Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in  Drosophila spermatogenesis, a process that resembles in many ways the generation of individual sperm cells in mammals, including humansKnown as executors of programmed cell death, several caspases are here shown to be involved in  Drosophila the apoptotic effector caspase DRICE is cleaved and activated throughout the length of elongated spermatids, and testis-specific expression of the baculovirus caspase inhibitor p35 results in male sterility, despite the fact that apoptosis is not an obligate step in spermatogenesis , in which 64 haploid spermatid nuclei descended from a diploid primary spermatogonial cell are connected by abundant cytoplasmic bridges  (Drosophila caspase-1 (DCP-1) were decreased specifically in the male germline using RNA interference (RNAi) (β2tub-Dcp-1-RNAi flies)  . Cysts uctively) . Testis ctively) A. In conctively) B and 1C.ctively) D was dractively) E. Cysts ctively) F. These ctively) , suggestctively) G and 1J.i flies) . Short pi flies) . Togethei flies)  below isDrosophila APAF-1 homolog ARK, which promotes activation of the apical caspase DRONC  showed a high level of male sterility (Ark under the control of the β2tub promoter (β2tub-Ark-RNAi flies)  I. To deci flies) . DRICE wi flies) A and 2C.i flies) B and 2D.i flies) F functioi flies) E and 2G.i flies)  below.E)  . In contE) A and 3B,E) C. The inE) C, and acE) D. As expE) E or in wE) F.reaper (XR38/H99) ( 05014hid/H99 cysts ( 05014hid/H99)   showed n99 cysts C, indica99 cysts A, but no014/H99) B. These hid allelic combinations,  A329hid/ A329hid (data not shown) and  05014hid/H99, showed defects in individualization similar to those observed in β2tub-DIAP1 or β2tub-p35 males, demonstrating a requirement for HID in this process  flies, consistent with the idea that HID promotes individualization, at least in part, by promoting DRONC activity  contained active DRICE, but often showed uncoordinated investment cone movement (data not shown). At the EM level, elongated cysts from testis of single  f02804dFadd/f02804dFadd  and  B118Dredd/ B118Dredd males showed a range of phenotypes. About 50% of cysts from  B118Dredd/ B118Dredd males and almost all cysts from  f02804dFadd/ f02804dFadd males (greater than 90%) displayed defects in individualization similar to those of β2tub-DIAP1 and β2tub-p35 flies  in the cyt-c-d gene were sterile and lacked active DRICE staining in testis  in which DRICE activation during spermatid individualization was completely eliminated   of ARK and DRONC; to complete loss of HID, DREDD, or FADD; and to expression of the potent general caspase inhibitors DIAP1, p35, or p49 see A–3D, sugn testis . Howeverotubules C and 5E,otubules F–5H. Theiminated A (see Maiminated B. In conely 50%) C. Howeveely 50%) D and 6E.driceless males also showed reduced levels of localized active DRONC staining (data not shown), raising the possibility that DRICELESS has at least some of its effects on individualization through regulation of DRONC activity. We do not favor a simple linear model in which DRICELESS mediates its effects on individualization only by promoting DRONC-dependent activation of DRICE. This is because removal of HID or inhibition of ARK or DRONC, each of which inhibited individualization, had no significant effect on DRICE activation. An attractive alternative is that DRICELESS-dependent activation of DRICE promotes individualization, at least in part, by indirectly facilitating local activation of DRONC and perhaps other caspases, such as DREDD  unclear. Interestingly, cysts from REDD see  below, tREDD see . DRICE cREDD see , providiAll together, our observations demonstrate that multiple caspases and caspase regulators, acting at distinct points in space and time, are utilized to promote spermatid individualization. In one pathway, whose mechanism of activation is unknown, active DRICE appears throughout elongated spermatids just as individualization begins. DRICELESS, which promotes individualization, is required for DRICE activation. But whether active DRICE mediates the requirement for DRICELESS is unknown. In a second pathway, HID, concentrated through unknown mechanisms in the cystic bulge, promotes the local ARK-dependent activation of the apical caspase DRONC, presumably at least in part through disruption of complexes between DRONC and DIAP1. As discussed above, active DRICE may facilitate this activation. Components of a second pathway for apical caspase activation, dFADD and DREDD, are also important for individualization. These proteins bind each other , and dFAArk, Dronc, and Hid; dFadd and Dredd; and Driceless) contained cysts in which individualization failed to occur. Interestingly, however, other cysts in the same testis, or from testis of sibling males, carried out individualization apparently normally. Thus, these flies were fertile, though in some cases at a reduced frequency . These observations suggest that no one of these caspase pathways is absolutely required for individualization. The stochastic nature of the defects observed complete failure of individualization in some mutant cysts and apparently normal individualization in others may reflect a requirement for a threshold level of caspase activity, which can be achieved through multiple pathways, or as a result of positive feedback between pathways, in order for a cyst to initiate individualization. Consistent with these possibilities, double mutants between components of the Ark and Dronc caspase cascade and mutants in the dFadd and Dredd cascade were almost completely sterile  or completely sterile , while single mutants for any of these components showed significant fertility .How do caspases contribute to spermatid individualization? Testis from flies mutant for any one of the above pathways . Second, spermatids in cysts with decreased levels of ARK often contained large fingers of excess cytoplasm despite the fact that in some cysts membrane encapsulation occurred apparently normally , such as we observed with DRONC, provides one possible solution. Others may also exist. In particular, it is important to recognize that while active caspase-specific antibodies recognize caspases that are in the cleaved and therefore activated conformation, these caspases may be kept inactive through interactions with other proteins or as a result of posttranslational modification.  compare . It is lCD4/Cyow1118, Ark ; β2tub-Ark-RNAi (three copies); β2tub-Dcp-1-RNAi (six copies).All crosses and stocks were maintained at 25°C. The following fly stocks were used: kCD4/Cyo , H99/TM3, both of which were conjugated with keyhole limpet hemocyanin as the immunogen . Active DRONC-specific antibodies were purified by sequential protein affinity purification. Antisera were first applied to a column bound with full-length inactive DRONC (C318ADronc) to eliminate antibodies reactive with uncleaved DRONC. The flowthrough was applied to a DRONC large subunit (residues 1–352) affinity column. Bound proteins were eluted using 100 mM glycine (pH 2.5). These antibodies detect the large fragment of active DRONC (cleaved after E352), but do not recognize full-length DRONC (see C318ADronc). Western blot analysis to demonstrate binding specificity was carried out with 100 ng of full-length C318ADronc and 1–352Dronc. These were detected using purified anti-DRONC peptide (1:100) or purified anti-active DRONC peptide (1:100) antibodies.Conditions for immunocytochemistry and confocal microscopy were as described in RONC see . Anti-DRIndividual male flies were placed with 4- to 5-d-old virgin females in vials for 3 d at 25°C. They were then transferred to fresh vials with four new females and allowed to mate for another 3 d. Males were scored as sterile if they failed to produce progeny by day 6.Testes extracts were prepared in 50 μl of cell lysis buffer  from 30–50 adults of the appropriate genotype. Total protein (70 μg) was used for Western blot analysis using rabbit anti-ARK  (generously provided by Lai Wang and Xiaodong Wang) or purified rabbit anti-DCP-1 (1:100). Filters were stripped using Restore Western blot stripping buffer  and reprobed with rabbit anti-full-length DRICE   as a loaTestes were dissected from adult 2- to 4-d-old males raised at 25°C and prepared for EM as described by Figure S1(A) EM section from β2tub-Dcp-1-RNAi testis. Individualization has failed to occur throughout the cyst.(B) The boxed area in M is shown at a higher magnification. Spermatid units sharing a common cytoplasm are outlined by the dashed line.(C) Western blot from wild-type (Wt) and β2tub-Dcp-1-RNAi (Dcpi) testis probed with anti-DCP-1 and anti-DRICE antibodies. DCP-1, but not DRICE, levels are greatly reduced in β2tub-Dcp-1-RNAi testis.(552 KB JPEG).Click here for additional data file.Figure S2 F118EGMR-Dronc larvae stained with purified anti-DRONC peptide antiserum (green). All cells posterior to the morphogenetic furrow labeled with this antiserum, as expected based on the pattern of GMR (glass multimer reporter)-dependent gene expression (C318ADronc). The second lane was loaded with a version of DRONC consisting of only residues 1–352. This protein terminates following glutamate-352, the DRONC autoactivation cleavage site, and is equivalent to the large subunit of cleaved and active Dronc. The anti-DRONC antibodies react well with both proteins.(A) Third instar eye imaginal disc from pression . Eye disF118EGMR-Dronc  larvae stained with anti-active DRONC antiserum extensively purified to select for antibodies that react only with versions of DRONC that have been cleaved at glutamate-352, as described in the Materials and Methods. Only cells in the most posterior region of the eye disc, which are presumably undergoing apoptosis, react with these purified antibodies. The inset shows a Western blot, similar to that in (A), which was probed with the purified active DRONC-specific antibodies. These antibodies react with the glutamate-352-cleaved version of DRONC, but not with full-length DRONC.(B) Third instar eye imaginal disc from (374 KB JPEG).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for p35 is P08160.The National Center for Biotechnology Information (NCBI) (http://flybase.bio.indiana.edu/search/) accession numbers of the sequences discussed in this paper are Ark (FBgn0024252), cyt-c-d (FBgn0000408), Dcp-1 (FBgn0010501), dFadd (FBgn0038928), Diap1 (FBgn0003691), Dredd (FBgn0020381), Drice (FBgn0019972), Dronc (FBgn0026404), Grim (FBgn0015946), Hid (FBgn0003997), and Reaper (FBgn0011706).The FlyBase (


