The medical needs of many of the world's population go unmet. A new treaty on essential health R&D could provide a binding framework to redirect today's scientific expertise to priority needs Biomedical science and technology are developing at a more rapid pace than ever. Investments in health research and development (R&D) have never been higher—global spending on health research increased from US$30 billion in 1990 to US$105.9 billion in 2001. But despite advances in technology and unparalleled research spending, the medical needs of many of the world's population go unmet. For example, only 1% of new drugs approved between 1975 and 1999 were specifically developed for tropical diseases and tuberculosis—diseases that account for over 10% of the global disease burden  [1].In recent years, some important steps have been taken to improve access to existing treatments in the developing world by increasing generic competition. Yet there continues to be a tension between promoting access to lifesaving medicines as a human right and maintaining a global trade regime that seeks to finance health R&D by allowing monopolies to charge high prices .There is a growing demand from many quarters for a new international policy framework . A new iUntil recently, providing patent protection for pharmaceuticals was a choice made by individual governments according to their level of industrial development. Today, pharmaceutical patents are globalized through the World Trade Organization's Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS Agreement) , and theLancet in 2002 showed that 68% of all new chemical entities marketed worldwide in the last 25 years were me-too products, representing little or no therapeutic gain [A study published in the tic gain .According to the United Nations Development Programme, less than 5% of drugs introduced by the top 25 pharmaceutical companies in the US represented true therapeutic advances; of these, 70% were developed with government involvement (, p. 69).Studies of drug development over the last decade in the US  and the 92% of medicines approved in 2002 by the US Federal Drug Administration were me-too drugs .www.unaids.org). The poor also dominate non-communicable disease tables, accounting for 59% of the 56.5 million annual global deaths [The poorest are hardest hit. While R&D of new therapies against tropical diseases has ground to a standstill, 14 million people die from infectious diseases each year, predominantly in developing countries [The patent system is also promoting new inequalities in high-income countries. Americans now spend a staggering $200 billion a year on prescription drugs. This figure is growing at a rate of about 12% per year . The avePatents, with their focus on maximizing profits, have at least three negative consequences. First, it has been argued that the patent system causes substantial welfare losses because consumers who would buy the product if it were priced at somewhere nearer production cost do not buy it at the monopoly price . Second,The only major international policy instrument that exists today to stimulate and finance health R&D is the TRIPS Agreement . The TRIIn 2001, the Doha Declaration on TRIPS and Public Health affirmed the sovereign right of WTO members to take measures to protect public health by overcoming patents whenever needed . The lasHealth R&D must be treated as an international problem that requires an international solution. It should be treated like other strategic sectors, as happens today for defense and space discovery—sectors that both benefit from very strong government support for innovation. When global public goods do correspond to national needs, governments should step in to mobilize and enforce the collective action required. For example, global cooperation in the sharing of infectious disease monitoring from 1890 onwards set a valuable precedent .The recent epidemic of severe acute respiratory syndrome—SARS—clearly shows that biomedical knowledge and the pharmaceutical sciences can be mobilized to achieve rapid advances relevant to social needs if sufficient resources and political will can be mustered. The SARS virus was completely sequenced in just six days, and a diagnostic test was developed in only three months. The public-sector funded, collaborative “public-goods model” used for the Human Genome Project shows that public collaborative research can be more efficient than the closed, monopolistic, private sector approach.An international treaty  would prA global, needs-driven R&D agenda: allowing policy makers, funding agencies, and the research community to set priorities for developing safe, effective, and affordable medicines according to health needs.Prioritization for neglected diseases: to ensure that immediate efforts are made toward finding new tools for lethal diseases that are currently difficult or impossible to diagnose and treat.Adequate international financing of health R&D: a new funding mechanism is urgently needed to support R&D on an ongoing basis, particularly for neglected diseases. All governments will need to participate according to their means.Equitable pricing: governments should ensure that the poor also have access to innovations resulting from government-funded or university research.Open access: governments should require access to the compounds and tools that result from public research in order to stimulate follow-on innovation elsewhere.International exchange: strengthening openness and transfer of technologies on a global basis will greatly help developing countries by improving access to information and ideas and accelerating the development of science and technology.The idea is to shift the discourse from trade to health. The treaty—focussed directly on R&D rather than patent rights or drug prices—would address the global management of publicly funded health R&D. Priorities for R&D would be defined through public-sector leadership and based on public health needs. R&D opportunities would be aimed at new lead compounds, new types of health tools  and new One of the main objectives of the treaty would be to encourage the broad dissemination of information and knowledge-sharing, and to support diversity, competition, and collaboration among researchers from developed and developing countries.www.tropicaldisease.org), for example, is a new, Internet-based, community-wide effort to develop new drugs for tropical diseases [http://parts.mit.edu/) at the Massachusetts Institute of Technology is exploring standardized tools and processes for DNA work, largely by computer. The Bios Initiative , launched by the Australian non-profit organization Cambia , is an effort to develop new innovation systems for market failures and for neglected priorities [There are already precedents for the free, public sharing of innovations with the aim of developing new drugs. The Tropical Diseases Initiative (diseases . The Bioiorities .www.edctp.org) [Among other incentives, technology exchange frameworks could include licensing agreements with developing countries, or affirmative commitments of research funds for collaborative projects with these countries. Such collaboration is currently being implemented in Europe, for example, through the European and Developing Countries Clinical Trial Partnership (ctp.org) . The parMuch of today's drug development know-how exists within the private sector. Further work is needed to define obligations and incentives in the treaty that maximize industry contributions to publicly funded R&D by providing in-kind contributions in areas where industry has the skills that public groups need. The treaty should also provide an expanded use of government rights against patent abuse on drugs developed with public support. This would include the right of a government to intervene if an invention is not made available to the public on reasonable terms, such as is included in the march-in rights clause of the United States's 1980 Bayh-Dole Act .There are a number of obvious difficulties in moving the treaty forward, and these should not be underestimated. A delicate issue is the treaty's relation with other binding agreements, particularly the TRIPS Agreement. Governments that join the treaty should be granted patent exceptions and should not be accused of “free-riding,” since they would be contributing to R&D through a different juridical avenue.Substantial government resources would need to be mobilized to finance the highest priority medical research. All governments should participate according to their means. Countries already contribute significantly to global R&D through the purchase of costly patented drugs. Among other measures, not-for-profit initiatives working to develop new drugs, vaccines, and diagnostic tools for neglected diseases should be funded at levels that enable them to reach their objectives. Recent examples clearly show that when political will is mobilized, resources are rapidly made available to generate R&D in a particular area. In 2001, the anthrax scare in the US led to increases in biodefense research spending at the US National Institutes of Health from US$53 million in 2001 to US$1.6 billion in 2004.A treaty on health R&D is certainly a feasible proposal—the successful adoption of a treaty on plant genetic resources shows that it can be done. After seven years of negotiations, the Food and Agriculture Organization of the United Nations adopted the International Treaty on Plant Genetic Resources for Food and Agriculture in November 2001 . This leThe Ottawa Convention to Ban Anti-Personnel Landmines and the 2003 Framework Convention on Tobacco Control show that international frameworks are essential to the regulation of the private sector for the good of global health. The World Health Organization, together with other relevant United Nations agencies, has full legitimacy to work with member states toward crafting challenging proposals, and provoking policy action. One lesson from these treaties is that support will have to be built from a strong coalition of like-minded countries that would steer the process internationally.While the development of the treaty is still at an early stage of discussion, the concept is already being aggressively opposed. As with tobacco, landmines, and more recently, sugar, the involvement of civil society will be crucial to defend these health improvement strategies where these may conflict with powerful vested interests in the private sector .It takes courage to change the rules. If governments are indeed persuaded to face up to their responsibilities in the coming years, it may very well be because of the many voluntary organisations that seek to promote the global public interest.


