, but In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes . HomologThe sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations  . TheA homology model of SfiI was constructed based on the alignments reported by threading methods, using the "FRankenstein's Monster" approach  , followed by local geometry optimization. One of the adenine residues in the mismatched A/A base pair in the middle of the DNA molecule was "mutated" to T/A. The curvature of the DNA remained unchanged. Essentially, the global structure of the protein-DNA complex for SfiI remains exactly as in the BglI structure, as reliable modeling of macromolecular interactions remains beyond the capabilities of the existing methods. The model of SfiI dimer is shown in Figure .BglI belongs to the "EcoRV-like" β-class of PD-DxK nucleases. The most typical features of REases from this class are: antiparallel orientation of the 5elements ,9. Most elements . Brieflyelements  and "mutIn the proposed model of SfiI, the spatial configuration of the catalytic residues is typical for an active site architecture conserved among PD-DxK nucleases. We predict that the active site of SfiI comprises residues: E55, D79, D100 and K102, which superimpose well on the catalytic residues of BglI: E87, D116, D142 and K144, respectively Figure , backbonInterestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63–80 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities.The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge  could disrupt the formation of the SfiI dimer and destroy the REase activity.SfiI is a Type IIF enzyme, i.e. a tetramer that binds simultaneously to two recognition sites and cleaves both sites concertedly , while BComparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: α (EcoRI-like) and β (EcoRV-like) -9. It waOur results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a β-lineage member, an "orthodox" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises α-lineage members for which crystal structures were solved  -41. ThusSequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST . SecondaThe alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the "FRankenstein's Monster" approach , comprisaa, amino acid(s); bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; MTase, methyltransferase; ORF, product of an open reading frame, RM, restriction-modification;JMB carried out the fold-recognition analysis for SfiI, built the preliminary models, drafted the manuscript and coordinated the whole study. AC built the final models and identified functionally important residues. KS participated in interpretation of the data and writing the manuscript. All authors have read and accepted the final version of the manuscript.


Leishmania (L.) mexicana-infected neonatal BALB/c mice. DCs were isolated and purified from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with L. (L) mexicana; b) Adult BALB/c mice infected during neonatal life; c) Healthy neonatal BALB/c mice; d) Healthy adult BALB/c mice. A neonatal model of infection, generated after inoculation with 5 × 105 promastigotes of L. (L) mexicana, was used as the infection control group. Sixteen hours after intraperitoneal transfer of DCs , neonatal recipient BALB/c mice were infected. The adoptive transfer of DCs diminished disease progression in neonatal mice. This reduction depends on the quantity and provenance of transferred DCs, since the effect was more evident with high numbers of DCs from adult mice infected during adulthood and healthy neonatal mice. Protection was significantly reduced in animals receiving DCs from healthy adult mice but it was absent in mice receiving DCs from adult mice infected during neonatal life. These results suggest that genetic susceptibility to Leishmania infection can be modified during neonatal life, and that the period of life when antigens are encountered is crucial in influencing the capacity of DCs to induce resistance or tolerance.We evaluated the adoptive transfer of DCs on  L. (L.) mexicana, and from healthy adult and neonatal mice. As in the L. major mouse model, we have shown that infection with L. (L.) mexicana strain MHOM/BZ/82/BEL21, generates a Th1 response associated to protective immunity in C57BL/6 mice, and a Th2 response related to non-healing disease in BALB/6 mice [Medawar et al.  showed aB/6 mice .Leishmania antigens to the lymph nodes and induce specific immune responses [Leishmaniasis is an excellent model to study the extremes of host/parasite relationships, particularly the diversity of the immune response associated to the genetic background of the host. In addition, mice can reproduce the distinct clinical forms observed in humans ,17. Thesesponses -24. Mollesponses .L. (L.) mexicana infection in neonatal BALB/c mice, after the inoculation with 5 × 104, 1 × 105, 2 × 105 or 5 × 105 promastigotes was determined by measuring the footpad thickness during 6 weeks. All 4 experimental groups developed lesions. Mice that received 1 × 105, 2 × 105 and 5 × 105 promastigotes respectively, showed a significant increase (p ≤ 0.05) on footpad thickness starting from the second week, reaching a maximal value on the sixth week of evaluation  mexicana-infected adult BALB/c mice inoculated with 1 × 106 promastigotes  two-tailed value and a very significant Spearman correlation . The starting footpad thickness in neonatal and adult BALB/c mice was 1.67 mm and 1.85 mm, respectively.The progress of ion Fig. . This ineek Fig. . Moreovetes Fig. . The sta5 promastigotes as the optimal concentration for L. (L.) mexicana infection in all the subsequent experiments including the infection control group. This neonatal murine model of L. (L.) mexicana infection used half the numbers of promastigotes previously described to infect adult BALB/c mice [Leishmania infection. Other studies have shown a similar pattern of Th2-biased immune response in other models of neonatal infection [Leishmania infection [We used 5 × 10B/c mice . A signinfection ,11. We anfection . Susceptnfection -31. Seconfection ,33.DCs obtained by our purification method showed characteristic dendritic cell morphology, and a 97% purity as determined by CD11c immunostaining and flow cytometry. A minor fraction of about 3.5 % expressed CD3 and NK1.1 Fig. . The expSplenic DCs were isolated for our adoptive transfer experiments since they are mobile antigen-presenting cells that migrate to peripheral lymph organs where they stimulate naive T cells, thus initiating primary T cell responses -36. Furt3, 1 × 105 or 1 × 106 DCs from adult BALB/c mice infected during adulthood with L. (L) mexicana on neonatal recipient mice modified the course of infection, showing a delayed lesion growth from the second week onward  mexicana fail to modify the course of infection of neonatal recipient BALB/c mice as compared with infection control animals  on weeks 2, 3 and 4. This effect disappeared from the fifth week onwards  in lesion growth from the second week of infection  smaller than those observed in mice transferred with 1 × 105 DCs, which also initiated their lesions on the third week  in lesion growth from the second week of infection. This reduction in footpad thickness was very similar in both tested concentrations  mexicana, healthy adult BALB/c mice and healthy neonatal BALB/c mice. The reduction in these 3 groups was statistically significant (p ≤ 0.05) as compared with the infection control group. This reduction in footpad thickness was absent or considerably diminished in mice receiving DCs from adult BALB/c mice infected during neonatal life with L. (L) mexicana  mexicana infection in neonatal BALB/c recipient mice. These results contrast with those of Moll and Berberich [Leishmania infection. In this study, the observed protection depends on the quantity and provenance of the transferred DCs, since the effect was more evident with high cellular numbers of DCs from adult BALB/c mice infected during adulthood and healthy neonatal mice, where lesions were about 40% smaller than in the infection control group. DCs from these two groups have the intrinsic capacity to induce protective or resistant immune responses very early in life. That neonatal DCs appear to be more protective, on a per cell basis, than adults DCs is a very striking result since only Dadaglio et al.[L. (L) mexicana during neonatal life. This result confirmed recent studies by Adkins et al. showing that animals initially immunized as neonates are unable to develop the expected Th1 memory effector function observed in adults [Our results showed that the preceding intraperitoneal adoptive transfer of DCs diminished the progression of erberich  showing io et al. have shoio et al.. Also, aio et al.