Chronic fatigue syndrome (CFS) is a disease which defined as medically unexplained, disabling fatigue of 6 months or more duration and often accompanied by several of a long list of physical complaints. We aimed to investigate abnormalities of hypothalamic-pituitary-gonadal (HPG) axis hormones and cortisol concentrations in premenopausal women with CFS and find out effects of depression rate on these hormones.We examined follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone and cortisol concentrations in 43 premenopausal women (mean age: 32.86 ± 7.11) with CFS and compared matched 35 healthy controls (mean age: 31.14 ± 6.19). Patients were divided according to menstrual cycle phases  and compared with matched phase controls. Depression rate was assessed by Beck Depression Inventory (BDI), and patients with high BDI scores were compared to patients with low BDI scores.There were no significant differences in FSH, LH, estradiol and progesterone levels in both of menstrual phases of patients versus controls. Cortisol levels were significantly lower in patients compared to controls. There were no significant differences in all hormone levels in patients with high depression scores versus patients with low depression scores.In spite of high depression rate, low cortisol concentration and normal HPG axis hormones of both menstrual phases are detected in premenopausal women with CFS. There is no differentiation between patients with high and low depression rate in all hormone levels. Depression condition of CFS may be different from classical depression and evaluation of HPG and HPA axis should be performed for understanding of pathophysiology of CFS and planning of treatment. Chronic fatigue syndrome (CFS) is a clinical presentation that primarily affects women and characterized by severe disabling fatigue, and other symptoms; including musculoskeletal pain, sleep disturbance, impaired concentration, and headaches, in the absence of organic illness or severe psychiatric disorder that would explain the fatigue. For diagnosis of CFS operational criteria have been developed by Centers for Disease Control (CDC) . AlthougIt has been reported that onset of CFS mostly following a significant stressor, most frequently a viral infection, and the course of the syndrome remits and relapses with occurrence of physical and psychological stressors . Stress Pertuberations of HPA axis function have been described in CFS ,12. StudCFS occurs more commonly in women . It was Interaction between HPA and HPG axes in stress-induced amenorrhea suggests that there may be perturbation of these axes in CFS. One important confound is co-morbid depressive illness, present in approximately 50% of CFS patients . RelatioIn this study, we aimed to investigate main hormones of HPG and HPA axes; FSH, LH, estradiol, progesterone and cortisol levels in premenopausal women with CFS. Furthermore to find out relationship between these hormones and depressive mood with comparing patients with high and low depression scores.A total of 43 premenopausal women diagnosed as CFS, according to the international CFS definition criteria , were re2, normal values 24.5 to 195 pg/mL for follicular phase and 40 to 261 pg/mL for luteal phase), progesterone , and cortisol  were evaluated in all patients and controls in both of menstrual periods.Blood samples were collected in the early morning (8.30–10.30 AM) after an all night fast and plasma was separated immediately by centrifugation; then sera obtained were stored at -20°C until hormonal assaying. All hormone values assayed by "Electro Chemil Luminescence Immunassay (ECLIA)"  method. Serum concentrations of follicle stimulating hormone , luteinizing hormone , estradiol (Ewere done by SPSS 8.0 PC program. Results were expressed as means ± SD (standard deviation). Statistical significances were tested using the independent student's t test for women with CFS and control group comparisons. Mann Whitney U test was used for comparison of hormonal data of women in both phases of menstrual cycle, and patients with high and low BDI scores groups comparison. The level of statistical significance was set at a two-tailed p-value of 0.05.All patients with CFS and healthy controls were reproductive premenopoausal women; and mean ages were 32.76 ± 7.07 and 31.14 ± 6.19 respectively for both groups. All of the patients had debilitating clinically evaluated and medically unexplained fatingue that does not resolve with bed rest and severe enough to significantly reduce daily activity for at least 6 months. The other clinical findings of patients with CFS were summarized in Table There wasn't significant differentiation between ages and BMI of groups (p > 0.05). There were no significant differences between the means of FSH, LH, progesterone and estradiol in all CFS patients compared to all controls (p > 0.05). Mean concentrations of cortisol were significantly lower in all CFS patients than all controls (p < 0.001) Table .There were no significant differences between the levels of FSH, LH, progesterone and estradiol in CFS patients compared to controls in follicular phase (p > 0.05). Mean concentrations of cortisol were significantly lower in CFS patients than controls in follicular phase (p = 0.001) Table .There were no significant differences between the levels of FSH, LH, progesterone and estradiol in CFS patients compared to controls in luteal phase (p > 0.05). Mean concentrations of cortisol were significantly lower in CFS patients than controls in luteal phase (p < 0.05) Table .Fifty patients (69.76%) with CFS had ≥17 BDI scores. Mean cortisol concentrations of patients with BDI scores ≥17 were higher than patients with BDI scores <17 but differentiation wasn't significant. There were no significant differences between these patients groups in HPG axis hormone levels Table .In this study, reproductive HPG axis hormone levels demonstrated no significant differences in women with CFS from controls during follicular and luteal phases. These findings are in agreement with Korszun et al.  who repoChronic fatigue syndrome is generally accepted stress related disease and disfunction of HPA axis has been reported in this syndrome . In thisHigh circulating cortisol is well replicated finding in major depression  and so pStudies in primates have demonstrated that intracerebroventricular infusion of CRH as well as proinflammatory cytokines such as interleukin-1 can decrease LH secretion ,28. StreIn this study was found significantly lower circulating cortisol levels in patients with CFS in contrast to high BDI scores. However, there was no significant differentiation in cortisol levels between the patients with low and high depression scores. This is in contradiction to those hypercortisolism of classical major depression and stress condition. More recent studies support this contradicton ,33. In rDetermining single basal levels of HPA and HPG axes do not reflect activity of these axes entirely. Dynamic test indicates differences in function of these axes. We did not carry out dynamic characteristics of these axes. This point is limit of our study.In conclusion, we detected low cortisol levels in patients with CFS in spite of their high depressive mood rate. However, in this study, we were unable to describe HPG axis hormone abnormalities in both menstrual phases. Hypocortisolism may be a biological factor that contributes fatigue chronicity and the reason of normality in HPG axis. Depressive mood of chronic fatigue syndrome may be different from classical depression. There are need to carry out future controlled and larger clinical trials to clarify these matters.The pre-publication history for this paper can be accessed here:


The capacity for self-generated movement is a defining characteristic of animal life. With the molecular components of cellular locomotion conserved in organisms from protozoa to vertebrates, directed cell motility appears to be an ancient cell process, likely dating back a billion years. Most directed motion relies on the assembly, or polymerization, of actin proteins into filaments. Actin is one of the most abundant proteins in cells; about half of the cellular concentration of actin is bound together in filaments at any given time while the other half floats freely as “monomers” in the cytoplasm. The erection and demolition of actin filaments directs the cell motility that lays down the remarkable million miles of nerve cells that form the nervous system and drives a variety of fundamental biological processes, from effective immune response to embryonic development. Mutations in proteins that regulate actin assembly can lead to the abnormal cell migration associated with metastatic cancer. The actin cytoskeleton also provides the structural support for animal cells that the cell wall provides for plants.The molecular mechanisms underlying actin assembly and cell motility remained obscure until 1994, when Thomas Pollard and his colleagues discovered the protein complex that initiates actin polymerization. Called actin-related protein 2/3 (Arp2/3) complex, this molecular machine consists of seven subunits, including the two actin-related proteins. Free actin monomers are primed for rapid polymerization, but polymerization must be initiated by the Arp2/3 complex in a process referred to as nucleation. To nucleate a new filament, the Arp2/3 complex must be activated, a job accomplished by a family of proteins called WASP . WASP proteins bind to both the Arp2/3 complex and an actin monomer. The Arp2/3 complex also binds two molecules of adenosine triphosphate (ATP) on the Arp2 and Arp3 subunits. ATP releases energy in a process called hydrolysis, which drives most energy-dependent processes, from actin polymerization to muscle contraction. The precise mechanisms governing Arp2/3 activation and nucleation are not known. Now Mark Dayel and Dyche Mullins show where hydrolysis occurs during this crucial first step in polymerization and use this finding to investigate the mechanisms that drive nucleation.In previous experiments, Dayel and Mullins found that Arp2/3 appears to require hydrolysable ATP to effect nucleation. To determine when and if ATP hydrolysis occurs on the Arp2/3 complex, Dayel and Mullins developed a technique that allowed them to analyze the Arp2 and Arp3 subunits separately. Dayel and Mullins discovered that hydrolysis occurs only on the Arp2 subunit of the complex and that it happens during the step when WASP initiates the nucleation of a new filament. The researchers then used ATP hydrolysis on Arp2 to dissect the mechanism by which WASP activates the Arp2/3 complex and develop a model of nucleation.  They find that WASP proteins activate the Arp2/3 complex by coordinating its interaction with an actin monomer—the first monomer of the new filament.By developing a novel technique to monitor activation of the Arp2/3 complex, the authors contribute a new tool for further investigations of this central part of the cellular motility machinery. And by showing how Arp2/3 is activated, they offer important insights into the workings of a multiprotein cellular machine and the mechanisms that cells enlist to control their shape and motility—which could suggest potential drug targets to inhibit the abnormal cell movement characteristic of cancer and other diseases.


Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement  results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. L. lactis does not produce LPS or any proteases as Escherichia coli or Bacillus subtilis do, respectively.Lactic Acid Bacteria (LAB) are anaerobic Gram positive bacteria with a GRAS  status. They are also food grade bacteria, and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. A last advantage compared to other well-known protein producers is that Lactococcus lactis, were developed: transformation protocols, cloning- or screening-vectors , th, thL. laen Table  19], an, anL. laus Table  [18]. Dinsertion . StrikinLeuconostoc mesenteroides dextransucrase, [Streptomyces tendae anti-fungal protein; Freitas et al., submitted) have been successfully secreted in L. lactis. This suggests that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis. In contrast to protein size, conformation may be a major problem for heterologous secretion in L. lactis as illustrated by some recent examples. The first example is the production of the non-structural protein 4 (NSP4) of the bovine rotavirus, the major etiologic agent of severe diarrhea in young cattle. In order to develop live vaccines against this virus, the NSP4 antigen was successfully produced in L. lactis [Usp45-NSP4 precursor and NSP4 mature protein were detected in the cell fraction. Two degradation products were detected in addition to the NSP4 precursor and mature protein. These results suggest that the cytoplasmic form of NSP4 was probably totally degraded inside the cell whereas fusion to the SPUsp45 protected NSP4 protein against intracellular proteolysis.Proteins with molecular mass ranging from 165 kDa  to 9.8 . lactis . DerivatB. abortus GroEL chaperone protein: only pSEC:GroEL plasmid was obtained and subsequently the fusion SPUsp45:GroEL was detected in Western blot experiments . In this case, B. abortus GroEL is likely to interact with lactococcal cytoplasmic proteins leading to severe cellular defects and thus to a lethal phenotype. On the other hand, fusion of SPUsp to GroEL might keep the chimeric protein in an unfolded and/or inactive state allowing thus its heterologous production.Similar results were obtained when pCYT and pSEC vectors were used to produce the L. lactis [L. lactis to test the immunomodulation of the allergenic response in mice when BLG is delivered by a bacterial vector [Usp45 although the SE was very low, with no detectable BLG in the supernatant of pSEC:BLG strains [Another example is the production of the bovine β-lactoglobulin (BLG) in . lactis ,31. BLG,l vector . Western strains . FurtherB. abortus GroEL, and BLG (which are medium-sized compared to DsrD or Afp1), either very low secretion yields or absence of secretion was observed in L. lactis. In all cases, fusion to a SP stabilizes heterologous protein production even though they are not efficiently secreted. These results could be due either to the SP itself that reportedly acts as an intramolecular chaperone or to the protection of the chimeric precursor from intracellular proteolysis by the cytoplasmic chaperones of the Sec-machinery. GroEL (a cytoplasmic chaperone), NSP4 , and BLG (a globular protein) have dramatically different primary sequences. A higher affinity of intracellular housekeeping proteases for these particular sequences cannot be hypothesized since the fusion of a SP leads to the stabilization of the protein. Change of conformation is therefore the predominant criterion involved in the stabilization of the precursors and the higher yields observed. On the other hand, these proteins might undergo rapid folding right after their synthesis, which interferes with (or hampers) the secretion process. Such interferences between protein conformation and SE were previously shown in E. coli and B. subtilis [L. lactis as well.For rotavirus NSP4, subtilis ,33. AltoL. lactis and allowed higher protein yields compared to E7 alone [L. lactis. Bernasconi et al (2002) fused the Lactobacillus bulgaricus proteinase PrtB to BLG, which was subsequently stabilized by the PrtB carrier [L. lactis strains for protein production, fusions can also facilitate purification (e.g. His-tag strategy). Protein fusion has also been successfully used to optimize the production of the two subunits of heterodimeric complexes as demonstrated with murine interleukin-12 in L. lactis [E. coli [L. lactis is used as an antigen delivery vector, fusions can be envisioned since it was demonstrated that both moieties of the chimerical protein are still recognized by the corresponding antiserum [It was clearly demonstrated that the secreted form of E7, a reportedly labile protein, can be stabilized by fusion to Nuc ,34. Nuc E7 alone . StabiliE7 alone . Recent E7 alone . These r carrier . It is t. lactis  or with [E. coli . In bothntiserum ,20,34 anntiserum .clpP deficient strain  strains: E7 was equally degraded in the cytoplasm and remained unchanged in supernatants samples. Altogether, these results indicate that E7 intracellular proteolysis is ClpP- and DnaK- independent. Until recently, only two cytoplasmic proteases, ClpP and FtsH [L. lactis. The existence of a third, as yet unidentified protease was postulated by studies of a clpP mutant suppressor [L. lactis protease that, as suggested by our data, is activated in stationary phase.Several of the results mentioned above suggest that secretion could be an efficient way to escape intracellular proteolysis. This hypothesis was particularly tested in E7 production . E7 was  lactis; ) and in d state; ). In exppP- Fig.  or dnaK-and FtsH , have beppressor . E7 may L. lactis [Besides the features of the precursor itself, these results also rise that host factors are involved in protein stability and SE Fig. . Researc. lactis .L. lactis possesses a wide range of enzymes  dedicated to intracellular proteolysis, it possesses only one extracellular housekeeping protease (HtrA) [versus cytoplasmic production. These results give clues and provide the research workers with target proteins to study intracellular proteolysis and protein stability inside and outside the host strain. Such studies already led to the development of htrA deficient L. lactis strains. Heterologous protein secretion and anchoring in a htrA deficient strain allowed higher protein stability at the cell surface for several heterologous proteins [Although e (HtrA)  and its e (HtrA) . Thus, aproteins .L. lactis. L. lactis complete genome sequence analysis revealed indeed that the Sec machinery comprises fewer components than the well-characterized B. subtilis Sec machinery. Notably, L. lactis does not possess any SecDF equivalent and complementation of the lactococcal Sec machinery with B. subtilis SecDF results in better secretion yields as determined for Nuc reporter protein . Random mutagenesis approaches also revealed that features of some cell compartment, such as the cell wall, play an important role in the secretion process [L. lactis .Current research works are now focusing on other host factors that affect protein production and secretion in  process . SimilarL. lactis and the list of heterologous proteins produced in this bacterium is regularly increased. The reports where cytoplasmic and secretion production can be compared mostly show that secretion allows better protein yields compared to intracellular production; and allow a better understanding of the protein production and secretion process in L. lactis.Many molecular tools are now available to direct heterologous protein secretion in L. lactis capacities for protein modifications. For example, we showed that proteins that require a disulfide bond (DSB) to acquire their native conformation can be efficiently produced and secreted in L. lactis [E. coli dsb or B. subtilis bdb, the genes involved in DSB formation, was found by sequence comparison in L. lactis. Similarly, other folding elements  are still to be identified and the L. lactis capacities for post-translational modifications are still to be investigated.Future works should investigate the . lactis ,22,27. HL. lactis [L. lactis and LAB in general, is a clear advantage for their use in production and secretion of therapeutic or vaccinal proteins.Altogether, these works will contribute to the development and the improvement of new food-grade systems for . lactis  and shou


Hist4h4. The proteins encoded by this transcript and the human H2afj mRNA isoform-2 have the highest amino acid similarity. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR.An uncharacterized histone H2a-coding transcript (E130307C13) has been cloned from a mouse full-length cDNA library. This transcript is encoded on chromosome 6, approximately 4 kb upstream of a histone H4 gene, E130307C13) is regulated in a replication-independent manner, and therefore it is H2afj. Certainly, H2afj transcript lacks a stem-loop structure at the 3'-UTR but contains a poly (A) signal. In addition, its promoter region has a different structure from those of the replication-dependent histone H2a genes.Quantitative RT-PCR indicated that the gene that encodes E130307C13 (E130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse.The bioinformatics imply that  Hist1 cluster on chromosome 13, 4 in the Hist2 cluster on chromosome 3 and 1 in the Hist3 cluster on chromosome 11 [Replication of the eukaryotic chromosomes requires the synthesis of histones to package the newly replicated DNA into chromatin. Control of the level of histone mRNA accounts for much of the control of histone protein synthesis . Mouse hosome 11 -7.H2afx on chromosome 9 and H2afz chromosome 3. These two genes encode polyadenylated mRNAs. H2afx mRNA has both a polyadenylated tail and a stem-loop structure [H2afx mRNA have a nuclear export element (5'-ACAACAAGAAGACGCGCATCAT-3') in the protein-coding region that functions to export the mRNA from the nucleus to the cytoplasm [Mouse also has two replication-independent histone H2a genes, tructure . Mouse rE130307C13 lies on chromosome 6, approximately 4 kb upstream of Hist4h4. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR. In addition to this, we compared the structure with the sequences deposited in the international DNA/protein database.An uncharacterized histone H2a-coding transcript  has been cloned from a mouse full-length cDNA library. E130307C13 with the amino acid sequences deposited in the international DNA/protein database showed that it has the highest similarity to that encoded by human H2afj mRNA isoform-2 , peaked at 2 h, and then decreased  signals at the middle and near the 3'-end. It indicates that the E130307C13 gives rise to two differentially polyadenylated mRNA transcripts. Considering the position at the 3'-UTR, it has a possibility that the poly (A) signal near the 3'-end is functional. Except for the pseudogene Hist1h2aj, the replication-dependent H2a mRNAs have the conserved stem-loop structure at the 3'-UTR. H2afx gives rise to a cell-cycle-regulated mRNA ending in the stem-loop when it is transcribed during S-phase, and a polyadenylated mRNA that is present in G1-phase cells [H2afz mRNA lacks the stem-loop structure and has poly (A) signals. H2afz contains four introns in the protein-coding region and needs a splicing mechanism to produce the mature mRNA. Interestingly, E130307C13 lacks introns. Comparing the transcript structure of E130307C13 with those of the other histone H2a genes suggests that E130307C13 has replication-independent characteristics.In addition, the 3'-UTR of the ure Fig. . But these cells . H2afz mE130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse.The bioinformatics imply that Multiple alignment of 44 amino acids sequences of histone H2a related proteins from human and mouse was created using the CLUSTAL W  on the DThe cell cycle of Hepa 1–6 cells was synchronized at the end of G1-phase by the addition of thymidine-hydroxyurea. The cell cycle arrest was released by washing out the thymidine-hydroxyurea, then the cells were harvested at intervals of 1 h from 0 h to 11 h.Total RNA was extracted by using the RNeasy mini kit (Qiagen) according to the manual for the cell line. After that, each sample was treated with DNase I.RNA (approximately 0.5 μg) and random hexamer primers were heated to 70°C for 10 min, followed by cooling on ice for 5 min. The cDNA was synthesized using Superscript III First Strand buffer (Invitrogen) according to the manual. The reverse transcriptase was inactivated by a 15-min incubation at 70°C.Hist2h2aa2; 5'-ACTCCGGAAAGGCCAAGACA-3' and 5'-GTTGTCCTAGATTTCAGGTG-3' for H2afz, product size 100 bp; 5'-CGTCCTGCCCAATATCCAG-3' and 5'-TCTGCACCCGTCTGTCG-3'  and 5'-AAGCAGGGCGGTAAGGTG-3' and 5'-TCCGCGTAGTTGCCCTTC-3'  for E130307C13; and 5'-TGTGTCCGTCGTGGATCTGA-3' and 5'-CCTGCTTCACCACCTTCTTGA-3' for GAPDH , product size 76 bp. Quantification of GAPDH mRNA was used as a control for data normalization. PCR amplification was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The PCR conditions were an initial step of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The SYBR premix Ex Taq (Takara) was used according to the manual. Expression was assessed by evaluating threshold cycle (CT) values. The relative amount of expressed RNA was calculated using Livak and Schmittgen's method [The following primers were used: 5'-AACTGTAGCCCGGCCCG-3' and 5'-TTCGTCTGTTTGCGCTTT-3'  and 5'-CAACAAGCTGCTGGGCAAA-3' and 5'-TCGCCTTATGGTGGCTCTCC-3'  for transcripts of s method .HN designed this study, carried out the molecular biological studies, and the molecular evolutionary studies. TS and YT carried out synchronization of cells and quantitative RT-PCR. YH participated in the design of this study.


We decided to investigate, in a group of patients from a geographical area with a low incidence of breast cancer, whether HER-2/neu positive tumours are correlated with established or suspected risk factors for breast cancer and thus to identify distinct subgroups of high risk women.Several researchers have claimed that classification of tumours on the basis of HER-2/neu protein, estrogen and progesterone receptor expression were performed using immunochemistry. Odds ratios and 95% confidence intervals were determined by chi-square test and logistic regression analysis. Case-case odds ratios were calculated in order to measure the risk heterogeneity between HER-2/neu+ and HER-2/neu-tumours. Separate analyses were performed for premenopausal and postmenopausal women and according to estrogen receptor status.This study analysed data from patients who attended the Breast Unit at the University Hospital of Heraklion, Crete, Greece between 1996 and 2002. 384 women with primary invasive breast cancer were compared with 566 screened women who were referred to the Unit and had not developed breast neoplasm by the time the data were analysed. Risk factor data were obtained from each subject by personal interviews using a structured questionnaire. The detection and scoring of the HER-2/neu stratification, an increased breast cancer risk was associated with only four of the factors examined: use of oral contraceptives , use of HRT , an age at first full pregnancy more than 23 years  and body mass index more than 29 kg/m2 . Additionally, a history of abortion or miscarriage  was correlated with a decreased risk of breast cancer. In the case to case comparison only BMI >29 kg/m2 revealed a relative connection that was stronger with positive than with negative HER-2/neu tumours . This may indicate evidence of heterogeneity of a rather significant degree for this factor. In the ER negative group an age at first full pregnancy >23 years and a BMI >29 kg/m2 were associated with an increased risk in both HER-2/neu groups, but the association was significantly stronger for the latter factor in the positive HER-2/neu tumours .In multivariate analysis without HER-2/neu+ versus HER-2/neu-breast tumours, with the exception of increased BMI. Further innovative studies with larger sample sizes are needed to examine how the status of these potentially modifiable breast cancer risk factors interacts with biological markers such as HER-2/neu oncoprotein.Our study did not confirm that the established or putative hormonal breast cancer risk factors differ regarding their relations with HER-2/ Indeed,survival . In addi lesions . As a regenesis. ,7-9.neu overexpression or amplification may define a subset of breast cancer in which the net effect of a risk factor could be rather more obvious and its impact on breast cancer development more clear [On the other hand, the contribution of even well established breast cancer risk factors to the aetiology of carcinogenesis in the breast remains obscure, ill-defined and tenuous, mostly because of the existence of different pathways for the initiation and the evolution of a breast tumour . In ordere clear ,7,8.neu overexpression could indicate either that a HER-2/neu alteration is the way that this risk factor evolves into the carcinogenesis or that there is a parallel interaction between them that leads to breast tumour initiation and development. Since the data in the literature supporting the above hypothesis are few and conflicting, we decided to investigate, in a group of patients from a geographical area with a low incidence of breast cancer, whether HER-2/neu positive tumours are correlated with established or suspected risk factors for breast cancer and thus to identify distinct subgroups of high risk women.Thus, a close correlation of a risk factor with HER-2/This study analysed data derived from the database of the Breast Unit of the Department of Surgical Oncology at the University General Hospital of Heraklion on the island of Crete, Greece. The study considered all women who were consecutively diagnosed with primary, invasive breast cancer in our unit from 1996 to 2002. Subjects of other races, ethnicity, with residence outside Crete or diagnosed with DCIS or LCIS were excluded. Finally, 384 women, all originating from the island of Crete, were eligible for analysis.An age-stratified random sample of 566 women was used as a control group, derived from the Breast Unit's database of screened patients who had not developed breast cancer after a median follow up period of 40 months (range 12–92 months). Personal interviews were conducted with each woman during her first visit (both patients and controls) by a consultant or a senior resident. The interview followed a structured questionnaire, which did not change during the study period. Anthropometric measures were also made during the first visit.Women were classified as postmenopausal if their menstrual cycles had ended naturally at least 12 months before the interview or from surgery or radiation therapy at any age. Those who reported not having menstrual cycles for the last 10 months were considered as perimenopausal and were combined with premenopausal women for the purpose of our analysis.2 vs. BMI>29 kg/m2, median value for the study population) and radiation history of the chest (yes/no).The following variables were analysed for all patients and controls: residence , age at interview (≤ 50 and >50 years), age at menarche (≤ 12 and >12 years old), age at first full birth (<23 vs. ≥ 23 years old), parity , lactation (yes/no), use of medications to suppress lactation (yes/no), abortions and miscarriages (yes/no), age at menopause for postmenopausal women (≤ 50 and >50 years old), use of HRT for more than 2 months (yes/no), use of oral contraceptives for more than 2 months (yes/no), family history of breast cancer in a first degree relative (yes/no), history of benign breast disease (yes/no), obesity on the day of the interview  and in 377 (98.17%) of the 384 interviewed cases for the immunohistochemical detection of HER-2/Immunohistochemistry with the monoclonal antibody CB11 , at a dilution of 1/50 with