Currently, there is no adequate animal model to study the detailed molecular biochemistry of fragile X syndrome, the leading heritable form of mental impairment. In this study, we sought to establish the use of immature neural cells derived from adult tissues as a novel model of fragile X syndrome that could be used to more fully understand the pathology of this neurogenetic disease.By modifying published methods for the harvest of neural progenitor cells from the post-mortem human brain, neural cells were successfully harvested and grown from post-mortem brain tissue of a 25-year-old adult male with fragile X syndrome, and from brain tissue of a patient with no neurological disease.The cultured fragile X cells displayed many of the characteristics of neural progenitor cells, including nestin and CD133 expression, as well as the biochemical hallmarks of fragile X syndrome, including CGG repeat expansion and a lack of FMRP expression.The successful production of neural cells from an individual with fragile X syndrome opens a new avenue for the scientific study of the molecular basis of this disorder, as well as an approach for studying the efficacy of new therapeutic agents. FMR1) gene to greater than 200 repeats (full mutation) [FMR1 protein (FMRP), an RNA-binding protein that is important for neural development and plasticity [Fragile X syndrome, the leading heritable form of mental impairment ,2, is geutation) . Such exutation) ,5 and thasticity ,7.FMR1 gene. The absence of a suitable model system is a consequence of the inability to clone full mutation alleles, and because no animal system has been found that carries native, full mutation FMR1 alleles. Thus, all animal or cell model systems for fragile X syndrome are based on FMR1 (homolog) knock-out constructs. While these models qualitatively recapitulate some of the features of the fragile X syndrome phenotype, they do not address any of the potential consequences of the expanded methylated CGG repeat.Although great strides have been made from animal models in our understanding of the neuropathology of fragile X syndrome -13, therAs with most disorders of the human nervous system, it has been impossible to directly study the detailed cellular pathogenic mechanisms that underlie fragile X syndrome, due to the absence of a suitable (human) cell model. However, this barrier may be overcome through the use of neural progenitor cells, which comprise relatively undifferentiated populations of cells in the central nervous system (CNS) that give rise to the broad array of specialized cells, including neurons and glial cells. Long thought to be an exclusive component of the developing CNS, these cells have been shown to exist in the adult CNS -19. ReceImportantly, we have previously shown that neural progenitor cells can be harvested from adult post-mortem brain tissue ,20, whicIn the current report, we describe the successful culturing of neural progenitor cells from an adult male with fragile X syndrome, and some of the characteristics of these cells. We further demonstrate initial efforts to differentiate these progenitor cells into both neuronal and astrocytic lineages. As expected, expression of FMRP is substantially reduced relative to its expression in neural cell culture from an unaffected control.Prior to tissue acquisition by the authors, informed consent for the donation of tissues was obtained under the auspices of the protocol for the National Human Neural Stem Cell Resource. This protocol is approved by Institutional Review Board (IRB) of Children's Hospital of Orange County, and follows a protocol approved by the UC Davis School of Medicine IRB. All tissues were acquired in compliance with NIH and institutional guidelines. Two patients were used for the present study: a patient with fragile X syndrome and a patient with no neurogenetic disease.The autopsy followed standard procedures as described . The perPortions of fresh brain, heart, and testicular tissue were received, and fixed in 10% formalin for 10 days prior to sampling and processing for paraffin sections in standard fashion. All tissue sections were stained with hematoxylin and eosin, with cardiac valves and aorta additionally stained for elastin and mucin. All sections were examined by standard light microscopy.The base medium was a high glucose 1:1 DMEM:F12 (Irvine Scientific). The basal medium (DGA) used for all other media was the base medium containing glutamine, penicillin, streptomycin, gentamicin, ciprofloxacin, and amphotericin as previously described . Medium 2 of fibronectin  overnight at 37°C, the fibronectin solution aspirated, and the plates allowed to air dry before the introduction of tissue homogenates.All procedures were performed as previously described , with mo2 total surface area).Approximately 300 mg fresh tissue was subjected to mincing and trituration, and the resulting crude tissue homogenate plated into six wells of a fibronectin-coated six-well tissue culture plastic plate , and these cells were cultured for two weeks before immunocytochemical analysis, or differentiation and immunocytochemical analysis, as previously described [At near confluence, cultures were passaged by lifting with a solution of Cell Dissociation Buffer (GIBCO) supplemented with trypsin. The cells were washed twice with DGF and plated in 1:1 (conditioned:fresh) medium into a fibronectin-coated T75 flask. Thereafter, and at approximately one week intervals, cells were lifted and similarly plated into twice the surface area from which they were removed. After the cells had reached a confluent surface area of 600 cmescribed .® Antifade Kit (Molecular Probes, Eugene, OR). Some cells were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma) before being rinsed and mounted. Pictures were imaged on an Olympus IX70 Microscope and digitally photographed via a Microfire digital camera (Optronics, Goleta, CA) using Image Pro Plus 4.5 with AFA plugin 4.5 software.Immunocytochemistry was performed as previously described . Primary6 neural progenitor cells and from post-mortem sections of about 500 mg of brain tissue using standard methods . For Southern blot analysis, 10 μg of isolated DNA were digested with EcoRI and NruI. The FMR1 genomic probe StB12.3, labeled with Dig-11-dUTP by PCR (PCR dig synthesis Kit, Roche Diagnostics), was used in the hybridization, as described in Tassone et al. [10. Southern blot and PCR analyses were both carried out using an Alpha Innotech FluorChem 8800 Image Detection System.Genomic DNA was isolated from approximately 5 × 10e et al. . Genomice et al. ; PCR pro6 neural progenitor cells and from post-mortem brain tissue using standard methods (Purescript, Gentra Inc. and Trizol). Reverse transcriptase reactions and quantitative fluorescence RT-PCR, using specific primers and probe set for the FMR1 gene and the control gene , were carried out as described in Tassone et al. [Total RNA was isolated from approximately 1 × 10e et al. .JS was a 25-year-old man with fragile X syndrome. His history included motor and language delays in childhood, with walking at 20 months, phrases at three years, and sentences at 6 years of age. He was diagnosed with fragile X syndrome at 11 years of age, by cytogenetic testing. His behavior included hyperactivity, anxiety, shyness, poor eye contact, hand flapping, finger biting, and perseverative speech. At age 24, cognitive testing with the WAIS III demonstrated a verbal IQ of 58, performance IQ of 51, and full scale IQ of 