-48. Thisio et al.-51. Our n adults . These in adults .Our results show that tolerizing DCs from animals initially immunized as neonates play a key role in the attenuation of Th1 responses. The present results may have a considerable epidemiological impact on leishmaniasis, where infection at early stages of life may impose a tolerogenic state that favors the development of visceral or diffuse cutaneous leishmaniasis, both characterized by Th2-type responses.L. (L.) mexicana infection.In this study, we have shown that intraperitoneal adoptive transfer of splenic DCs is able to surpass the genetic bias of the mice, allowing the development of an immune response that modifies the progression of Adult (6 weeks) and neonatal (about 24 hour newborn) female BALB/c mice  were raised in the Animal House of the Instituto de Biomedicina, under appropriate conditions of temperature, water and feeding.d  and CD86  purchased from BD Pharmigen .The following rat monoclonal antibodies were used to isolate and characterize dendritic cells: CD19 , MOMA-2 (Macrophages/Monocytes), CD45R , CD3 , CD11c , CD19 (clone 6D5) conjugated to phycoeritrine (PE), NK1.1 (clone PK136) conjugated to PE, Macrophages-Monocytes (MOMA-2) conjugated to fluorescein isothiocyanate (FITC), CD3 (clone KT3) conjugated to FITC. All were purchased from Serotec Ltd.  except CD205  donated by Georg Kraal, Vrije Universiteit, Amsterdam, The Netherlands; I-ALeishmania (Leishmania) mexicana (MHOM/BZ/82/BEL21) were extracted from footpad nodules of hamsters infected a month earlier with 1 × 106 amastigotes. The nodules were aseptically dissected out and washed in phosphate-buffered saline  with 100 U/ml penicillin and 100 μg/ml streptomycin, and finely cut and ground in a Petri dish containing cold PBS. The suspension was filtered through a sterile sieve to remove large debris. These parasites were cultured on blood agar base  at room temperature for 7 days (the stationary growth phase) to obtain infective promastigotes. For an enriched population of parasites, free of erythrocytes and cellular debris, 100 μl of that sample were cultured in 2 ml Schneider's insect cell culture medium  for one week at room temperature. Promastigotes were isolated after 3 washes with sterile PBS and centrifugation at 1000 g at 4°C for 15 min. Pellets were resuspended in 1 ml of sterile PBS. Viable parasites were counted by trypan blue exclusion. Parasite concentration was adjusted to 5 × 104, 1 × 105, 2 × 105 and 5 × 105 per μl to be used in the different experimental groups.Amastigotes of L. (L.) mexicana infection to that established in adult mice [4, 1 × 105, 2 × 105, or 5 × 105 promastigotes suspended in 10 μl sterile PBS, applied with a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette . For comparison, adult BALB/c mice were infected the standardized optimal parasite load of 1 × 106 promastigotes of L. (L.) mexicana [A similar pattern of ult mice  was detemexicana . The cou7 cells/ml in complete RPMI-10 and 8 ml plated in tissue culture flasks. The flaks were incubated for 2 hr at 37°C in a 5% CO2 incubator , allowing DCs to adhere. Non adherent cells were carefully removed and placed in sterile 50 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. Adherent cells were covered with 10 ml complete RPMI-10 and incubated as before for 16–18 hours, allowing DCs to detach. After gently washing the surface of the flasks with a plugged Pasteur pipette and complete RPMI-10, pools of the eluted cells were placed in sterile 15 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. For each tube, the cell pellet was resuspended in 6 ml complete RPMI-10. This volume was carefully layered over a 3 ml NycoPrep™ density gradient  and centrifuged at 600 g, 20°C for 20 min. Mononuclear cells were removed from the interface ring using a Pasteur pipette, transferred to a sterile 15 ml centrifuge tube and spun down in 10 ml complete RPMI-10 at 400 g, 20°C for 15 min three times. The final pellet was resuspended in 1 ml of cold (4°C) Hanks balanced salt solution supplemented with 10% decomplemented FBS and 2 mM HEPES. Cells were quantified and viability assessed by trypan blue exclusion.DCs from adult and neonatal BALB/c mice were isolated from the spleen. Under sterile conditions, spleens were minced on a metallic mesh with RPMI-1640  supplemented with 10% of decomplemented fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 100 U/ml penicillin (complete RPMI-10). The cell suspension was filtered on a nylon sieve and transferred to 15 ml centrifuge tubes  and spun at 250 g at 4°C for 10 min. Viable cells were counted by trypan blue exclusion. Cell concentration was adjusted to 1 × 106 cells). After incubation, cells were washed three times in Hanks centrifuging at 250 g, 4°C for 10 min. The pellet was resuspended in 1 ml cold Hanks in a sterile 15 ml centrifuge tube and incubated under continuous agitation at 4°C for 1 hour with a secondary sheep anti-rat IgG polyclonal antibody coupled to magnetic microspheres  at a 7:1 sphere/target ratio. Non-dendritic magnetic-coated cells were removed by positive selection in three sequential depletions using a magnetic gadget  at 4°C for 6 min.The final purification stage consisted of an immunomagnetic negative selection of DCs. The cell suspension obtained above was incubated under continuous agitation at 4°C for 1 hour, with primary rat anti-mouse monoclonal antibodies recognizing B and T lymphocytes, NK cells and monocytes/macrophages (1.5 μg/ml antibody per 1 × 105 cells were suspended in PBS (1% FBS) and incubated with 10 μl primary monoclonal antibodies directly coupled to PE or FITC recognizing T and B lymphocytes, NK cells and monocytes/macrophages. DCs were characterized by an indirect method using primary monoclonal antibodies to CD11c and a secondary antibody, hamster anti-rat IgG1conjugated to FITC . The incubations were carried out in the dark at 4°C for 45 min, followed by 3 washes and centrifugation at 250 g, 4°C for 10 min. The cell pellet was resuspended in 500 μl PBS and the percentage of labeled cells determined in a flow cytometer . The control consisted of an antibody of irrelevant specificity conjugated to FITC.DC purity was determined by flow cytometry and immunocytochemistry. For flow cytometry, 1 × 105 cells were suspended in PBS (1% FBS) and spun down at 50 g in a Cytospin . Sample slides were hydrated in PBS, fixed in fresh acetone for 5 min. and sequentially incubated for 90 min with primary rat monoclonal antibodies to CD11c and CD205, biotinylated goat anti-rat IgG (50 μg/ml)  for 45 min., and Vectastain® Elite ABC kit  at 1:100, 30 min. Five-minute washes with PBS were done between incubations. The reactions were developed for 3 minutes in Vector® NovaRed™ substrate. The slides were then washed and counterstained with methyl green. Omissions of the primary antibody and incubation with an antibody of irrelevant specificity at the same protein concentration were used as controls.For immunocytochemistry, 1 × 10L. (L.). mexicana (n = 4); b) Adult BALB/c mice infected during neonatal life with L. (L.). mexicana (n = 4); c) Healthy neonatal BALB/c mice (n = 4); d) Healthy adult BALB/c mice (n = 4). The infection control group consisted of neonatal BALB/c mice infected with 5 × 105 promastigotes of L. (L) mexicana.DCs were isolated from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with 3, 1 × 105, or 1 × 106 cells/ml in sterile PBS for intraperitoneal transfer to neonatal recipient BALB/c mice. Cells, at the mentioned concentrations, were injected in 20 μl volumes using a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette . After sixteen hours of adoptive transfer, neonatal recipient BALB/c mice were infected with 5 × 105 promastigotes of L. (L) mexicana.DCs from the 4 experimental groups were adjusted to 1 × 10Isolation of DCs and adoptive transfer experiments were done in duplicates.).The results were expressed as mean ± standard error of the mean (SEM). Each experimental group consisted of 4–5 individuals. Comparisons between groups were made with Student t test and Welch t test for unpaired samples. Any value of p ≤ 0.05 was considered significant. All tests were performed using GraphPad InStat 3.02  declare that they have no competing interests.LVP carried out most of the experimental work and drafted the manuscript. JC developed the experimental design, carried out part of the experimental work and drafted the manuscript. NLD participated in the in experimental design and evaluated the progression of infection in the mice. FJT conceived the study, participated in the experimental design and coordinated the work. All authors read and approved the final manuscript.