incubation period of 60 min, was performed using the OPTIMAX automated system with the Super Sensitive Link-Label Detection System RTU Multilink AP/Fast Red, QA200OXE , following antigen retrieval by microwave pre-treatment at 500 Watts for 3 × 5 min in citrate buffer . Sections from breast cancer of known positivity were used as positive controls. Negative controls were processed by omitting the primary antibody and substituting non-immune serum. Scoring was based on the criteria recommended by DAKO A/S for the HercepTest . Only meThree (3) μm-thick sections taken on negatively-charged (SuperFrostPlus) slides were dewaxed in xylene, and rehydrated through graded alcohols. Following antigen retrieval by microwave pre-treatment at 500 W for 3 × 5 min in citrate buffer , estrogen receptor (ERa) and progesterone receptor (PR) expression was detected by immunohistochemistry using the same automated system and detection kit as above, and primary monoclonal antibodies to ERa (DAKO M7047) and PR (Biogenex code # MU 328-UC) at dilutions of 1/50 and 1/20, respectively, with incubation time 60', at room temperature. Positive and negative controls were processed as above. Positive nuclei counting was performed at a final magnification of 400× . After scanning at a final magnification of 100× for locating the areas with highest density of ER+ or PR+ carcinoma cell nuclei (hot spots), a 40X/¥/0.17 WD 0.65 objective lens was used for cell counting. All carcinoma cells in three hot spots per immunostained slide were evaluated by two pathologists working simultaneously, though independently, and the mean of the two independent counts was considered the final counting value for each field and hot spot. The ratio of the ER+ or PR+ carcinoma cell nuclei was recorded separately for each of the hot spots. The final immunoreactivity index (score) was calculated as the mean percentage of ER+ or PR+ carcinoma cell nuclei in the three hot spots. Specimens were interpreted as positive for ER or PR if at least 10% of the cells demonstrated nuclear staining of any intensity of reactivity, from 1+ to 3+. Staining intensity was graded as negative (0), weak (1+), intermediate (2+) or strong (3+), and reported separately. A mean value of intensity was assigned for specimens in which the staining intensity varied from field to field, and/or from hot spot to hot spot.2) test. A p value <0.05 was defined as significant. The potential association between breast cancers stratified by HER-2/neu status and well known predisposing factors was further investigated by using a stepwise logistic regression analysis (backward LR), testing the independent effect of breast cancer risk factors (independent variables) on breast cancer (dependent variable) for all women and also separately for premenopausal and postmenopausal females. In addition, we undertook further stratification with estrogen receptor status by using the same multivariate logistic regression model. These patient-controls odds ratios helped us to detect the pattern of heterogeneity and to explore plausible aetiological correlations between patient subgroups. Additionally, case-case odds ratios were calculated in order to measure the risk heterogeneity between HER-2/neu+ and HER-2/neu-tumours. It seems that the departure of the OR from unity can reveal the degree of heterogeneity between these subgroups [Odds ratios (OR) and 95% CI , as approximators of relative risk, were calculated to measure the association of the groups of breast cancer and the risk factors, using the chi-square , use of HRT , an age at first full pregnancy more than 23 years  and body mass index more than 29 kg/m2 . Additionally, a history of abortion or miscarriage  was correlated with a decreased risk of breast cancer. However, the number of oral contraceptive and HRT users was too small for reliable estimates of risk.Risk factor distributions in breast cancer patients and matched controls are presented in table neu protein overexpression. HER-2/neu positive tumours were not related with menopausal state, age at interview, tumour size, grade and stage, nodal and estrogen receptor status, but there was a modest positive association between HER-2/neu and progesterone negative tumours.Tumour characteristics of breast cancer patients are shown in table premenopausal group of women, an increased risk for HER-2/neu-tumours was observed for those women who reported an age at first full pregnancy ≥ 23 years , a BMI>29 kg/m2, first degree family history  or use of oral contraceptives , while an age at menarche less than 12 years was the only factor which slightly increased the risk in premenopausal HER-2/neu+ patients . Abortion played a less protective role (p = 0.068) for HER-2/neu-breast cancer in premenopausal than in postmenopausal women (p = 0.038). However, the intercase comparison in the premenopausal subgroup showed an evidence of heterogeneity only for the HER-2/neu+ women who had ever had an abortion , while use of oral contraceptives  and a positive first degree family history  showed a stronger association for HER-2/neu negative tumours.Subgroups of women stratified by menopausal status were further analysed by a multivariate stepwise logistic regression model adjusted for the remaining variables table . In the all women and the postmenopausal groups of patients due to the large sample size. Patients with an age of menarche ≤ 12 years , first degree family history , use of HRT  or OC  were at increased risk of developing HER-2/neu-breast cancer only, although the significance of the latter two factors was of little value due to the limited sample size. On the other hand, an age at first full pregnancy ≥ 23 years and a BMI greater than 29 kg/m2 increased breast cancer risk independently of HER-2/neu status, while a history of abortion decreased risk in the same way. In the case to case comparison only BMI >29 kg/m2 revealed a relative stronger connection with positive than with negative HER-2/neu tumours  and this may indicate an evidence of heterogeneity of a rather significant degree for this factor. The stronger association between an age at menarche ≤ 12 years, use of HRT and negative as opposed to positive HER-2/neu status did not reach statistical significance .The results of logistic regression were identical for neu positivity and obesity already shown in the analysis so far. In more detail, although BMI >29 kg/m2 elevated risk for both ER negative and positive tumours independently of HER-2/neu status, the association was significantly stronger for positive HER-2/neu tumours in the ER negative group . Additionally, in the same group an age at first full pregnancy >23 years revealed an increase of risk in both HER-2/neu groups, while first degree family history , age at menopause >50 years  and birth of 1–2 children  elevated risk for HER-2/neu negative tumours only. In the ER+ group of women the direct comparison between cases revealed no associations with any factor at all, while abortion showed a protective pattern against breast cancer which expressed estrogen receptors independently of HER-2/neu status.A different stratification pattern of our study's population is presented in table neu oncoprotein. In fact, increased BMI elevated risk in both groups, but the comparison between HER-2/neu+ and HER-2/neu- tumours revealed a much stronger association with HER-2/neu+ breast cancers.This epidemiological study, conducted in a low incidence Mediterranean population,  found thneu status can help discriminate aetiologically distinct subgroups of breast cancer cases, and none of them has identified the effect of increased BMI with HER-2/neu positive tumours [Very few studies have examined the possibility whether HER-2/ tumours ,5,7-9.neu+ tumours with an early age at first full pregnancy, we found a strong elevated risk with a late age regardless of HER-2/neu protein expression [neu+ breast cancers, while we also found this inverse association but independently of HER-2/neu status [neu+ tumours only in the premenopausal group of women. Early contraceptive use has been positively associated with HER-2/neu+ breast cancer in two studies [neu negative tumours. However, because the number of oral contraceptive (and HRT) users in this study was small, this subgroup analysis was hindered by decreased power to detect associations of any magnitude. The slightly protective effect of parity found in the age-adjusted analysis was diminished after logistic regression and did not reveal any association with HER-2/neu status, in contrast with previous findings [neu positive tumours in one study while other investigators reported opposite results [More specifically, in contrast with other investigators who have shown an elevated risk for HER-2/pression . Previouu status . Interes studies ,8, but ofindings . Breastf results ,9. Althoneu status. This inconsistency may reflect differences in study design, populations, and laboratory methodology. In this study we used immunochemistry  to assess the HER-2/neu protein overexpression, which is highly correlated with gene amplification according to previous reports [neu protein overexpression found here was within the limits reported elsewhere [Our findings have similarities and differences with respect to previous reports that examined the associations of breast risk factors with HER-2/ reports . Also, tlsewhere ,5-9.neu protein overexpression and most of the hormone-related breast cancer risk factors does not completely agree with several hypotheses which have maintained that combined estrogen and HER-2/neu activation is closely involved in the same pathway in breast cancer carcinogenesis [This lack of relationship between HER-2/ogenesis ,18.neu positive tumours in our study was high body mass index, which is an established risk factor that has an estrogen-mediated oncogenic effect on the mammary gland. More specifically, obesity is associated with higher