51. He did not have autism; childhood autism rating scale (CARS) score was 28.5 (below autism range).Previous medical history included severe mitral valve prolapse; echocardiography revealed moderate thickening and redundancy of both mitral leaflets, with central mitral regurgitation (grade 2 to 3+ by Doppler), mild tricuspid regurgitation, and moderate left ventricular and mild left atrial enlargement. Upon physical examination at age 24, JS had a long face, prominent ear pinna, high arched palate, and macroorchidism with testicular volume of 60 ml bilaterally. Blood pressure was 142/74, and a grade III/IV systolic and diastolic murmur with click was heard on examination. He died unexpectedly at age 25, presumably due to a cardiac arrhythmia secondary to mitral valve prolapse.A complete autopsy was performed 16 hours after death. Abnormal findings included increased brain weight , an enlarged heart, , with features of mitral valve prolapse , and increased testicular weight of 73 g . Our laboratory did not receive the intact brain, but portions of it, therefore detailed gross and histological evaluation was not possible. In particular, neither the hippocampus nor the amygdala was available for histopathological examination. Evaluation of available brain sections showed diffuse, acute early ischemic damage corresponding to the manner of death; no intraneuronal inclusions were seen, nor was there spongiosis of white matter in the cerebrum, cerebellum, or middle cerebellar peduncles. Cerebellar folia showed moderate patchy absence of Purkinje cells; because of the manner of death, loss due to ischemic damage cannot be ruled out. However, the majority of Purkinje cells present appeared histologically normal.2 of confluent adherent cells had been produced. Cells were then further expanded under serum-free conditions for immunocytochemical analysis, or under growth-factor-free conditions for biochemical analysis.Minced brain tissue, derived from the periventricular zone in the area of the head of the caudate nucleus and maintained under proliferation conditions in serum-containing medium with growth factors, yielded viable cells that formed an adherent monolayer on fibronectin-coated plates. When lifted and plated without serum or fibronectin substrate, the cells grew in suspended clusters/spheres Figure , similari) The cells were grown from cryopreserved, rather than fresh, tissue. This modification allows pathologists with no local access to a stem cell culture laboratory to preserve tissues for later stem cell harvest at a remote collaborating laboratory. (ii) No enzymatic digestion, only trituration, was used to generate the crude tissue homogenates. Preliminary studies showed that enzymatic digestion, typically used with fresh tissue, adversely affected our ability to harvest living cells from cryopreserved tissues. (iii) Serum (20%) was maintained in the culture until it was expanded to 600 cm2 of confluent adherent cells, after which the cells were cultured in serum-free medium. Preliminary studies indicated that, unlike cells harvested from fresh tissues, cells harvested from cryopreserved tissues required application of serum for a longer time in culture to sustain a sufficient rate of proliferation. (iv) The non-adherent fraction was transferred to new fibronectin-coated plates after one week. Preliminary studies showed that the non-adherent fraction from cryopreserved tissues retained a significant population of viable cells for a longer period of time than that from fresh tissue. These plates were cultured for an additional week before the remaining non-adherent fraction was finally discarded. Cells grown from both sets of plates were combined for expansion.Although the methodology used was similar to that previously reported , four coImmunocytochemical analysis of fragile X neural progenitor cells grown under expansion conditions demonstrated the expression of a range of developmental and mature neural markers Figure . Many ofFMR1 gene (GenBank accession number L29074). Southern blot analysis of DNA isolated from neural progenitor cells revealed the presence only of hypermethylated full mutation alleles (536 and 591 CGG repeats). No premutation alleles were detected by PCR analysis.Southern Blot analysis of DNA isolated from brain tissue  showed the presence of a mosaic pattern. Specifically, full mutation alleles were present in 82% of the cells (435, 528, 652, 727, 847 CGG repeats) with the remaining 18% harboring a premutation allele. Sizing of the CGG repeat number by PCR analysis demonstrated the presence of a premutation allele of 90 CGG repeats and an allele with the deletion of the CGG element and the flanking region, the latter of which was not detected by Southern Blot analysis. Sequence analysis of this allele indicated the presence of a deletion extending from nucleotide 13742 to 13915 of the FMR1 mRNA level  relative to the reference gene (glucuronidase) was low . In agreement with the observed lack of FMRP expression, no FMR1 message was detected after 40 cycles of PCR in total RNA isolated from the neural progenitor cells.The brain In the current work, we have successfully isolated and cultured neural progenitor cells from post-mortem brain tissue of an adult male with fragile X syndrome, which, to our knowledge, is the first example of the production of adult, human neural progenitor cells for any neurodevelopmental disorder. This result is of particular importance for the study of fragile X syndrome, since the disease-causing CGG repeat expansions have thus far been refractory to cloning into any animal or human cell model. With these cells, we hope to better understand the mechanistic link between the CGG expansion and the disease phenotype, which is known for the donor of the cells.We have demonstrated the feasibility of generating neural progenitor cell cultures from post-mortem brains of patients with neurogenetic disease. Moreover, as the cultures were generated from cryopreserved tissue, our data suggest that cells can be harvested and processed from the required tissues in locations that are remote from the stem cell culture laboratory. This last point is of particular importance for the study of neurogenetic diseases, which generally affect only 1:20,000 to 1:200,000 live births; a technique that can be implemented at multiple institutions will be necessary to generate sufficient numbers of specimens for statistical analysis.in vitro and in vivo pathophysiology. Due to the broad variability in phenotypic expression in fragile X syndrome , any such correlations are problematic using tissue obtained at fetopsy.A major determinant of the proliferative capacity of neural progenitor cells in culture is donor age, with younger donors (particularly fetuses and infants) having greater proliferative capacity than adults . AlthougThe method used to isolate the neural progenitor cells in the current study was adapted from the neuro-selective methods developed for culturing CNS stem cells from the brain , spinal The successful production of neural cells from an individual with fragile X syndrome opens a new avenue for the scientific study of the molecular basis of this disorder, as well as an approach for studying the efficacy of new therapeutic agents.The author(s) declare that they have no competing interests.PHS conceived of the study, participated in its design and coordination, and drafted the manuscript. FT carried out the molecular genetic studies, participated in the