Prosaposin is a potent neurotrophic molecule. Homozygous inactivation of We have recently reported that prosaposin is expressed at a higher level by androgen-independent (AI) prostate cancer cells as compared to androgen-sensitive prostate cancer cells or normal prostate epithelial and stromal cells. In addition, we have demonstrated that a synthetic peptide (prosaptide TX14A), derived from the trophic sequence of the saposin C domain of prosaposin, stimulated cell proliferation, migration and invasion and activated the MAPK signaling pathway in prostate cancer cells. The biological significances of saposin C and prosaposin in prostate cancer are not known.Here, we report that saposin C, in a cell type-specific and dose-dependent manner, acts as a survival factor, activates the Akt-signaling pathway, down-modulates caspase-3, -7, and -9 expression and/or activity, and decreases the cleaved nuclear substrate of caspase-3 in prostate cancer cells under serum-starvation stress. In addition, prosaptide TX14A, saposin C, or prosaposin decreased the growth-inhibitory effect, caspase-3/7 activity, and apoptotic cell death induced by etoposide. We also discovered that saposin C activates the p42/44 MAP kinase pathway in a pertussis toxin-sensitive and phosphatidylinositol 3-kinase (PI3K) /Akt-dependent manner in prostate cancer cells. Our data also show that the anti-apoptotic activity of saposin C is at least partially mediated via PI3K/Akt signaling pathway.We postulate that as a mitogenic, survival, and anti-apoptotic factor for prostate cancer cells, saposin C or prosaposin may contribute to prostate carcinogenesis at its early androgen-dependent or metastatic AI state. We used 10% FBS as a positive control. Cells were lysed and 10 μg of clarified protein samples was subjected to SDS-PAGE under reducing conditions. Phospho-specific p42/44 antibody  was used as the primary antibody and as a loading control. Filters were also probed or reprobed with anti-p42/44 antibody. Additional tissue culture plates that had been treated with or without inhibitors were also tested for cell viability by trypan blue dye-exclusion assay.Cells were cultured up to 70% confluency in their complete media and after washing with PBS, they were serum-starved for 24 h, and then treated with 10% FBS or saposin C at 0.1, 1 or 10 nM for 10 min. A representative tissue culture plate was also pretreated with the PI3K-inhibitor  before treating cells with saposin C (at 1 nM for LNCaP and at 10 nM for PC-3 and DU-145 cells). After preparation of the cell lysate, 15 μg of protein per sample was subjected to SDS-PAGE under reducing conditions. Immunoblotting was performed using phospho-specific Akt antibodies against serine 473 or threonine 308 . A loading control was evaluated with anti-Akt antibody. Each experiment was performed in duplicate, and the assays were repeated three times.3VO4, and 10 mM MgC12 supplemented with 200 μM ATP and 1 μg glycogen synthase kinase-3  of fusion protein (GSK-3α/β) and incubated 30 minutes at 30°C, allowing immunoprecipitated Akt (if activated) to phosphorylate GSK-3. The kinase reaction was terminated by adding 20 μl of 3 × SDS sample buffer. Phosphorylated GSK-3 was then detected by western analysis using phospho-GSK-3α/β (for Ser 21 of GSK-3α and ser 9 of GSK-3β) antibody. The above in vitro kinase assay is based on the fact that phosphorylated-Akt (active) regulates GSK-3α/β kinase activity via phosphorylation at ser 21/9. For control loading, 10 μg protein per sample from the same whole cell lysates were subjected to western analysis using monoclonal anti-Akt antibody or actin. Each experiment was performed in duplicate, and the assays were repeated three times.A non-radioactive Akt kinase assay kit  was used to determine whether saposin C treatment of cells under serum-starvation stress would lead to Akt-activation. For Akt-kinase assays, cells were grown up to 70% confluency in their maintenance media, serum-starved for 24 h, and then treated for 5 or 10 min in the presence or absence of saposin C at 0.1, 1, or 10 nM. Cells were washed once with ice-cold PBS and harvested under nondenaturing conditions using 1X ice-cold cell lysis buffer (from the Kit) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 5 (or 10) minutes. Akt was selectively immunoprecipitated from 250 μg protein (whole cell lysates) using 20 μl of immobilized Akt 1G1 monoclonal antibody, and then incubated with gentle rotation for 4 h at 4°C. Samples were then centrifuged briefly  and pellets were washed twice with 1X lysis buffer and once with 1X kinase buffer. Immunocomplexes (pellets) were resuspended in 40 μl 1X kinase buffer [composed of 25 mM Tris (pH 7.5), 5 mM β-glycerolphosphate, 2 mM DTT, 0.1 mM NaCells were cultured up to 60% confluency in their complete culture media. After washing with PBS, they were incubated with their respective serum-free media in the presence or absence of saposin C at 0.1, 1, or 10 nM for 48 h; whole cell lysates were prepared as described above. Clarified protein samples (75 μg) were subjected to SDS-PAGE under reducing conditions. Western analyses were carried out using monoclonal antibodies against non-cleaved and cleaved caspases-3, -7, and -9 and poly (ADP-ribose) polymerase (PARP) provided in an Apoptosis Sampler Kit . Each experiment was performed in duplicate, and the assays were repeated three times.We next examined the effect of an apoptogenic agent, etoposide, on cell growth and caspase activity in the presence or absence of saposin C, prosaposin, prosaptide TX14A, or an analogous inactive mutant peptide . Cells were seeded at 1,000 per well in 96-well plates in their complete culture medium for 3 to 4 days. After this period, cells were treated in complete culture media for 3 days with etoposide  to find the lowest concentration that led to the highest growth inhibition, as measured by MTS assay (described above). Using the optimal cell type-specific etoposide concentration (20 μM for PC-3 and LNCaP and 2 μM for DU-145), cells were treated with the vehicle (DMSO), saposin C , TX14A (10 nM), mutant peptide , or prosaposin (at 1 ng/ml). Using the cell type-specific OD/cell number calibration curve as obtained by MTS assay , cell number per well was determined for the above treatment conditions.Parallel tissue culture plates were also used to determine caspase-3/7 activity using the Apo-ONE™ Homogeneous Caspase-3/7 assay . This assay provides a homogeneous Caspase-3/7 reagent  which performs a dual function, by rapidly and efficiently permeabilizing the cultured cells and at the same time exposing the intracellular space to the profluorescent caspase-3/7 substrate, rhodamine 110 (Z-DEVD-R110). After cleavage and removal of the DEVD peptides by caspase-3/7 activity, the fluorescence in each well was quantitated at an excitation wavelength of 485 + 20 nM and an emission wavelength of 535 + 25 nM and after correction based on blank control  or the homogeneous caspase-3/7 reagent. Final fluorescent intensity was depicted as endpoint relative fluorescent unit, RFLU.Using similar experimental conditions but as an independent study, the effect of LY294002 (a PI3-kinase inhibitor) was also evaluated on growth and caspase-3/7 activity of cells treated with saposin C ± etoposide. After initial studies to find the optimal concentration, we used a non-toxic tolerable dosage of 1.5 μM for LY294002. In addition, we chose the most effective  concentration of etoposide (20 μM for PC-3 and LNCaP and 2 μM for DU-145) and saposin C (1.0 nM for PC-3 and LNCaP and 10 nM for DU-145) for this study.In situ determination of apoptosis by Terminal dUTP nick-end labeling (TUNEL) was performed using an ApopTag Peroxidase In Situ kit as recommended by the manufacturer . The ApopTag Kit detects single- and double-stranded DNA breaks associated with apoptosis. Drug-induced DNA damage is not identified by the TUNEL assay unless it is coupled with the apoptotic response. Briefly, at the end of the incubation period, cells were fixed in 1% paraformaldehyde in PBS, pH 7.4 for 10 min at room temperature, washed with PBS-twice, and permeabilized in pre-cooled ethanol: acetic acid (2:1) for 5 min at -20°C. After washing twice in PBS, 5 min each time, endogenous peroxidase activity in the cells was quenched in 3% H2O2 in PBS for 5 min at room temperature, incubated with terminal deoxynucleotidyl transferase (TdT enzyme) and then with peroxidase-conjugated anti-digoxigenin antibody. Nuclear staining of the apoptotic cells was detected by 3',3'-diaminobenzidine tetrahydrochloride dihydrate substrate, as recommended by the manufacturer. Cells were then counterstained in 0.5% (w/v) methyl green and slides were mounted under a glass coverslip in permount mounting medium.Cells were cultured in multiwell chamber slides and treated with etoposide in the presence or absence of saposin C at 0.1, 1, or 10 nM as indicated above. For control staining, the enzyme incubation step was deleted. Microscopic examination of cells was carried out using a phase contrast microscope. Cells were counted by choosing ten random fields and the percentages of apoptotic cells were determined. Apoptosis was indicated by the presence of apoptotic bodies, exhibiting brightly labeled punctuated nuclei.p < 0.05 or 0.01. Statistical analyses were performed using GraphPad Prism version 3.00 for Windows .For cell survival and other quantitative data, a one-way analysis of variance (ANOVA) was employed to evaluate the influence of one variable on multiple independent groups. Bonferroni's corrections were also applied whenever a significant group effect was observed. To compare a control group with a single experimental group of interest, we used the Student's t-test. For cell survival studies, each treatment concentration was examined three times and in triplicate samples. The effect of saposin C or other effectors in the presence of etoposide on cell growth, apoptosis, or caspase-3/7 activity was studied in twelve replicates and repeated three times. Statistical significance was set at Author 1 (T-JL) carried out experiments described in figure