breast cancer risk among postmenopausal women through greater lifetime exposure to higher levels of estrogens produced in adipose tissue and lower SHBG production [neu gene [neu receptor, and so promote carcinogenesis through common means [The only hormone-related factor that was found to be related with HER-2/oduction ,20. Highneu gene ,21. Accoon means ,17,18.neu-positivity and lack of hormone receptor expression in breast tumours [neu positive tumours were weakly related with the absence of estrogen receptors, although this was not statistically significant .Numerous epidemiological and experimental studies have shown the strong relationship between HER-2/ tumours ,18,22. IThe interview was conducted during the subjects' first visit to the unit and before clinical examination or any other intervention took place. This constitutes an advantage, because there was no chance that the subjects (both cases and controls) would be influenced by the diagnosis and might therefore falsely inflate the relative risk. Thus, the likelihood of recall bias is not high, improving the comparability of several covariates in both groups, and the selection bias is lessened since all subjects had taken the same route through the Breast Unit's standard routine procedures.neu status subgroup. Some caution regarding our findings is related to the size of the study group. In the analyses stratified by HER-2/neu and menopausal or ER status numbers are quite small and for some risk estimates the confidence intervals are wide and the estimates of risk unstable.Since each case group was compared with the same control group, any selection bias would be expected to have a similar effect on the estimates in the tumour subgroups. Thus, it is extremely unlikely that recall bias issues would apply only to those cases in a specific HER-2/neu+ versus HER-2/neu-breast tumours, with the exception of increased BMI. Further innovative studies with larger sample sizes are needed to examine how the status of these potentially modifiable breast cancer risk factors interacts with biological markers such as HER-2/neu oncoprotein. Their findings will provide us with greater insight into breast cancer aetiology and will help us identify any association that would help discriminate subgroups of women at higher risk.In conclusion, our study did not confirm that the established or putative hormonal breast cancer risk factors differ regarding their relations with HER-2/EGFR: epidermal growth factor receptor, HRT: hormonereplacement therapy, BMI: body mass index, SHBG: sex hormone-binding protein, ER: estrogen receptor, PR: progesterone receptor, OC: oral contraceptives.The author(s) declare that they have no competing interests.NT conceived the study, participated in its design and drafted the manuscript.ES participated in the design of the study, assisted in writing and reviewed the final article.EfS and KM scheduled and performed the laboratory analysis.NA performed the statistical analyses.VG and DDT participated in the design and coordination of the study and reviewed the final article.All authors have read, discussed and approved the manuscript.The pre-publication history for this paper can be accessed here:


A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P). However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line.RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments.We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA) or all-trans-retinoic acid (ATRA), and strongly inhibited by N-(4-hydroxyphenyl) retinamide (HPR). The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that the potency of retinoids on cell growth inhibition may be increased through combination of estrogen and progesterone treatment.Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of  It is well documented that women who experience a full-term pregnancy early in their lives have a significantly reduced risk for developing breast cancer ,2. The mRetinoids are a promising class of chemopreventive agents against breast cancer because of their antiproliferative and proapoptotic properties ,6. RetinThe 76N TERT cells were derived from a reduction mammoplasty specimen ,9. They In this study, we investigated whether E/P induce growth inhibition in 76N TERT cells; and the molecular mechanisms by which E/P inhibited 76N TERT cell growth. For comparison purpose, the anti-growth effect of both natural and synthetic retinoids was examined in parallel in this immortalized mammary epithelial cell line. We also investigated whether we could increase the responsiveness of retinoids by using retinoids in combination with E/P. Our studies demonstrate that 1) inhibition of cell growth by E/P and retinoids could be partially mediated through a p53-dependent mechanism; 2) induction of p21 expression, inhibition of telomerase activity, or up-regulation of estrogen receptor beta (ERβ) by E/P and retinoids may contribute to their anti-growth effects; 3) combination of retinoids with E/P lead to increased inhibitory effects on cell growth.As the ability of estrogen, progesterone and retinoids to influence cell proliferation is mediated by their respective receptors in most cases, we first examined the expression of these receptors in 76N TERT cells. Using Western blot analysis, the proteins of RAR , RXR , and ER (ERα and ERβ) were observed as suggested by the manufactures Figure . The ant3H]thymidine incorporation assay. The concentrations of E/P and retinoids used in the experiments were chosen based on previous studies and are clinically or physiologically relevant thymidine  for 24 hours, cells were harvested by washing with PBS and 10% TCA, solubilizing with the mixture of 0.1% SDS and 0.1N NaOH. Aliquots were taken for the quantification of radioactivity by the Tri-Carb 2900 TR Liquid Scintillation Analyzer . Incorporation of [3H]thymidine was expressed as a fold change from vehicle control under the same conditions.Cells were seeded into 24-well plates at 5 × 104 cells per well. Cells were transiently transfected with 0.5 μg of either a RARE-luciferase or a p53-luciferase plasmid along with 0.05 μg of pCMV-Renilla luciferase using the SuperFect transfection reagent , following the manufacturer's recommended procedure. Twenty-four hours after transfection, triplicate cultures were treated with retinoids for 24 h in the presence or absence of E/P. The cells were then washed and lysed. The luciferase activities were measured using the DUAL-luciferase Assay System , and normalized by pCMV-Renilla luciferase activity for each sample.Cells were seeded in 24-well plates at 5 × 10g for 20 min at 4°C, and the supernatant was collected. The protein concentration was measured in each extract using the BCA protein Assay Reagent Kit . Telomerase activity was determined in duplicates by a quantitative real-time PCR telomerase detection Kit  according to the manufacturer's protocol, using Mx4000 Multiplex Quantitative PCR System .Cells were lysed in CHAPS lysis buffer  and incubated at 4°C for 30 min. The lysate was then centrifuged at 12000 × Cells at subconfluence were treated with retinoids in the presence or absence of E/P for 72 hours. Total RNA was extracted with TRIZOL Reagent from Gibco  according to the instructions of the manufacture. Single-stranded cDNA was made from 1 μg of total RNA with the Cells-to-cDNA kit  at 42°C for 15 min. The primers for ERβ were 5'CGA TGC TTT GGT TTG GGT GAT 3' (forward) and 5'GCC CTC TTT GCT TTT ACT GTC 3' (reverse). The primers for GAPDH were 5'CCA TGG AGA AGG CTG GGG 3' (forward) and 5'CAA AGT TGT CAT GGA TGA CC 3' (reverse). All primers were from Integrated DNA Technologies, Inc. . cDNA (1 μl) was amplified in duplicates in Mx4000 Multiplex Quantitative PCR System  by using Brilliant SYBR Green QPCR Master Mix from Stratagene . The reaction was carried out at 95°C for 10 min to denature, 40 cycles of 95°C for 30 sec, 55°C for 60 sec, 72°C for 60 sec. ERβ gene was quantified and normalized with external standard GAPDH.2, and protease inhibitor cocktail , followed by centrifuged at 12,000 rpm for 20 min. Protein content was determined using the BCA Protein Assay Reagent Kit . Cell lysates (~30 μg protein) were separated on 10% polyacrylamide gels in the presence of 0.1% SDS with prestained low-range molecular-weight standards . After transfer, the membranes were blocked and then probed with antibodies against interested proteins as suggested by manufactures, followed by incubation with a peroxidase-conjugated secondary antibody. Immunoreactive bands were developed with an ECL reagent from Amersham . All blots were probed with anti-actin to normalize for loading differences. Quantification of gels was carried out using ImageJ software .Cells were treated with retinoids in the presence or absence of E/P for 72 hours. Cell lysates were obtained by incubating cells for 30 minutes at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 100 mM NaF, 10 mM MgClThe p53 (DO-1), p21 (F-5) and RARγ (G-1) mouse monoclonal antibodies, and RXRα (D-20) polyclonal antibody were from Santa Cruz Biotechnology Inc . Anti-ERβ (Ab-2), ER (Ab-1), PR (Ab-1), RARα and RARβ mouse monoclonal antibodies, and Bax (Ab-1) polyclonal antibody were from Calbiochem . Monoclonal anti-actin, RXRβ and RXRγ were from Sigma .Statistical differences were analyzed by T-test. Levels of statistical significance were set at p < 0.05.9-cis RA: 9-cis-retinoic acid; ATRA: all-trans-retinoic acid; E/P: ?β-estradiol and progesterone; ERβ: estrogen receptor beta; HPR: N-(4-hydroxyphenyl) retinamide; PR: progesterone receptors; RARs: retinoic acid receptors; RXRs: retinoid X receptors.The author(s) declare that they have no competing interests.JZ designed and carried out most of the assays and drafted the manuscript. YT carried out the real-time PCR experiments. SS conceived of the study and participated in its coordination. All authors read and approved the final manuscript.