design of the study, and helped to draft the manuscript. CMG performed the neuropathological analyses. HEN carried out the cell culture. BZ performed the immunocytochemical analyses and imaging. RJH performed the clinical evaluation. PJH participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Between 2% and 5% of malignant germ-cell tumors in men arise at extragonadal sites. Of extragonadal germ cell tumors, testicular carcinoma in situ (CIS) are present in 31–42% of cases, and CIS are reported to have low sensitivity to chemotherapy in spite of the various morphology and to have a high likelihood of developing into testicular tumors. A testicular biopsy may thus be highly advisable when evaluating an extragonadal germ cell tumor.A 36-year-old man was diagnosed as having an extragonadal non-seminomatous germ cell tumor, that was treated by cisplatin-based chemotherapy, leading to a complete remission. In the meantime, testicular tumors were not detected by means of ultrasonography. About 4 years later, a right testicular tumor was found, and orchiectomy was carried out. Microscopically, the tumor was composed of seminoma.We herein report a case of metachronous occurrence of an extragonadal and gonadal germ cell tumor. In the evaluation of an extragonadal germ cell tumor, a histological examination should be included since ultrasonography is not sufficient to detect CIS or minute lesions of the testis. Between 2% and 5% of malignant germ-cell tumors in men arise at extragonadal sites . Cytogenrd July. Pathology showed the resected tumor was a seminoma with CIS .2. The side effect of CIS therapy (whether irradaition, orchiectomy or chemotherapy) are significant, especially concerning fertility and androgen production.3. Testicular tumors early detected by adequate surveillance respond well to treatments.4. Testicular biopsy is not entitled to detect all the CIS.The author(s) declare that they have no competing interests.IK, MU, HY, KN, TT and ND carried out clinical treatments.TM carried out histopathological studies.The pre-publication history for this paper can be accessed here:


We have examined genetic diversity at fifteen autosomal microsatellite loci in seven predominant populations of Orissa to decipher whether populations inhabiting the same geographic region can be differentiated on the basis of language or ancestry. The studied populations have diverse historical accounts of their origin, belong to two major ethnic groups and different linguistic families. Caucasoid caste populations are speakers of Indo-European language and comprise Brahmins, Khandayat, Karan and Gope, while the three Australoid tribal populations include two Austric speakers: Juang and Saora and a Dravidian speaking population, Paroja. These divergent groups provide a varied substratum for understanding variation of genetic patterns in a geographical area resulting from differential admixture between migrants groups and aboriginals, and the influence of this admixture on population stratification.ST = 0.010), the contemporary caste and tribal groups formed distinct clusters in both Principal-Component plot and Neighbor-Joining tree. In the phylogenetic tree, the Orissa Brahmins showed close affinity to populations of North India, while Khandayat and Gope clustered with the tribal groups, suggesting a possibility of their origin from indigenous people.The allele distribution pattern showed uniformity in the studied groups with approximately 81% genetic variability within populations. The coefficient of gene differentiation was found to be significantly higher in tribes (0.014) than caste groups (0.004). Genetic variance between the groups was 0.34% in both ethnic and linguistic clusters and statistically significant only in the ethnic apportionment. Although the populations were genetically close (FThe extent of genetic differentiation in the contemporary caste and tribal groups of Orissa is highly significant and constitutes two distinct genetic clusters. Based on our observations, we suggest that since genetic distances and coefficient of gene differentiation were fairly small, the studied populations are indeed genetically similar and that the genetic structure of populations in a geographical region is primarily influenced by their ancestry and not by socio-cultural hierarchy or language. The scenario of genetic structure, however, might be different for other regions of the subcontinent where populations have more similar ethnic and linguistic backgrounds and there might be variations in the patterns of genomic and socio-cultural affinities in different geographical regions. Human society in a geographic area develops when colonizing populations bring along with them different languages, cultures and technological advancements over a period of time. As more populations migrate to settle in the same area, they are either eliminated, subjugated or absorbed . In Indith – 5th century B.C. from southeast Asian countries of Java, Sumatra, Brunei and Indonesia [This study aims to understand the genetic diversity of populations of Orissa and examines the role of language and genetic origin on structure of populations inhabiting the same geographic region and evaluates some of the suggested population histories from a molecular perspective. Orissa is a coastal state in the southeast region of India, which is occupied by population groups having varied ethnicity, belonging to different strata of the hierarchical caste system and speaking languages belonging to different linguistic families. Its strategic geographic location between Northern plains and peninsular Southern India and cultures assimilated during the 4ndonesia  have enrndonesia . Other tndonesia . Of thesndonesia ,11,12. Hndonesia .Microsatellites or short tandem repeats (STRs) are most extensively used for elucidating the genetic diversity and evolution of human populations because of their abundance and prevalence in the genome, high level of polymorphism and amenability to automation -23. HighIn this study, we have examined variation at 15 autosomal STR loci in a sample of 404 individuals from Orissa  on the seven populations of Orissa have been previously reported ,31. ExceST was calculated separately for caste groups and tribes. The coefficient of gene differentiation was found to be significantly higher in tribes (0.014) than caste groups (0.004). Combining all seven populations yielded an FST of 1%, demonstrating low level of population differentiation within Orissa. All values of FST were significantly different from zero (p < 0.05). Analysis of molecular variance (AMOVA) presented in Table To quantify the amount of genetic diversity that exists among populations, Fibes 0.01 than casBecause the amount of genetic variance between groups was found to be low, we also used clustering algorithm implemented in STRUCTURE analysis Figure  to exploThe inter-population genetic relationship among Brahmins, Khandayat, Karan, Gope, Juang, Saora and Paroja was determined using principal component analysis. The plot Figure  of princThe Neighbour-Joining (NJ) tree Figure  gives a India is a remarkable representation of a large segmented society that harbours rich genetic diversity within its human populations and offers myriads of attributes to study the various factors influencing demographics of human populations. It is of particular interest to study patterns of genetic affinities among endogamous groups inhabiting small geographical regions within the subcontinent