However, the α7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.Expression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1β, nitric oxide, interleukin-10 and prostaglandin E2.Rat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by α7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.Since prostaglandin E The inflammatory response is in the first instance a mechanism of self-defense, set by the innate immune system against endogenous and exogenous insults, and essential for the survival of the organism. Inflammation must be tightly regulated as deficiency as well as excess in its response will result in pathological conditions, such as immunodeficiency or chronic inflammatory diseases . In the Among the endogenous mechanisms that regulate the inflammatory response, cross-talk between the immune and nervous systems play an important role. In particular, it has been shown that electric stimulation of the vagus nerve attenuates the inflammation during endotoxemia in rats , and thaA, glycine and 5-HT3 receptors [Neuronal acetylcholine receptors (nAChRs) are ligand-gated ion channels, which belong to a large family of neurotransmitter receptors that includes the GABAeceptors . Each nAeceptors . At leaseceptors .2A-oligodendrocyte precursor cells but not in adult differentiated oligodendrocytes, suggesting that receptor expression is developmentally regulated [In addition to neurons and peripheral macrophages, several studies have demonstrated the expression of nAChRs in cell types both within and outside the nervous system . In the egulated . Cultureegulated  and cortegulated . A functegulated . In periegulated ,14. Acutegulated . Furtheregulated . Lymphocegulated . These o2 synthesis. Other important microglial products, such as interleukin-1β (IL-1β), nitric oxide (NO) and interleukin-10 (IL-10) were not affected or moderately decreased.Epidemiological studies indicate that nicotine, besides its immunosuppressive effects, may be protective against the development of neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson's disease (PD) , in whicAll cell culture reagents were from Gibco  and virtually endotoxin free (less then 10 E.U./ml as determined by the manufacturer). BCA protein assay was from Pierce . ELISA-kits for rat TNF-α and IL-10 were from Endogen Inc. . ED-1 monoclonal antibody was from Serotec . (±) Nicotine, α-bungarotoxin, FITC-α-bungarotoxin and lipopolysaccharide LPS  were from Sigma Chemical . Rabbit polyclonal antibody against alpha 7 subunit was from Santa Cruz Biotechnology.5 cells/cm2. Cells were allowed to adhere for 20 min and then washed to remove non-adhering cells. After a 24 h of incubation, the medium was replaced with fresh medium containing the substance(s) under study. Cell viability was greater than 95%, as tested by Trypan Blue exclusion. Immunostaining, performed as previously described [Microglial cultures were prepared from 10–14 day mixed primary glial cultures obtained from the cerebral cortex of 1-day-old rats, as previously described  and in a2 content was quantified using a specific radioimmunoassay [2 with other prostaglandins less than 0.25%.At the end of the incubation time, cell supernatants were collected, centrifuged, and stored at -70°C until tested. The levels of TNF-α and IL-10 were assayed by specific ELISAs, following the manufacturer's instructions. The ranges of determination were: 31–2500 pg/ml for TNF-α, 10–1000 pg/ml and 8–500 pg/ml for IL-10. The production of NO by measuring the content of nitrite, one of the end products of NO oxidation, as previously described . PGE2 counoassay . The ass5 cells/cm2), allowed to adhere for 20 min and then washed to remove non-adhering cells. After a 24 h of incubation, the complete BME medium was replaced with fresh BME medium without serum. Cells were incubated at 4°C for 15 min with FITC-labeled α-bungarotoxin at 1.5 μg/ml. Where indicated, nicotine was added at the concentration of 500 μM for 10 min, in order to saturate all the binding sites before the addition of FITC-labeled α-bungarotoxin. Cells were washed 3 times with BME medium and then fixed with 4% paraformaldehyde at room temperature for 15 min. After fixation, coverslips were washed twice with PBS solution, mounted in PBS:glycerol and examined using a fluorescent microscope. Cell culture lysates from microglial cells and PC12 cells (used as positive control) were analyzed for α7 subunit expression. Total protein content was estimated using the Bio-Rad protein assay. An aliquot corresponding to 50 μg (microglia cells) and 20 μg (PC12 cells) of total protein for each sample was separated by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and transferred electrophoretically to nylon membranes. Membranes were blocked with 10% non-fat milk and incubated with a rabbit policlonal antibodies against α7 subunit (1:2000) overnight at 4°C. Horseradish peroxidase conjugated anti-rabbit IgG  and ECL reagents were used as detection system.Microglial cells were plated on uncoated glass coverslips  in a total reaction volume of 20 μl following the conditions provided by the manufacturer's protocol.Oligonucleotide primers with similar Tm were designed to generate a PCR fragment of 754 bp for the α7 subunit. PCR conditions (number of cycles and cDNA and primer concentration) that ensure the data to be obtained within the exponential phase of amplification of each template were carefully assessed. The amplification of the β-actin, COX-2 and α7 subunit within the exponential phase of amplification was achieved with 25, 30 and 40 cycles respectively.4 (2 mM), 1 Unit of Platinium Taq DNA polymerase High Fidelity (Invitrogen). The primers were: α7 subunit (Gene bank accession number S53987), sense 5'-TCT GTG CCC TTG ATA GCAC, antisense 5'-CTT CAT GCA ACC AGG ATC AG, product length 754; COX-2 [NM031144) sense 5'-GTC GAC AAC GGC TCC GGC ATG; antisense 5'-CTC TTG CTC TGG GCC TCG TCGC, product length 158 bp. A sample containing all reaction reagents except cDNA was used as PCR negative control in each experiment. The absence of genomic DNA was verified using 2 μg of RNA from microglia that was reverse-transcribed without the enzyme (-RT). The PCR conditions for COX-2 were as follows: initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C for 30 sec, 58°C for 45 sec, 68°C for 1 min, and an additional cycle with extension at 72°C for 7 min. The PCR conditions for β-actin were as follows: initial denaturation at 94°C for 5 min followed by 25 cycles of 94°C for 30 sec, 68°C for 30 sec, 68°C for 45 sec and an additional cycle with extension at 72°C for 1 min. The PCR conditions for α7 subunit were as follows: initial denaturation at 94°C for 5 min followed by 40 cycles of 94°C for 30 sec, 57°C for 1 min, 68°C for 45 sec and an additional cycle with extension at 72°C for 7 min.Five μl, 15 μl and 40 μl of diluted cDNAs were amplified for β-actin, COX-2 and α7 respectively. PCR-amplification was done in a final volume of 50 μl containing 1x PCR buffer, the four dNTPs (0.2 mM), MgSO4; COX-2 , sense 5PCR products were analyzed by electrophoresis, stained with ethidium bromide and photographed. Transcript levels were analyzed by Fluor-STM Multimager analyser (Biorad). For each experiment, the ratio between optical density (arbitrary units) of bands corresponding to COX-2 and β-actin  was calculated to quantify the level of the transcripts for COX-2 mRNAs.t-test. A two-tailed probability of less than 5 % (i.e. p < 0.05) was taken as statistically significant.Data are expressed as mean ± SEM with the number of independent experiments, run in duplicate, indicated in figure legends. Comparison between treatment groups was made by Student's The expression of the mRNA for α7 subunit in rat microglial cells was investigated by RT-PCR. As shown in Figure Once we had demonstrated the presence of α7 subunit mRNA and protein in microglial cells, we studied the functional consequences of receptor activation using the specific agonist nicotine. Microglial cells were pre-treated for 30 min with increasing concentrations of nicotine and then incubated for 4 or 24 h in the absence or the presence of 10 ng/ml LPS. In resting microglial cultures nicotine did not affect the basal level TNF-α(data not shown).As previously demonstrated using mouse microglial cultures, nicotine pre-treatments dose-dependently inhibited the release of TNF-αFig. . At 1 μMTo verify that the effect of nicotine was mediated by α7 subunit, we measured the level of TNF-α in activated microglial cells exposed to nicotine in the presence or in the absence of α-Bgtx. The addition of 0.01 μM α-Bgtx almost totally prevented the inhibitory effect of nicotine Fig. .In addition to TNF-α, we also analyzed the release of two important microglial mediators such as NO and IL-1β and we found that nicotine pre-treatment only slightly reduced the release of NO  and did not modify the release of IL-1β (data not shown).2 (PGE2), two important local mediators with anti-inflammatory and immunoregulatory functions. Nicotine pre-treatment only moderately reduced  the level of IL-10 in the culture media of microglia cells stimulated for 24 h with LPS (data not shown).We then analyzed the effects of nicotine on the production of interleukin-10 (IL-10) and prostaglandin E2 in LPS-activated microglial cells. The presence of 0.01 μM α-Bgtx, blocked the nicotine-dependent increase of PGE2 released by LPS-activated microglia  positive fibers around β-amyloid plaques in transgenic APPSW mice suggests a close connection between cholinergic terminals and microglial cells [PGE in vivo ,37. We h and NGF ,28. In ticroglia , consisticroglia . It has g tissue . It is tal cells . A deficIn the last few years, several lines of evidence have suggested that activation of α7 subunits plays an important role in the maintenance of cognitive functions in several neurodegenerative disorders . EpidemiAs mentioned in the introduction, the presence of α7 subunit on immune cells as well as on other non-excitable cells has provided a molecular basis for a non-neuronal cholinergic pathway that might function as an essential regulator of inflammation as well as immune responses ,5. PrimaIt should be noted that although our study focused on the effects of nicotine on the process of microglial activation induced by LPS, our findings may have broader implications since other microglial activators, such as pro-inflammatory cytokines and fibrillogenic peptides, share some common signaling pathways with LPS ,53. In a2 up-regulation is under investigation. Shytle et al. [2 synthetic pathway [At present, the signaling pathways downstream to α7 subunit activation and leading, in particular, to COX-2 and PGEe et al.  have rep pathway ,56, thusActivation of α7 nicotinic receptors in microglial cells by nicotine controls some important microglial functions, thus preventing chronic inflammation. Since microglial activation and chronic inflammation have been associated with most neurodegenerative pathologies  the undeLipopolysaccharide (LPS)Acetylcholine (ACh)Neuronal acetylcholine receptors (nAChRs)Tumor necrosis factor-α (TNF-α)2 (PGE2)Prostaglandin EInterleukin-1β (IL-1β)Nitric oxide (NO)Interleukin-10 (IL-10)The author(s) declare that they have no competing interests.RDS conceived of the study, participated in its design and coordination, produced the primary microglial cultures, performed the ELISA and the immunofluorescence staining, was primarily responsible of the RT-PCR review the data and drafted the manuscript. MAAC participated in the design and coordination of the study, produced the primary microglial cultures, performed the ELISA and the immunofluorescence staining, was primarily responsible for western blot analysis and review the data. DC participated in the production of the primary microglial cultures and in RT-PCR. LM contributed to the design of the study, guided data interpretation and presentation and assisted in the preparation of the manuscript.