The aim of this study is to validate the questionnaire ECOS-16  for the evaluation of health related quality of life (HRQoL) in post-menopausal women with osteoporosis.An observational, prospective and multi-centre study was carried out among post-menopausal women with osteoporosis in primary care centres and hospital outpatient clinics. All patients attended 2 visits: at baseline and at 6 months. In addition, the subgroup of outpatients attended another visit a month after the baseline to assess the test-retest reliability. The psychometric properties of the questionnaire were evaluated in terms of feasibility, validity  and internal consistency in baseline, and in terms of test-retest reliability and responsiveness to change in visit at month and visit at 6 months, respectively. In all visits, ECOS-16, EUROQoL-5D (EQ-5D) and four 7-point items about health status  were administered, whereas only outpatients were given MINI-OQLQ , besides all clinical variables; and sociodemographic variables at baseline.Feasibility: 94.3% of patients answered all items of the questionnaire. The mean administration time was 12.3 minutes. Validity: factor analysis suggested that the questionnaire was unidimensional. In the multivariate analysis, patients with vertebral fractures, co-morbidity and a lower education level showed to have worse HRQoL. Moderate to high correlations were found between the ECOS-16 score and the other health status questionnaires (0.47–0.82). Reliability: internal consistency (Cronbach's α) was 0.92 and test-retest reliability (ICC) was 0.80. Responsiveness to change: ECOS-16 scores increased according to change perceived by the patient, as well as the effect size (ranges between 1.35 to 0.43), the greater the perception of change in patients' general health status, the greater the changes in patients' scores. The Minimal Clinically Important Difference (MCID) suggested a change of 0.5 points in the ECOS-16 score, representing the least improvement in general health status due to their osteoporosis: "slightly better".316 women were consecutively included, 212 from primary care centres and 104 from hospital outpatient clinics. ECOS-16 has been proven preliminarily to have good psychometric properties, so that it can be potentially a useful tool to evaluate HRQoL of post-menopausal women with osteoporosis in research and routine clinical practice. Osteoporosis is characterised by low bone mass and a deterioration in bone tissue micro-architecture, leading to increasing bone weakness and consequently risk of fracture. The most common clinical complications of osteoporosis are hip fracture, vertebral deformity and wrist fracture. According to bone densitometry values, osteoporosis affects approximately 2 million women in Spain ,2. The mTo date, clinical trials on osteoporosis have been based on outcomes measured by imaging tests. But these measurements do not adequately reflect the extent to which the patient is affected in their usual daily activities, and are not appropriate to assess patients' disability and symptoms . NeverthDisease specific questionnaires for osteoporosis are available, such as the Osteoporosis Quality of Life Questionnaire (OQLQ)  or the QThe aim of the present study is to evaluate the psychometric properties of the ECOS-16 in post-menopausal women with osteoporosis.316 post-menopausal women with primary osteoporosis attended in Primary Care Centres or in outpatient clinics were included in the study. Diagnosis was confirmed by a Bone Mineral Density (BMD) using Dual Energy X-Ray Absorptiometry (DEXA). Furthermore, outpatients should have at least one prevalent vertebral fracture confirmed by Genant's radiological criteria due to osteoporosis, a requirement which was not essential in patients from Primary Care Centres. 212 patients from 49 Primary Care Centres and 104 patients attended in outpatient clinics from 14 hospitals were consecutively selected and evaluated from March 2000 to August 2001.All patients attended two visits, a baseline and a follow-up visit 6 months after the inclusion. In order to evaluate test-retest reliability, outpatients were also attended in another follow-up visit one month after the baseline. All patients received the study information and gave their informed consent.An observational, prospective and multi-centre study was carried out for the validation of the ECOS-16 in post-menopausal women with vertebral fractures due to osteoporosis in conditions of clinical practice. At the baseline, feasibility together with the content and construct validity of the ECOS-16 were evaluated. At the visit after 6 months, responsiveness to change with regard to ECOS-16 was evaluated. Outpatients also attended another visit a month after the inclusion so that the test-retest reliability of the ECOS-16 could be evaluated.In the baseline, data on the patients' sociodemographic characteristics  and clinical variables  were collected as well as the ECOS-16, the EUROQoL-5D and four 7-point items which refer to general health status, back pain, limitation in daily activities and emotional status. These items were used in a previous study and showed its validity . OutpatiIn the two follow-up visits, any modifications to the specific treatment for osteoporosis prescribed in the baseline, the number of concomitant treatments, and patients' withdrawals causes were registered. Moreover, the ECOS-16, EUROQoL-5D and the four 7-point change items were again administered. Outpatients were again administered the MINI-OQLQ questionnaire.In the present study, the EQ-5D and MINI-OQLQ questionnaires were used in order to assess the validity of the ECOS-16.The ECOS-16  where it was acceptable for the questionnaire to be administered by health care personnel or experienced interviewers.The EUROQoL-5D (EQ-5D) is a generic HRQoL questionnaire. The EQ-5D consists of two parts: a descriptive system and a Visual Analogue Scale (VAS) . The desThe VAS of the EQ-5D is a vertical scale divided into millimetres along a 20 centimetres long thermometer where the two ends are labelled "worst imaginable health state" and "best imaginable health state" with a score of 0 and 100, respectively. The respondent should mark the point on the thermometer which, in their opinion, best describes their actual overall health status.The MINI-OQLQ is a specific HRQoL questionnaire for women with vertebral fractures due to osteoporosis. The MINI-OQLQ is based on a selection of the two highest impact items from each of the five domains of the Osteoporosis Quality of Life Questionnaire (OQLQ). The MINI-OQLQ is, therefore, composed of 10 items grouped into the same five HRQoL dimensions  .Each item has seven response options ranging from 1 (worse HRQoL) to 7 (better HRQoL). The scoring is obtained per dimension, by calculating the mean score of the response items for each dimension, so that the higher the score in each dimension, the better the resulting HRQoL.Changes in four health status items were assessed through four different items regarding the change in the patient's overall health status: the change in the patient's general health status due to osteoporosis, the change in the patient's back pain, the change in the patient's limitation in daily activities and the change in the patient's emotional status, all of them due to osteoporosis, with reference to the baseline. The items have seven possible response options, ranging from "Much better" to "Much worse" and including the category "More or less the same". These items were designed to be self-administered and validated in previous studies ,14.® for Windows version 10.0  was used to carry out the entire data analysis.Double data entry was carried out with a subsequent validation to guarantee the quality and consistency of the data. A statistical significance level of p < 0.05 was used in all statistical tests performed. The statistical program SPSSPreviously the statistical analysis, a Kolmogorov-Smirnov test was conducted to assess the distribution of the variables in order to use a parametric or non-parametric tests.With the aim to describe the study sample characteristics and to evaluate differences among patients with and without vertebral fractures a descriptive and comparative analysis was done on patients' sociodemographic characteristics  and clinical variables (Body Mass Index (BMI), number of years with menopause, presence or absence of non-vertebral fractures, concomitant diseases and received treatment during the previous year) according to vertebral fracture presence. In order to compare the two groups, the chi-squared test was used, given that the variables were categorical, and the Bonferroni correction for multiple tests.The feasibility of the ECOS-16 was analysed on the basis of missing data, time and method of administration. In order to evaluate the missing data, the number and percentage of missing response items in the whole questionnaire as well as the number and percentage of patients who failed to respond to any of the questionnaire's items were both calculated. The time spent administering the questionnaire was evaluated according to the method of administration (self-administered or administered by an interviewer).The floor (percentage of patients with the lowest score) and ceiling (percentage of patients with the highest score) effect were calculated for each one of the ECOS-16 items and for the overall score.An exploratory factor analysis was used to assess the content validity of the ECOS-16, using the scores obtained during the baseline by all patients for all 16 questionnaire items. Factors were extracted using principal-axis factoring method and varimax rotations. The adequacy of the factor analysis was assessed with the Kaiser-Meyer-Olkin measure and the Bartlett's test of sphericity .To evaluate the construct validity, the correlations between the ECOS-16 scores and the patients' sociodemographic and clinical characteristics  were analysed. Pearson's correlation coefficient was used for continuous variables and the analysis of variance (ANOVA) for categorical variables. A multivariate analysis was also carried out taking the ECOS-16 score as a dependent