because of their diverse origins and interethnic admixtures.We have typed a set of fifteen polymorphic autosomal microsatellite markers in linguistically and socially divergent populations with different histories of origin to elucidate the genetic diversities and affinities among them and to understand the role of genetic origin and language on the genetic structure of populations living in geographic contiguity. The most distinctive feature of our study was the clear delineation between castes and tribes, as was evident from both multivariate and phylogenetic analyses Figure , 2b and The significantly low coefficient of differentiation among the seven populations (Fst: 0.010, p < 0.05), along with the number of alleles shared between them, confirms admixture and suggests an increased genetic affinity among populations residing in geographic proximity irrespective of their socio-cultural affiliation ,38,39. Tet al 1998, where populations studied from widely separated geographic areas were found to exhibit closer genomic affinities with their geographic neighbors than with those sharing similar social ranks. It also substantiates the suggested origin of Khandayat from skilled individuals drawn from peasantry and aboriginals of the region [Among the caste groups, Orissa Brahmin showed close affinity to the other upper caste populations of North India rather than to its geographic neighbors. The affinity between Bihar Brahmin and Orissa Brahmin was supported with moderate bootstrap values in the phylogenetic tree Figure , which ce region . BecauseOur study on linguistically distinct but geographically contiguous populations of Orissa using autosomal microsatellite markers reveals a significant amount genetic homogeneity in them. AMOVA results suggest that linguistic differences probably play a negligible role in the present day scenario in restricting gene flow between these populations. The middle-order caste groups shared genetic affinity with the local people of the area, while the Brahmins were similar to those from northern regions. Tribal populations, on the other hand, because of their long-term isolation and mating patterns, were well differentiated from the upper caste groups. This paper provides evidence that for populations living in geographic contiguity, ancestry is the governing factor in fine-tuning of genetic differentiation.Blood samples were collected from randomly chosen consenting volunteers, distributed across 17° -48' and 22° -34' North latitude and 81° -24' and 87° -29' East longitude of Orissa . STR loci analyzed in the study included thirteen tetranucleotides D3S1358, THO1, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPOX, FGA and two pentanuleotides, PentaD and PentaE.ST estimates, genetic distance and the analysis of molecular variance [A distance measure [The genetic structure of the populations was analyzed at two hierarchical levels – within populations and among populations. The intrapopulation variability was estimated by analyzing the number of alleles and most common allele at individual loci and by estimating the average gene diversity  across tvariance  were calvariance . Genetic measure , and wer measure . The deg measure  assumingSS carried out laboratory experiments, statistical analysis and drafted the manuscript and VKK conceptualized the paper, provided important intellectual inputs in intrepretation of data and preparation of the manuscript. Both authors read and approved the final manuscript.


Cerebrospinal Fluid Research aims to provide a specialist platform for high quality articles on all aspects of the cerebrospinal fluid (CSF), bringing together experts working in the field and promoting synthesis and dialogue. This, launch Editorial provides an overview of the field, some history, and highlights some of the journal policies. Cerebrospinal Fluid Research is an open access, online journal that publishes manuscripts on cerebrospinal fluid (CSF) in health and disease in the broadest sense. The CSF, its composition, circulation and absorption play vital roles in normal and abnormal brain function. The fluid within the CSF system is important for normal chemical signaling, physical and chemical buffering, and for neurodevelopment. In disease states, the CSF impacts on neurodevelopmental disorders such as hydrocephalus and neural tube defects, brain inflammation, brain injury and repair, normal pressure hydrocephalus and neurodegenerative diseases such as Alzheimer's, Parkinson's and multiple sclerosis. The CSF can be used as a tool for diagnosis, through composition analysis, and as a window for drug delivery to the brain.th century B.C. recorded the presence of fluid and is thought to have tapped the ventricles in a patient with hydrocephalus. Subsequently, in the 2nd century A.D., Galen described the ventricles and his dogma that the ventricles contained a gaseous vital spirit, lasted for over 1000 years. The return to the fluid hypothesis occurred in 1543 with Vesalius who made detailed observations of the anatomy and noted the presence of a watery humor. Further studies in the 17th and 18th centuries by Valsalva, Haller, and Contugno elaborated and extended this knowledge. Magendie in 1825 made chemical and physiological studies on the fluid and coined the name liquide cephalo-rachidien or fluid cerebrospinal. He saw pulsatile movement and concluded the fluid was under positive pressure. Later in the 19th century the anatomists Key and Retzius made extremely detailed studies of the cavities and the membranes of the brain, and provided a foundation for many 20th century investigations starting with Dixon and Haliburton, 1913 [Fluid within the brain cavities was known of in ancient times. Hippocrates in the 4Physiological studies began in the 1950's with numerous investigations led by Hugh Davson, the 'father' of CSF physiology . It was th century include the measurement and control of CSF secretion by Pappenheimer et al [Cerebrospinal Fluid Research [Particular milestones in the latter half of the 20er et al , and upter et al  and Poller et al , the demer et al , and theer et al . FurtherResearch . The jouCerebrospinal Fluid Research is supported by an international Editorial Board [al Board . Each maCerebrospinal Fluid Research will consider the publication of research articles, reviews, commentaries, book reviews and meeting proceedings. All articles will be published immediately upon acceptance (after peer review) and listed in PubMed. The intention is to publish, as supplements, the proceedings of scientific meetings in relevant subject areas.Cerebrospinal Fluid Research's Open Access policy changes the way in which articles are published. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [Cerebrospinal Fluid Research's articles are archived in PubMed Central [troduced . Third,  Central , the US  Central  in Germa Central  in Franc Central , the NatOpen Access has broad benefits for science and the general public. Most importantly, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access their work. In addition, authors are free to reproduce and distribute their work, for example by placing it on their institution's website. Furthermore, there is evidence that free online articles are more highly cited because of their easier availability  and publCerebrospinal Fluid Research looks forward to receiving your submissions.This is an exciting opportunity to disseminate our science in the new world wide medium of electronic publishing.