Alterations in carbohydrate metabolism are frequently observed in cirrhosis. We conducted this study to define the prevalence of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in Iranian patients with chronic liver disease (CLD), and explore the factors associated with DM in these patients.One hundred and eighty-five patients with CLD were enrolled into the study. Fasting plasma glucose and two-hour plasma glucose were measured in patients' sera. DM and IGT were diagnosed according to the latest American Diabetes Association criteria.The subjects included 42 inactive HBV carriers with a mean age of 42.2 ± 12.0 years, 102 patients with HBV or HCV chronic hepatitis with a mean age of 41.2 ± 10.9 years, and 41 cirrhotic patients with a mean age of 52.1 ± 11.4 years. DM and IGT were diagnosed in 40 (21.6%) and 21 (11.4%) patients, respectively. Univariate analysis showed that age (P = 0.000), CLD status (P = 0.000), history of hypertension (P = 0.007), family history of DM (P = 0.000), and body mass index (BMI) (P = 0.009) were associated with DM. Using Multivariate analysis, age , family history of DM , chronic hepatitis , and cirrhosis  remained as the factors independently associated with DM. When patients with cirrhosis and chronic hepatitis were analyzed separately, higher Child-Pugh's score in cirrhotic patients  and older age , higher fibrosis score , and higher BMI  in patients with chronic hepatitis were found to be associated with higher prevalence of DM.Our findings indicate that patients with cirrhosis and chronic hepatitis are at the increased risk of DM occurrence. Older age, severe liver disease, and obesity were associated with DM in these patients. Alterations in carbohydrate metabolism are frequently observed in liver cirrhosis. Glucose metabolism impairment in cirrhotic patients, both in fasting state and in response to oral glucose or meals has been widely documented in the literature -5. ThereIn this study, for the first time in Iran, we reported the prevalence of DM and IGT in patients with chronic liver disease (CLD) using the latest generally accepted criteria, and investigated independent correlation of CLD with DM, considering other known possible DM risk factors. Moreover, we explored the factors that might be potentially associated with DM in patients with cirrhosis and chronic hepatitis.From October 2002 to March 2003, all consecutive patients with chronic liver disease referred to "Tehran Hepatitis Center" were enrolled into the study. Tehran Hepatitis Center is a referral specialized clinic for liver diseases where many patients suffering from liver diseases and hepatitis from around Iran are referred to in order to receive consultation and clinical medical care. Pregnant females, patients under 20 years old, those who were on regular corticosteroid or hydrochlorothiazide therapy, known or suspected cases of hemochromatosis, or autoimmune disease were excluded. Having a clinical, biochemical (serum amylase and/or lipase elevation) or ultrasonographic evidence for chronic pancreatitis was also considered an exclusion criterion. No patient had a history of habitual drinking. All of the patients were Moslem and belonged to the white race.5 copies/ml (ROCHE Cobas Amplicore) and normal liver enzymes. The mean age in this group was 42.2 ± 12.0 years ranging between 23–84 years. Eight patients with nonalcoholic steatohepatitis (NASH) were initially enrolled but were not entered in analysis because of the low case number. Therefore analysis was finally performed for 185 patients.A total number of 193 patients who met the criteria were eligible to enter the study. First group included 41 cirrhotic patients. Mean age was 52.1 ± 11.4 years ranging between 26–74 years. Diagnosis of cirrhosis was confirmed by histology in 17 (41.5%) patients. The occurrence of signs or biochemical evidences of liver decompensation, ultrasound features of portal hypertension and/or esophageal varices in gastroscopy were used for the clinical diagnosis in the remaining cases. No cirrhotic patients had evidence of hepatocellular carcinoma, screened by serum alpha-fetoprotein level test and abdominal ultrasonography. Severity of liver cirrhosis was graded according to Child-Pugh's classification. Second group included 102 patients with chronic hepatitis B, or C. Mean age was 41.2 ± 10.9 years ranging between 22–67 years. All patients had adequate documentation of elevated serum aminotransferases for more than 6 months, and viral hepatitis molecular assays by qualitative RT-PCR. In 91 (89.2%) patients liver biopsy reports were accessible. The histological staging and grading in liver biopsy was scored according to Knodell scoring. Third group included 42 inactive hepatitis B virus (HBV) carriers who were hepatitis B s antigen (HBsAg) positive, quantitative HBV DNA level less than 10Informed consent in writing was obtained from each patient involved and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as approved by institutional review board. Initial data were collected by chart review and interview. Then, two venous blood samples were taken: First sample in the morning after a 12-hour overnight fast to measure fasting plasma glucose (FPG) level, triglycerides (TG) level, and cholesterol (Chol) level; and a second sample following 2 hours after eating 75 gr glucose to measure two-hour post-loaded glucose (2-hr PG). FPG and 2-hr PG measurement were repeated on two other samples in second session.Patients receiving therapy with insulin or oral hypoglycemic medications were considered as diabetics. In other patients, FPG ≥ 126 mg/dl or 2-hr PG ≥ 200 mg/dl on more than one occasion was used as diagnostic for DM in agreement with the latest American Diabetes Association (ADA) criteria . FPG bet2).Body mass index (BMI) was calculated using the standard formula of weight in kilogram divided by square of height in meters (kg/mResults were expressed as mean ± standard deviation (SD). Comparison between diabetic and non-diabetic groups was made using Students T test for continuous variables and the Chi-square or Fisher exact test for categorical variables. At univariate analysis the factors possibly associated with DM development were evaluated. A multivariate analysis based on a stepwise logistic regression model was used to assess the independent effect of all variables found significant at the univariate analysis. P-value less than 0.05 was considered significant. All statistical analyses were performed using SPSS for Windows software .The sample population contained 150 (81.1%) males and 35 (18.9%) females. The overall mean age was 43.8 ± 12.0 years ranging between 22 and 84 years. In the total of 185 patients, DM was found in 40 (21.6%) patients, and IFG and/or IGT was found in 21 (11.3%) patients. Thirty patients were already aware of their problem whereas the diagnosis of DM was new in 10 patients.By univariate analysis a number of variables including the factors mentioned by ADA as risk factors for type II diabetes  in additAt multivariate analysis age , family history of DM , chronic hepatitis , and cirrhosis  showed up as the only factors independently associated with DM prevalence rate  patients were diabetic and 7 (17.1%) patients had IFG and/or IGT. Univariate analysis showed that only two factors were associated with DM rate. Mean of age in diabetic cases was significantly higher than that of non-diabetic ones (P = 0.003). Moreover, DM was significantly more prevalent in patients with Child-Pugh's score B than score A (P = 0.04). Since there was no score C in study sample, we could not evaluate this class of cirrhosis. After applying logistic model, only Child-Pugh's score kept its significance  as an independent predictive factor for DM in cirrhotic patients  patients and IFG and/or IGT was found in 11 (10.8%) patients. There was no significant difference between diabetic and non-diabetic cases in regard with sex, etiology of chronic hepatitis, hypertension, interferon therapy, smoking, and serum TG and Chol levels. On the other hand, diabetic patients had higher mean age compared with non-diabetic cases (P = 0.001). More cases of diabetic patients compared with non-diabetic ones had a family history of DM (P = 0.01). The mean BMI of patients with DM was higher than that of patients without DM (P = 0.02). Furthermore, liver biopsy showed significantly more fibrosis activity in diabetic patients compared with non-diabetic cases (P = 0.003), whereas no difference in diabetic vs. non-diabetic patients was seen with respect to histological grading. Multivariate analysis revealed that older age , higher BMI , and more severe fibrosis activity  were the predictive variables for DM in patients with chronic hepatitis  declare that they have no competing interests.Dr. Alavian participated in the design of the study and coordination and drafted the manuscript. Dr. Hajarizadeh conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. Dr. Nematizadeh designed the questionnaire and helped in data collection. Dr. Larijani facilitated the study progress and participated in coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. Furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes.mBmal1, mNpas2, mRev-erbα, mDbp, mRev-erbβ, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak) showed robust circadian expressions of mRNAs in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. The bioinformatics analysis revealed that these genes have one or a combination of 3 transcriptional elements , which are conserved among human, mouse, and rat genome sequences, and indicated that these 3 elements may be responsible for the biological timing of expression of canonical clock genes.We systematically analyzed the mRNA expression of clock and clock-controlled genes in mouse peripheral tissues. Eight genes (The observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. Our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including 3 known elements, E-box, RORE, and DBPE. HS carried out the in silico study. MA carried out the real-time luciferase reporter assay. TM participated in the coordination, and provided financial support. TT conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