variable and the sociodemographic and clinical variables that were significant in the bivariant analysis as independent variables, so that any possible confounding factors could be controlled. Secondly, the relationship between the ECOS-16 and the EQ-5D scores, the four 7-point items  and the MINI-OQLQ (only outpatients) were all analysed using Spearman's correlation coefficient, apart from the VAS which was analysed using Pearson's correlation coefficient. Higher correlations were expected between dimensions that measure the same HRQoL aspects.Because the lowest score in ECOS-16, in EQ-5D and in 7-point general health status item represent the best HRQoL, the expected correlations among their dimensions would be positive. The opposite applies to the dimensions of MINI-OQLQ and the other three 7-point items , where the lowest score represent the worst HRQoL and, therefore, the expected correlations between ECOS-16 would be negative.The reliability of the ECOS-16 was evaluated in terms of internal consistency and test-retest reliability. Internal consistency was calculated by Cronbach's α coefficient using the baseline scores of all questionnaire items. Test-retest reliability was evaluated only for outpatients who did not perceive a change in their general health status due to osteoporosis after a month, as shown by the change in 7-point general health status item (response category: 'More or less the same'). The Intraclass Correlation Coefficient (ICC) between the scores for both visits was used for this analysis. The hypothesis that the standard psychometric recommendations for Cronbach's α and ICC were greater than or equal to 0,7 was taken as a starting point for both internal consistency and test-retest reliability [Longitudinal validity of the ECOS-16 was evaluated by analysing the correlations among changes registered in the ECOS-16, and changes in the EQ-5D, changes in the MINI-OQLQ and changes in the four 7-point change items from baseline to visit at 6 months. For this purpose the Spearman's correlation coefficient was used. The expected correlations are the same as in the construct validity.To assess responsiveness to change, first of all, attention has been drawn to whether the questionnaire detects the changes which are perceived by patients between the baseline and the visit at 6 months. In order to do so, the Student's t-test for paired data was used. To assess the magnitude of changes, the effect size was calculated, thus it was establish that the changes in the patients' scores increased at the same time that the changes perceived by patients. In order to calculate the effect size, the change in 7-point general health status item is used .The Minimal Clinically Important Difference (MCID) has been defined as the smallest difference between the scores in a questionnaire that the patient perceives to be beneficial . The MCITable Statistically significant differences (p < 0.01) were found for age, concomitant diseases (patient-reported), and length of time with menopause, it being the case that those patients with vertebral fractures were older and had had menopause for a longer time. In addition, those patients with vertebral fracture were those that had received more treatments during the previous year (p < 0.01) and also had a greater BMI (p < 0.05). However, those patients without a vertebral fracture presented concomitant diseases more frequently (p < 0.01). Only 14.4% of patients showed some type of non vertebral fracture and about 70% had completed at least primary education level.After the Bonferroni correction, the analysis that were still significant  were age, years with menopause, concomitant disease and previous treatment.At baseline, all osteoporotic patients were prescribed or changed their treatment by the physicians, some having changed his previous treatment but others having received no treatment before.The mean (SD) administration time of the questionnaire was 12.3 (7.8) minutes for all patients, with a median of 10 minutes. In 55.1% of cases, the questionnaire was self-administered, the remainder being administered by health care personnel (due to eyesight difficulties or illiteracy). No statistically significant differences were observed in administration times according to the type of administration.The 99.7% of the patients answered all items of the questionnaire. Only one patient failed to respond to any item.A third of the items had a floor effect greater than or equal to 20%, 38.3% in item "Do you have problems with dressing?". The maximum ceiling effect was observed in 55.7% of the patients in item "How often have you had back pain in the last week?". In the remaining items, the ceiling effect was lower, ranging from 1.3% to 20.3%, the highest being in item "Are you afraid of getting a fracture?". Two patients (0.7%) recorded the highest scores for all the items .In the factor analysis, the Kaiser-Meyer-Olkin measure was 0.916 indicating a good sampling adequacy. The Bartlett's test of sphericity (p < 0.001) made it possible to accept the identity of the matrix correlations for the ECOS-16 items, thus indicating the suitability of the factor analysis.Table A multivariate analysis was carried out to identify patients' characteristics that were related to the ECOS-16 score . All these correlations were also statistically significant.The internal consistency of the ECOS-16 was very high, with a Cronbach's α coefficient of 0.92. Test-retest reliability was analysed for 44 outpatients who declared that their general health status due to osteoporosis had not changed after a month, with an Intraclass Correlation Coefficient of 0.80 and a mean (SD) score change of 0.2 (0.5) points.Figure The measurement of HRQoL has attracted increasing attention as a clinically relevant outcome of research and clinical practice. HRQoL questionnaires reflect the impact of health care interventions on health aspects such as physical, mental and social well-being. However, either in clinical research and in practice, a lengthy questionnaire is problematic for both the health care personnel and the patient. Shortish measure attempt to minimize time and effort as well as to increase patient interest . Thus, sThe ECOS-16 originates from the reduction of two validated and widely used HRQoL questionnaires in osteoporosis patients with vertebral fracture . HoweverAlmost all the patients responded to all the questionnaire items. The presence of a floor effect for one third of the questionnaire items could lead to the conclusion that the questionnaire detects changes only when the severe disease status occurs. However, according to a previous qualitative division , the floAs some previous studies, this study shows that the variables usually used to evaluate patients with osteoporosis, such as Bone Mineral Density (BMD) and the presence of vertebral fractures, have low or no correlation with HRQoL scores. This finding is not new ,20 and sIn this study, the fact that the presence of vertebral fractures does not have a negative effect on HRQoL  deserves special attention. There seems to be some discrepancy regarding this issue in literature. Several studies have shown that HRQoL progressively deteriorates in relation to the presence and number of vertebral fractures ,24. HoweRecently, a significant number of studies have highlighted the importance of the site of the vertebral fracture and its effect on HRQoL. In this regard, it seems that the site of vertebral fracture has a much greater effect on HRQoL than the presence and number of vertebral fractures -30. ThisAlthough in the present study, the bivariate analysis does not discriminate between patients with and without vertebral fracture, the multivariate model shows that the presence of fractures is indeed significant even if the percentage of explained variation is small. This is not surprising given the fact that such analysis is dealing with prevalent fractures in a short term observational study . The incThe results obtained for internal consistency and test-retest reliability showed high levels of homogeneity among questionnaire items and good reproducibility over time. Moreover, the outcomes, have also been shown that the new questionnaire effectively detects changes in patients' perceived health status due to osteoporosis. Expressing responsiveness to change is an important characteristic of the new instrument, one which will doubtless allow its use in clinical research. The high correlation between the ECOS-16 and generic (EQ-5D) and specific (MINI-OQLQ) HRQoL questionnaires corroborates this hypothesis. It also highlights the new questionnaire's validity by demonstrating that it measures concepts which are closely related to already validated HRQoL questionnaires ,13.The mean change in score per question corresponding to the effect size in general and to the MCID in particular is consistent, in terms that the larger the change assessed by the ECOS-16, the larger the effect size. Moreover, MCID is consistent with the results of other HRQoL questionnaires, and it is useful to compare the magnitude of changes detected between them. MCID will also be useful in the planning of new trials, as sample size depends on the magnitude of the difference investigators consider clinically important and are not willing to risk failing to detect .The potential limitations of this study are mainly due to it being unable to rely on certain variables which have shown a clear influence on HRQoL. In particular, the "time spent since the fracture occurred"  was not The low education level of the study sample must also be taken into account. It is consistent with previous studies ,9, and aIn conclusion, the ECOS-16 is a HRQoL questionnaire which is short, easy to administer  and with adequate preliminary psychometric properties. This makes the ECOS-16 potentially very useful during routine clinical practice or/and research for the treatment and follow-up of post-menopausal women with osteoporosis. Nevertheless, its actual potential must be proven in future clinical trials in order to recommend its use in research and clinical practice.ECOS Appendix1.doc, the ECOS-16 questionnaireClick here for file