The purpose of this study was to further explore this hypothesis by examining the effects of TGF-β2 on primary cell cultures derived from patient-matched disease and normal palmar fascia tissue using a three-dimensional collagen contraction assay.Dupuytren's disease (DD) is a debilitating fibro-proliferative disorder of the hand characterized by the appearance of fibrotic lesions (nodules and cords) leading to flexion contractures of the fingers and loss of hand function. Although the molecular mechanism of DD is unknown, it has been suggested that transforming growth factor-β2 and neutralizing anti-TGF-β2 antibodies.Fibroblast-populated collagen lattice (FPCL) contraction assays using primary cell cultures derived from diseased and control fascia of the same DD patients were studied in response to exogenous TGF-β2 enhanced the rate and degree of collagen contraction in a dose-dependent fashion for both control and diseased cells. Neutralizing anti-TGF-β2 antibodies abolished exogenous TGF-β2 stimulated collagen contraction, but did not inhibit the enhanced basal collagen contraction activity of disease FPCL cultures.Contraction of the FPCLs occurred significantly faster and to a greater extent in disease cells compared to control cells. The addition of TGF-β2 stimulated both disease and control FPCL contraction, neutralizing anti-TGF-β2 antibodies did not affect the elevated basal collagen contraction activity of disease FPCLs, suggesting that the differences in the collagen contraction activity of control and disease FPCL cultures are not due to differences in the levels of endogenous TGF-β2 activity.Although exogenous TGF-β Dupuytren's disease (DD) is a fibro-proliferative disorder of the palmar fascia (PF) characterized by the formation of fibrous nodules and cords . The disin-situ hybridization [in vitro [Since Baron Guillaume Dupuytren's classical description of the disease in 1831, multiple clinical associations have been described, however, no clear molecular mechanism for the disease has been established . Histochdization , and immdization -18, whildization  and promin vitro . As a re2 and its effects on collagen contraction in vitro using a three-dimensional fibroblast populated collagen lattice (FPCL) contraction assay and DD patient-matched disease and control primary cell cultures. Previous reports have examined the role of TGF-β in DD by comparing disease fibroblasts to 'control' fibroblasts obtained from transverse carpal ligament material obtained from patients undergoing carpal tunnel release (CTR). By contrast, the control fibroblast cultures used in this study were established from unaffected PF tissue from the same patient, thus providing us with unique patient- and tissue- matched control cultures. The observed phenotypic differences between patient/tissue-matched control and disease FPCL cultures, specifically elevated collagen contraction activity, and β-catenin and fibronectin (Fn) expression in disease cells [2, may be regulating these disease-associated events in vitro, since TGF-βs are known to promote fibroblast mediated collagen contraction [2 could significantly stimulate 'normal' and disease FPCL contraction in a dose-dependent manner. While neutralizing anti-TGF-β2 antibodies completely blocked exogenous TGF-β2 stimulated FPCL contraction they had no effect on the enhanced basal collagen contraction activity of disease FPCL cultures.In this study, we chose to focus on TGF-βse cells -24, raistraction ,25,26 antraction ,27-30. A2. Medium was changed every 4–5 days and the cells sub-cultured using 0.05% Trypsin-EDTA (Gibco, Invitrogen Corporation, Grand Island, NY) when confluent.Our study protocol was cleared through the UWO Ethics Committee for Research Involving Human Subjects. Areas of diseased fascia and uninvolved normal (control) PF tissue were collected during surgery. DD explant cultures were initially cultured in starter media consisting of α-MEM (Gibco, Invitrogen Corporation) supplemented with 20% fetal bovine serum , and antibiotics (Penicillin G and streptomycin sulfate) and fungizone (Gibco, Invitrogen Corporation) as previously described . Establi4 cells/ml of matrix. The cell-collagen mixture was then aliquoted into 24 well culture dishes (0.5 ml/well) that were pre-treated with a PBS solution containing 2% (w/v) bovine serum albumin (BSA). Following FPCL polymerization (1 hr, 37°C) culture medium (0.5 ml) consisting of α-MEM + 10% fetal bovine serum (FBS) was added atop each lattice. After 2 days of culture the attached FPCLs were mechanically released from the sides of the culture plates. Digital images of the contracting FPCL were captured at various time points over a 5-day assay period using a conventional flatbed scanner. Collagen lattice areas were then quantified using the Image J program [Collagen contraction was carried out using patient-matched disease (D) and control (C) primary cultures (passages 2 – 6) established from patient-matched DD lesions and uninvolved palmar fascia (control). Collagen lattices were prepared by mixing cell suspensions with a neutralized solution of collagen type I matrix (8 parts Vitrogen100 collagen type I, 2.9 mg/ml, Collagen Corp, Santa Clara, CA, USA + 1 part 10 × α-MEM + 1 part HEPES buffer, pH 9.0). The cell-collagen concentrations were adjusted with sterile phosphate buffered solution (PBS) to attain a final collagen concentration of 2.0 mg/ml and a final cell concentration of 8.6 × 10 program . Each as2 (expressed in NSO murine myeloma cells) was acid activated in a solution of 4N HCl + 0.1% (w/v) BSA according to the manufacturer's instructions (Product # T 2815, Sigma, St. Louis, MO). Acid activated human recombinant TGF-β2 was then aliquoted and frozen for extended storage at -70°C. The indicated concentrations of activated human recombinant TGF-β2 were added to complete culture media immediately following FPCL polymerization. Neutralizing anti-TGF-β2 antibodies (R&D Systems, Minneapolis, MN) were added to complete culture media either immediately following FPCL polymerization or added to the cell suspension prior to mixing with the neutralized Vitrogen collagen type I solution. Control FPCL cultures were treated with appropriate carrier solutions.Commercially available human recombinant TGF-β® Aqueous One Solution cell proliferation assay according to the manufacturer's instructions (Promega, Madison, WI, USA). This cell proliferation assay is a colorimetric method that uses a tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) in combination with a stable electron coupling reagent PES (phenazine ethosulfate). This produces a chemically stable MTS tetrazolium compound that can be bio-reduced by cells to form a soluble colored formazan product [2 (1 ng/ml) atop each FPCL. At each of the designated contracting time points 100μl of CellTiter 96® Aqueous One solution reagent was added to the FPCL cultures and incubated for 3 hours (37°C, 5% CO2 atmosphere, humidified chamber). FPCL media was then collected and aliquoted into a 96-well culture plate for absorbance reading at 450 and 650 nm (background reference λ) using a 96-well BIO-RAD microplate reader. Media was also collected from FPCLs containing no cells (negative control) for background absorbance readings. The assay was conducted over 5 days during the course of FPCL contraction. A standard curve was also generated to calculate relative cell numbers per FPCL (range of 4 × 103–105 cells/FPCL).FPCL cultures were assayed using a commercially available CellTiter 96 product ,33. Briet test was used to compare data between two groups. Values were expressed as mean ± standard deviation of the mean (SDM). P values < 0.05 were considered