Possessing two parkin mutations leads to early-onset parkinsonism, while having one mutation may predispose to late-onset disease. This dosage pattern suggests that some parkin families should exhibit intergenerational variation in age at onset resembling anticipation. A subset of familial PD exhibits anticipation, the cause of which is unknown. The aim of this study was to determine if anticipation was due to parkin mutation dosage.parkin analysis included sequencing and dosage analysis of all 12 exons.We studied 19 kindreds that had early-onset parkinsonism in the offspring generation, late-onset parkinsonism in the parent generation, and ≥ 20 years of anticipation. We also studied 28 early-onset parkinsonism cases without anticipation. Patients were diagnosed by neurologists at a movement disorder clinic. parkin mutations, but contrary to our postulate, the affected relative with late-onset parkinsonism did not have a parkin mutation. In effect, none of the anticipation cases could be attributed to parkin. In contrast, 21% of early-onset parkinsonism patients without anticipation had parkin mutations.Only one of 19 cases had compound parkin, and may signify a distinct disease entity.Anticipation is not linked to  Howeverecessive -5, nor iecessive -9. Severse onset -10. Collth decade of life, whereas homozygotes show symptoms at much younger ages, sometimes at birth (OMIM *143890).Several groups have reported what appears to be genetic anticipation in parkinsonism -13. In tparkin mutation dosage? We hypothesized that some parkin mutations are dominant: heterozygotes have incomplete penetrance and may develop late-onset parkinsonism, whereas homozygotes and compound heterozygotes have accelerated disease leading to early-onset parkinsonism. This unifying hypothesis was attractive because if true, it could explain the reported variations in mode of inheritance of parkin , the range in age at onset from juvenile to late onset, and the significantly earlier onset in some of the children of affected parents. To test this hypothesis, we studied parkin in 19 kindreds with early-onset parkinsonism in the index generation, late-onset parkinsonism in the parent generation, and exhibited ≥ 20 years of anticipation.Is anticipation in parkinsonism related to Families were recruited from a movement disorder clinic. The probands and affected family members had the clinical diagnosis of idiopathic Parkinson's disease (here referred to as parkinsonism due to lack of autopsy confirmation). Diagnosis was made by a neurologist according to the British Parkinson's Disease Brain Bank criteria except that family history was not an exclusion criterion . These fparkin exon amplifications were multiplexed under standard conditions with an 84-bp fragment of a single-copy human β-actin gene (Genbank accession number XM_004814) as an internal control. A standard curve was generated for each parkin exon and for β-actin using 0, 5, 15, 55 and 220 ng of control human genomic DNA. The number of PCR cycles required before the ABI 7700 detects each parkin exon product  was plotted against the corresponding exon standard curve, thus calculating the relative parkin copy number. The copy number for each exon was normalized to the single-copy actin gene within each multiplexed reaction and to a normal control reference individual, allowing an estimate of the number of copies of parkin. Optimal threshold levels for each primer set were maintained between plate analyses. All samples were analyzed in triplicate.DNA was extracted from blood using standard protocol. Genotyping was blind to phenotype. To identify point mutations, we sequenced both DNA strands of all 12 exons. Exons and 50–100 bp of flanking intronic sequences were PCR-amplified ,18. parkparkin analysis based on having parkinsonism in two consecutive generations, late-onset parkinsonism in the parent generation , early-onset parkinsonism in the offspring generation , and ≥ 20 years of anticipation as measured by the difference in mean ages at onset in two generations. In the younger generation, 2 probands had onset before age 20, 1 was in his twenties, and 16 were over 30 years old. It was hypothesized that the affected individuals with early-onset parkinsonism are homozygous or compound heterozygous, and the parents and other relatives with late-onset parkinsonism are heterozygous. For parkin analysis, we began with the family member from the generation with the earlier onset, so as to enrich for parkin mutations. We expected the majority of the index cases to be compound heterozygous. At the minimum, 16–49% of index cases should have had mutations, since this is the range reported for early-onset sporadic and familial parkinsonism [parkin mutations. This patient had compound deletions in exon 3 with onset at age 8. Her sister, onset at age 15, also had the compound mutation. The relative with late-onset was an uncle with onset age of 64. Contrary to our hypothesis, the uncle did not have a parkin mutation. The cause of late-onset parkinsonism in the uncle was different from the nieces. In effect, we did not find any cases where we could attribute the intergenerational difference in age at onset to parkin dosage.Nineteen kindreds were chosen for insonism . Howeverparkin in 28 additional patients with early-onset parkinsonism , either with (n = 4) or without (n = 24) family history, but with no evidence of anticipation. In this group, 3 probands had onset at or before age 20, 5 were in their twenties, and 20 were over 30 years old. Nine mutations were found in six individuals. Two subjects were compound heterozygous (onset ages 31 and 37) and four were heterozygous . The frequency of parkin carriers in early-onset parkinsonism without anticipation was 21% which is in the range 16% – 49% reported in the literature . It was of interest to determine if any of the parents of the three early-onset parkinsonism patients with compound mutations had developed late-onset parkinsonism. All six parents were heterozygous , and all had remained free of parkinsonism to the ages of 53, 60, 63, 74, 76 and 78 yrs. These individuals may still develop parkinsonism.We also analyzed Familial parkinsonism with anticipation may be more common than classical dominant and recessive subtypes combined. In our clinic population, among 487 patients studied, 145 had a first or second degree relative with PD; that is 30% which is in line with the published figures for other referral clinics. Among the 145 familial cases, 110 had parkinsonism in consecutive generations, which is compatible with autosomal dominant inheritance, and 35 had an affected sibling or cousin, which is suggestive of recessive inheritance. However, among the 110, only 26 were compatible with a classical dominant pattern ; while 63 exhibited 10–68 years of anticipation, and 7 had 10–17 years of reverse anticipation (14/110 had unknown onset ages). Our clinic is a referral center, which explains the relatively high proportions of early-onset and familial cases. The interesting finding was the relative proportions of autosomal dominant, autosomal recessive and anticipation cases within the familial subtype. Despite its relatively high prevalence, at least in our clinic, familial parkinsonism with anticipation has been largely overlooked in genetic research. A few parkinsonism families have been attributed to expansions in known SCA loci, but in the majority of the kindreds, the cause remains unknown. The more obvious possibilities are mitochondrial inheritance, modifier genes, parent-child exposure to environmental triggers, as yet unidentified triplet repeats or dominant genes with dosage effect, and in some cases, artifactual appearance of anticipation due to ascertainment bias. None of these are mutually exclusive; more than one may be true and operative in different families.parkin literature suggest that possessing two parkin mutations is fully penetrant and leads to early-onset parkinsonism, whereas having only one mutation may be incompletely penetrant and lead to later disease onset. While the causative link to early-onset parkinsonism is widely accepted, the association of parkin with late-onset parkinsonism remains controversial [parkin heterozygotes are at risk for late-onset disease, then some parkin families should exhibit intergenerational variation in age at onset resembling anticipation, where heterozygous parents develop late-onset parkinsonism and children who inherit two mutations develop early-onset parkinsonism. We hoped to explain the appearance of anticipation in relation to parkin dosage, but the findings do not support this postulate. While the results rule out a link between parkin and anticipation in these families, they do not negate the association of parkin with late-onset parkinsonism.The current oversial ,10,20,21parkin mutation dosage. The underlying mechanism for anticipation may be genetic or environmental. Identification and a-priori classification of pedigrees that exhibit significant intergenerational age at onset variation, as being distinct from families that display classical dominant pattern, may facilitate gene mapping studies by reducing heterogeneity. Anticipation in parkinsonism merits investigation in its own right, not only because it is a common phenomenon and may account for a large subset of familial parkinsonism, but it may also uncover a novel mechanism in parkinsonism.The phenomenon of anticipation is not due to The author(s) declare that they have no competing interests.PP carried out the molecular genetic studies. LM interviewed the subjects and gathered family histories and medical records. JN performed the neurological examinations. GS participated in study design and supervised molecular genetic studies. JM performed molecular analysis of one large pedigree. HP conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