statistically significant.Student two-tailed 2 on FPCL contraction. Initial experiments showed a typical dose-dependent response for TGF-β2 stimulated FPCL contraction .Exogenous TGF-β2 on collagen contraction using primary FPCL cultures derived from patient-matched disease and control unaffected PF tissue. Here, we found that disease FPCL cultures contracted collagen faster and to a greater degree than patient-matched control FPCL cultures. Although neutralizing anti-TGF-β2 antibodies effectively blocked exogenous TGF-β2 stimulated FPCL contraction for both control and disease cultures, the same neutralizing antibodies had no effect on the basal collagen contraction activity of either disease or control FPCL cultures, suggesting that enhanced disease FPCL contraction is not due to elevated levels of endogenous active TGF-β2. These results, however, do not exclude the possibility that there may be differences in the levels of latent TGF-β2 produced by these cell cultures that may be subsequently activated in vivo. Recent work by Kuhn et. al. (2002) reported reduced DD FPCL contraction in response to tamoxifen, which was associated with a decreased TGF-β2 production [2 assayed in this study required an acid-activation step, suggesting that TGF-β2 produced by these cells is largely in its latent, non-activate form. Thus, it is possible that TGF-β2 produced by DD fibroblasts and/or other resident cell types in vivo may be important to disease cord contraction, provided appropriate regulators of latent TGF-β activation are also present and active. However, it does not explain the enhanced basal FPCL contraction rates of disease cell cultures, suggesting that other signalling factors may be regulating this disease cell function in vitro. It is interesting to note that both TGF-β and β-catenin signalling pathways show some degree of 'cross-talk' [Members of the TGF-β family are potent fibrogenic factors that play an important role in the patho-physiology of numerous fibro-proliferative disorders, including DD . The stuoduction . Howeverss-talk' ,30,36-381 stimulated myofibroblast differentiation, while Thy-1 negative fibroblasts appear to be only capable of lipofibroblast differentiation [1 had a pro-apoptotic effect on non-palmoplantar (keloid) fibroblasts and an anti-apoptotic effect on palmoplantar fibroblasts. Similar to what we have observed for detached or contracting DD FPCL cultures, they showed that TGF-β1 treated (in the presence of serum) keloid FPCL cultures underwent the most extensive apoptosis response upon mechanical release compared to dermal fibroblasts from different body sites [2 did not appear to have any significant pro-apoptotic effect on relaxed 'control' FPCL cultures. In light of these and other findings, it appears that the patient-matched control fibroblast cultures employed in these studies may truly represent a suitable 'control' phenotype, with the added advantage of having the same PF origins as the disease cell cultures. While this does not exclude the possibility that the control cultures may harbour some residual disease cells, this is not supported by the distinct phenotypic differences we have observed between these two types of patient-matched PF cultures in the current and previous studies. In these earlier studies, we reported elevated levels of β-catenin and fibronectin isoforms in disease FPCL cultures [2 on disease FPCL cultures described here, further support the notion that the patient/tissue-matched 'control' cultures have a non-disease phenotype that is suitable for these types of investigations.Although previous DD studies have used different 'control' primary fibroblast as 'disease-free' controls, namely fibroblasts derived from transverse carpal ligament material obtained from patients undergoing carpal tunnel release (CTR), they have the disadvantage of being of different anatomical origin than PF derived 'control' fibroblast cultures. This is an important consideration given that fibroblasts exhibit functional heterogeneity depending on their origin -41. For ntiation . Phenotyntiation . For exady sites . Perhapscultures -24, as w2 does not play a role in regulating these phenotypic differences in vitro. Nevertheless, aberrant expression of various TGF-β signalling components have been previously shown to trigger this 'synthetic' myofibroblast phenotype in other fibro-proliferative disorders, specifically keloids and burn hypertrophic scarring [2 antibodies [2, tension and other important intersecting signaling pathways.Although the 'synthetic' myofibroblast features of the disease cells described in previous studies are known to be stimulated by TGF-β ,25-30,35scarring ,44,45, ttibodies ,47. Hope2 antibodies can block exogenous TGF-β2 stimulated collagen contraction for both control and disease FPCL cultures, it had no effect on the basal contraction rates of either control or disease FPCL cultures. We, therefore, conclude that the enhanced collagen contraction activity of disease FPCL cultures is not due to differences in the levels of endogenous active TGF-β2.Primary disease FPCL cultures contract collagen faster and to a greater extent than control PF-matched FPCL cultures. While neutralizing anti-TGF-βDD, Dupuytren's disease; PF, palmar fascia; CTR, carpal tunnel release; TGF-β, transforming growth factor-beta; FPCL, fibroblast populated collagen matrix; PBS, phosphate buffered saline; RT, room temperature; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; α-MEM, alpha-minimal essential medium; FBS, fetal bovine serum; Fn, fibronectin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; PES, phenazine ethosulfate; RT-PCR, reverse transcriptase polymerase chain reaction; SDM, standard deviation of the mean.The authors declare that they have no competing interests.RT and YW carried out the experiments. RT drafted the manuscript. JCH and BSG designed and coordinated the research, and reviewed and edited the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The envelope (Env) protein of jaagsiekte sheep retrovirus (JSRV) can transform cells in culture and is likely to be the main factor responsible for lung cancer induction by JSRV in animals. A recent report indicates that the epithelial-cell transforming activity of JSRV Env depends on activation of the cell-surface receptor tyrosine kinase Mst1r . In the immortalized line of human epithelial cells used (BEAS-2B cells), the virus receptor Hyal2 was found to bind to and suppress the activity of RON. When Env was expressed it bound to Hyal2 causing its degradation, release of RON activity from Hyal2 suppression, and activation of pathways resulting in cell transformation.Due to difficulty with reproducibility of the transformation assay in BEAS-2B cells, we have used more tractable rodent fibroblast models to further study Hyal2 modulation of RON/Stk transforming activity and potential effects of Hyal2 on RON/Stk activation by its natural ligand, macrophage stimulating protein (MSP).We did not detect transformation of NIH 3T3 cells by plasmids expressing RON or Stk, but did detect transformation of 208F rat fibroblasts by these plasmids at a very low rate. We were able to isolate 208F cell clones that expressed RON or Stk and that showed changes in morphology indicative of transformation. The parental 208F cells did not respond to MSP but 208F cells expressing RON or Stk showed obvious increases in scattering/transformation in response to MSP. Human Hyal2 had no effect on the basal or MSP-induced phenotypes of RON-expressing 208F cells, and human, mouse or rat Hyal2 had no effect on the basal or MSP-induced phenotypes of Stk-expressing 208F cells.We have shown that RON or Stk