This study reports on clinical data from an 8-week open-label study of 20 HIV-seropositive individuals, diagnosed with Major Depressive Disorder (DSM-IV), who were treated with SAM-e (S-Adenosylmethionine). SAM-e may be a treatment alternative for the management of depression in a population reluctant to add another "pill" or another set of related side effects to an already complex highly active antiretroviral therapy (HAART) regimen.The Hamilton Rating Scale for Depression (HAM-D) and the Beck Depression Inventory (BDI) were used to assess depressive symptomatology from 1,2,4,6 and 8 weeks after initiation of treatment with SAM-e.Data show a significant acute reduction in depressive symptomatology, as measured by both the HAM-D and the BDI instruments.p < .001), with progressive decreases in depression symptom rating scores throughout the 8 week study.SAM-e has a rapid effect evident as soon as week 1 ( The incidence of depression in people living with HIV (PLWH) has consistently been reported to be higher than the 12–15% rates reported for the general population .In New York City, 27% of all people living with HIV are over 50 years of age and 66% are over 40 years age [Under-treated depression in the HIV population may be the result of multiple factors. People of color, women, substance abusers, and those living in poverty are less likely to have access to or to accept mental health outpatient services -7. Many SAM-e is a compound that has been used for many years in Europe as an antidepressant. It became available in the USA as a non FDA-regulated compound in 1999. SAM-e resembles a naturally occurring compound in the human body, formed by methionine and adenosine triphosphate. SAM-e donates an active methyl group to several acceptor molecules, including catecholamines, fatty acids, phospholipids, proteins and nucleic acids . SAM-e aThe efficacy of SAM-e as an antidepressant has been reported in a number of open-label trials  and in tth International Conference on AIDS. A total of 20 patients were recruited between April 2000 and November 2000.This study is an analysis of the complete dataset from an independently funded, IRB-approved project conducted at ACRIA, a 12-year-old, community-based HIV research and education agency in New York City. A preliminary report, based on a subset of these data, was presented at the 14Recruitment activities included distribution of information brochures to community physicians, announcements at public ACRIA treatment education workshops, and direct calls to persons listed in the ACRIA database. Individuals who responded to recruitment materials were screened via phone by the research coordinator using the PRIME-MD . HIV-posTreatment was initiated at 200 mg of SAM-e twice a day with daily supplementation of 1,000 mcg Vitamin B12 and 800 mcg Folic Acid, which were provided by Pharmavite, LLC. Over the course of the study, the dose of SAM-e was individually adjusted up to 800 mg, twice daily. Patients reporting nausea or insomnia were not increased above 800 mg daily, while patients with no reported side effects could be increased to a total daily dose of 1600 mg. Dosing was adjusted according to the severity of symptoms and clinical treatment response. We defined clinical treatment response as an improvement in depressive symptomatology of greater than 50% reduction on patient scores on the BDI and HAM-D as our treatment endpoint ,26. A stAt each study visit , patients completed a clinical interview and a series of structured questionnaires on medical/physical symptoms, substance abuse, dementia scales, as well as two instruments designed to measure depressive symptoms, self-administered Beck Depression Inventory (BDI and the clinician-administered HAM-D).The BDI is a self-report measure for depressive symptoms . Total sThe HAM-D is a 17-item, clinician-rated instrument. It was designed to assess the changes in severity of depressive symptomatology over time in patients who had been diagnosed with Major Depressive Disorder . Scores Two separate analyses  were identified as the most critical indicators of SAM-e efficacy. The first analysis represented evidence for acute onset of SAM-e, and the second indicated effectiveness of SAM-e over time. BDI and HAM-D scores were centered and the resulting Z-scores were used in a series of t-tests to assess the agreement between patient and investigator ratings of depression. Mean scores from both depression measures (BDI and HAM-D) were compared between time points, using a series of paired t-tests. Finally, an intent-to-treat analysis, using the last score carried forward, was conducted on patient BDI scores for all subjects who initiated treatment.Thirty (30) subjects were screened by telephone and twenty (20) entered into the study. Of these, fifteen 15) were male, five (5) were female and the majority were people of color . Table 5 were maM = 33.5, SD = 11.1; Range: 15 – 55) to Week 1  was significant, t = 5.15, p < .001. Mean HAM-D scores declined in a parallel manner, from Baseline  to Week 1 . This change was also statistically significant, t = 3.58, p < .01. Both depression assessment instruments reached the defined clinical treatment response threshold of a greater than 50 percent reduction in depression symptom scores.Table M = 35.1, SD = 12.0; Range: 15 – 55) to Week 8 , t = 8.28, p < .001. Similarly, HAM-D scores were reduced from Baseline  to Week 8 . The reduction in HAM-D scores reached statistical significance, t = 9.92, p < .001.The 15 patients who completed the study also demonstrated clinical treatment response over time. Mean BDI scores dropped significantly from Baseline  were higher than Week 8 . HAM-D scores dropped from Baseline  to Week 8 . The reduction in self-reported  depressive symptomatology was significant, t = 8.85, p < .001. The results were equivalent for the psychiatrist-rated HAM-D, t = 7.23, p < .001.The intent-to-treat analysis yielded similar results. The last reported BDI and HAM-D scores for each of the 20 patients who initiated treatment  were used in this analysis. Baseline BDI scores (The intent-to-treat remission rate was 79%. Fifteen (15) of the 20 patients achieved a HAM-D of 7 or lower, while one patient did not receive a valid HAM-D rating and four (4) received HAM-D ratings greater than 7 . The intent-to-treat response rate was 74%, with 14 of the 19 patients achieving 50% or greater reduction in HAM-D scores.No patients terminated their participation due to side effects. Two (2) patients reported nausea and one (1) reported diarrhea; all resolved spontaneously. One (1) patient had insomnia and a subjective feeling of 'high energy,' but did not meet criteria for hypomania.Recent data from the HIV Cost and Services Utilization Study (HCSUS) indicate that almost half the nation's adult HIV patients suffered from symptoms of mental disorders. The report indicates that the prevalence of mental disorders range from four to eight times higher than those found in the general populations. Over 60 percent of HIV-positive adults used mental health or substance abuse services during the period studied; 26% visited mental health specialists, 15% had group mental health treatment, 40% discussed emotional problems with medical practitioners and 30% used psychotherapeutic medications .Depression remains under-reported and under-treated in the HIV population. Patient factors include stigma, fear of more medication, and perhaps the assumption that depression is a normal part of HIV disease, or due to HAART. Physicians tend to focus more on physical symptoms, and may be unaware of how to screen for depression in this population. Both physicians and patients are disadvantaged in accessing gay-affirmative, well-trained HIV mental health specialists who can disentangle HIV medical problems from substance abuse and mental illness. Risk factors for depression  are prevalent in those populations who are disproportionately affected by HIV. Many patients are reluctant to add more medication to their complex treatment regimens. The need for effective and safe treatments for depression is clear.Results of this study demonstrate that SAM-e significantly reduces depression in people living with HIV. This finding supports previous research demonstrating the efficacy of SAM-e for use in treating depression. More importantly, the therapeutic effect of SAM-e had an acute onset. There were significant reductions in the severity of depression among study patients within the first week of treatment. Unlike most antidepressants that require several weeks to reach maximum therapeutic efficacy, previous research has shown that serum levels of SAM-e peak within 24 hours of treatment. This may account for the rapid, effects of SAM-e seen on the HAM-D and BDI scores.SAM-e is a safe, effective treatment for depression among people living with HIV. SAMe has few reported side effects, and in our population of PLWH, was extremely well tolerated. SAM-e in this group had rapid onset of clinical efficacy. Both of these results make it an appealing alternative therapy for the management of depression in the HIV population.The results of the current study must be interpreted in the context of the study's limitations. Limitations of this study include the lack of a placebo group, the lack of a double-blind design and small sample size. The criteria for participation and the small sample size may limit the generalizability of our findings to other patient populations. Without a placebo control group, we cannot assess two factors that might have affected our results. Although patients were treatment-experienced with other anti-depressants, we cannot rule out the potential placebo-response that may have contributed to the rapid therapeutic effect of SAM-e. Also, meeting with a study psychiatrist may have contributed to improved depressive symptom ratings. Nonetheless, the study results provide useful and clinically relevant information for treating depression in HIV-positive individuals. Future studies employing a double-blind, placebo-control design are warranted.The absence of depression does not imply wellness. Many people living with HIV/AIDS report feelings of sadness and depression but do not receive treatment for depression. Future studies should also assess health-related quality of life. Such protocols would determine if people with significant, but subclinical depressive symptomatology would benefit from SAM-e.RASrecoded study data, conducted statisticalanalyses andco-wrote the final manuscript. DM provided details of the study design and procedures andwas responsible for extractingclinical data from patient records. IC and KJ served as clinical investigators and provided valuable comments on the final paper. SEKwrote the first draft of the paper and co-wrote the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Faced with new and ongoing threats to public health, researchers are becoming increasingly resourceful in their quest to discover new drugs. Drug researchers have long looked to living organisms for inspiration, either mimicking or extracting chemical formulas from naturally occurring compounds. Bacteria and fungi, for example, produce a wide range of compounds—some of which give them a selective advantage in their own environments—that provide important pharmaceutical activities. One class of these natural compounds are the polyketides, which make up a large portion of the antibiotics (including erythromycin and tetracycline) and antitumor drugs (such as doxorubicin and epothilone) that have been isolated from various microorganisms.Polyketides are synthesized by bacteria and fungi by the appropriately named polyketide synthases (PKSs). PKSs can be thought of as large molecular factories containing a series of enzymes working on an assembly line: each enzyme in the line adds molecules to a primer, or starter, unit—which is usually an acetate molecule—and then hands off the growing chain to the next enzyme. The specific enzymes set all the characteristics of the polyketide, including the chain length, the building blocks used, and the branching pattern of the molecules. Although microorganisms generate polyketides with a variety of characteristics, one goal of drug discovery research is to increase this diversity even further—a larger pool of polyketides promises more drugs with enhanced pharmaceutical applications.Early attempts at creating artificial polyketides focused on altering the functional characteristics of naturally occurring polyketides—the length of the chain, the building blocks, and the patterns of the branches. Chaitan Khosla and colleagues have taken this approach one very large step further. Rather than changing the machinery to modify the growing structure of a polyketide, they engineered bacteria to use an alternative, nonacetate primer molecule. This has important practical implications because some medicinally significant compounds do not use the usual acetate primer unit. By dissecting out the specificities of the “starter” and longer, multiunit “elongation” PKS enzymes and by mixing and matching modules, they have produced novel polyketide analogs  with more effective medically relevant properties. One of the compounds they engineered shows enhanced efficacy in blocking the growth of breast cancer cells that depend on the activity of the estrogen receptor, while a second polyketide inhibits an enzyme linked to adult-onset diabetes, demonstrating just two possible new therapeutic applications for synthesized polyketides. But, as the authors propose, this method promises to reveal new pharmaceutical agents that haven't even been discovered yet.


To examine the relationship between mitochondrial DNA (mtDNA) alterations and colorectal tumorigenesis, we used high-resolution restriction endonucleases and sequencing to assess the mitochondrial genome from three histologic subtypes of colorectal adenomas , colorectal cancer (CRC) tissues = 27, and their matched surrounding normal tissue (MSNT) = 52. The mitochondrial genomes were amplified using 9 pairs of overlapping primers and systematically analyzed by means of high-resolution analysis. DNA fragments showing a shift in banding patterns between the three adenomas, CRC, in comparison to the MSNT were sequenced to identify the mtDNA alterations. A total of thirty-eight germ-line mtDNA variants were observed in this study. Twenty-two of the thirty-eight were identified as mutations and 59% (13 of 22) were silent mutations and one was a 1-bp insertion. Sixteen of thirty-eight were distinct SNPs in flanking regions of the restriction sites and, 6 of the 16 (37%) SNPs were not previously reported. Most of these mutations/SNPs were homoplasmic and distributed in various regions of mitochondrial genes including the 16S and 12S rRNA. Based on our results, mtDNA germline variants increased in prevalence with adenoma CRC progression. To the best of our knowledge, this is the first report to show an increased prevalence of mitochondrial gene variants in CRC tumorigenesis. Colorectal polyps are a frequent occurrence in the general population and adenomatous changes in these polyps are associated with the overwhelming majority of CRC. These adenomas are the precursor lesions of colon cancer . CurrentThis study was approved by the Institutional Review Board of Morehouse School of Medicine and the Research Oversite Committee of the Grady Memorial Hospital, Atlanta, Georgia and the University of Alabama at Birmingham School of Medicine, Birmingham, Alabama. Primary fresh frozen and paraffin embedded tissues from 3 histologic subtypes of adenomatous polyps , 27 CRC tissues, and histologically MSNT, n = 52 were obtained from the Tissue Procurement Network at the University of Alabama at Birmingham and the Department of Surgery of Morehouse School of Medicine/Grady Hospital. Adenomas were defined by histologic type, degree of dysplasia, and presence of infiltrating adenocarcinoma in adenoma and classified as tubular, tubulovillus, and villous. CRC diagnosis was confirmed by histological examinations of biospied specimens for all patients and pathological tumor staging for these was based on American Joint Committee on Cancer [Genomic DNA was extracted from both frozen and paraffin embedded remnant tissues using Tri-Reagent  and Purgene DNA purification kits  according to the manufacture's protocol. The mtDNA from each tissue sample was amplified by PCR using nine overlapping mtDNA primer pairs as previously described ,11, whicThe analysis of mtDNA samples by restriction digestion and sequencing of the DNA fragments showed non-predicted banding patterns either as homoplasmic single bandshifts or heteroplasmic multiple bands on the gel. The PCR product data point in overlapping regions were counted only once. However overlapping regions were used as internal controls for the identification of the variants. Results of the high-resolution restriction analyses and sequencing of the entire mitochondrial genome yielded 38 sequence variants (including 16 SNPs from the flanking restriction sites) for the precursor adenomatous polyps and cancer tissues. In no instance was a variant detected in the adenomatous tissue found in the histologically matched adjacent surrounding tissue, indicating that these were germ-line origin.Val). However, one was a 9-bp Ins5892C in a non-coding region between MTTY and MTCOI. Some of these mutations are located in ATP synthase genes that are involved in mtDNA genome maintenance and integrity in yeast [Asp), 7335insC , G7256A , T7146A ), G8206A , A16325G (D-loop), T16309G (D-loop), G16294A (D-loop), G16266A (D-loop), G16233A (D-loop), 8269-9bp del (noncoding). Worth mentioning is also the high frequency of 16S rRNA gene variants (> 65%) in the mtDNA among CRC tissue versus the < 25% in the precancerous tissues. Mutations in the 16S rRNA gene were commonly found in different cancers, except those of the thyroid [Based upon our assessment of the mitochondrial DNA from non-cancer, precancerous (adenomatous polyps), and colorectal cancer tissues using a high-resolution restriction analysis, we have identified sequence variants. To our knowledge this is the first assessment of the mitochondrial genome using solely primary tissue rather than cell lines. This is important since tissues in culture can undergo clonal evolution which can distort frequency data. Although, the restriction analyses identified a number of band shifts indicating site gains or losses, these data only indicated that the predicted sequence had changed. They did not identify specific base pair changes. We therefore used the original primers and a series of nested primers to sequence the band shifts in order to identify these specific changes. A total of thirty-eight germ-line mtDNA variants were observed and all mutations/SNPs were considered as germ-line origin since the variants found in the colorectal adenoma polyps or cancerous tissues were also found in the MSNT tissues. Twenty-two of the thirty-eight were identified as mutations and 59% 13 of 22) were mostly silent mutations of T-to-C. or a G-to-A transition, which is consistent with the mutagenic spectra of oxidative, damage ,13. Amon of 22 wein yeast . Seventyin yeast . SixteenWe then sought to determine whether or not any of mtDNA variants observed in flanking regions of these restriction site variants may prove to be informative biomarkers. Although, a total of 38 of the mtDNA variants were found, none of the variants appeared to be a marker for a particular adenoma CRC tumor tissue type. Nevertheless, cumulative frequencies of mtDNA variants in the different tissue types resulted in a high prevalence of mtDNA sequence variants in CRC tissue and the trend was for the number of variants to be lowest in the precancerous Fig. , suggestThese data should be interpreted cautiously as they are based on small number of sample size. Similar studies have looked at precancerous tissue types , howeverMtDNA, mitochondrial DNA; ATP, adenosine triphosphate; CRC, colorectal cancer; MSNT, matched surrounding Normal tissue; SNPs single nucleotide polymorphisms; PCR polymerase chain reaction.FOA performed few assays while MK performed almost all assays. DC provided the statistical support. JO provided some tissue samples and suggestions for finalization of the manuscript. RG provided some technical supervision to MK and suggestions for finalization of the manuscript. All authors read and approved the manuscript.