expression in 208F rat fibroblasts results in a transformed phenotype that is enhanced by addition of the natural ligand for these proteins, MSP. Hyal2 does not directly modulate the basal or MSP-induced RON/Stk activity, although it is possible that adaptor proteins might mediate such signaling in other cell types. JSRV is an acutely oncogenic retrovirus that can induce lung tumors in newborn sheep in as little as 10 days -5. The oAt least two mechanisms of transformation by JSRV Env have been identified in cultured cells. JSRV Env can transform immortalized mouse, rat, and chicken fibroblasts -4, and tA second pathway for JSRV Env transformation involves activation of the transmembrane receptor tyrosine kinase Mst1r . RONWe have had difficulty in further exploring transformation through the RON pathway in BEAS-2B cells due to difficulty with reproducibility of the transformation assay. Others have found that Stk can transform immortalized rodent fibroblasts ,24, and 2-air atmosphere at 100% relative humidity.Cell lines used here include 208F Fischer rat embryo fibroblasts , a morphNotI site of the pAlter-Max expression plasmid (Promega, Madison, Wisconsin). Human RON was expressed by using a plasmid  that contains the human RON cDNA inserted into the pCI-neo expression plasmid (Promega). The expression cassette in both of these plasmids is identical and consists of a human cytomegalovirus immediate early promoter followed by splicing signals, the cDNA, and the simian virus 40 late polyadenylation signal. Mouse Stk was also expressed using the previously described pcDNA3-based expression vector [Mouse Stk was expressed by using a plasmid  that contains the mouse Stk cDNA cloned into the n vector   and neomycin phosphotransferase (Neo). LAPSN virus was made using PT67 (10A1-MLV pseudotype) or PJ4 (JSRV pseudotype) packaging cells. Retroviral vectors encoding human Hyal1, human Hyal2, mouse Hyal2 cloned from NIH 3T3 cells, and rat Hyal2 cloned from 208F rat cells (GenBank accession numbers U03056, U09577, AF535140, and AF535141, respectively) were made by insertion of the cDNAs into the LXSN vector  and by gNIH 3T3 and 208F cells were transfected using calcium phosphate as previously described  and wereRecombinant human MSP activated by treatment with kallikrein (R&D Systems, Minneapolis, MN) was dissolved at a concentration of 10 μg/ml in phosphate buffered saline with calcium and magnesium containing 0.2% bovine serum albumin and was stored at -70°C. Cells were seeded at 10,000 per well (d = 16 mm) of 24-well plates. Two days after seeding, the cells were treated with 0.06 to 20 μl of MSP stock in 0.5 ml of fresh medium (MSP concentration of 1.2 to 400 ng/ml) and cellular morphology was evaluated the day after MSP addition. Treatment of cells with 20 μl buffer containing 0.2% bovine serum albumin in 0.5 ml of fresh medium had no effect on cell morphology.We tested plasmids encoding RON or Stk (pCIneoRON and pAlter-Max-Stk, respectively) for transforming activity in NIH 3T3 mouse and 208F rat fibroblasts. The cDNAs in both plasmids were driven by a strong human cytomegalovirus immediate early promoter and each contained an intron to promote high-level expression. In these assays, strong oncogenes induce transformed foci of cells in less than a week while less active oncogenes take longer to induce foci. In both cases the numbers of foci generally increase with time after transfection.Neither the RON nor the Stk expression plasmid induced transformed foci in NIH 3T3 cells (<0.2 foci per μg plasmid DNA at one month after transfection), while a plasmid expressing JSRV Env (pSX2.Jenv) induced 33 well-developed foci per μg plasmid DNA under the same conditions. This result is consistent with previous reports showing a lack of transformation by human RON -32, but Next we tested for transformation of 208F rat fibroblasts by the RON and Stk plasmids. The Stk plasmid (pAlter-Max-Stk) induced 3 clear foci in a total of 7 dishes (0.2 foci per μg plasmid DNA), and the RON plasmid (pCIneoRON) induced 1 clear focus in a total of 5 dishes (0.1 foci per μg plasmid DNA) (foci counted 1 month after transfection, 2 μg plasmid DNA per dish, 2–3 separate experiments for each plasmid). The Stk expression plasmid pcDNA3Stk used in the earlier studies ,24 did nFoci of transformed 208F cells were isolated from plates of cells transfected with the RON or Stk expression plasmids and were tested for their response to MSP. Cells transformed by the RON plasmid were not responsive to MSP and were discarded. Cells transformed by the Stk plasmid were responsive to MSP, and two clones (208F/Stk c12 and c13) were used for further study. Both of these clones made Stk protein of the appropriate molecular weight as judged by western analysis using RON/Stk-specific antibodies (data not shown).The response of 208F/Stk c13 cells to overnight treatment with MSP is shown in Fig. Since we were unsuccessful in generating 208F cells expressing functional RON by isolation of transformed cells following transfection, we isolated RON-expressing cells by transfection with a RON expression vector that also expressed a selectable marker (pMSCVpuroRON), grew the cells in puromycin to select for cells expressing the marker, and isolated multiple cell clones that showed a weakly-transformed phenotype. These clonal lines were screened for scattering response to MSP, and two independent clones that had the most transformed appearance and that responded to MSP (208F/RON c9 and c10) were identified and used in further analyses. Both clones produced RON protein of the appropriate size band by western analysis using a RON/Stk-specific antibodies, and both exhibited clear surface expression of RON by FACS analysis using a RON-specific antibody while control 208F cells showed no expression (data not shown). The 208F/RON clones had a less obvious transformed phenotype than the 208F/Stk clones but still showed convincing scattering in response to MSP (see below).We tested whether overexpression of Hyal2 would reverse the altered phenotype of 208F cells expressing RON or Stk. Since the original work in BEAS-2B human cells showed inhibition of RON activity by human Hyal2 , we testWe exposed 208F cells that expressed RON to retroviral vectors encoding human Hyal2, AP, or human Hyal1, grew these cells in G418 to select for cells expressing the vector, and quantitated the morphologies of the cells that grew out. The experiment was repeated by replating the G418-resistant cells at low density and repeating the morphologic quantitation. The results were similar for both cell lines in both experiments so the results for the two experiments with each of the two cell lines were averaged Fig. . There wNext we exposed the 208F/Stk c12 and c13 clonal cell lines to retroviral vectors encoding mouse, rat or human Hyal2, or human Hyal1, grew these cells in G418 to select for cells expressing the vector, and quantitated the morphologies of colonies that grew after plating the cells at low density  and the next day the cell morphology was examined. We measured the minimum MSP concentration at which an alteration in morphology was observed and the MSP concentration above which no further morphologic changes were observed. The right panels of Fig. The original studies showing an interaction between RON/Stk and Hyal2 studied the human proteins , so we fWe next explored the possible effects of Hyal2 on the MSP response of 208F/Stk cells. We tested human, mouse, and rat Hyal2 proteins to address the possibility that only the rodent Hyal2 proteins would interact with Stk. Identical responses to MSP were observed for 208F/Stk c12 cells that expressed mouse, rat, or human Hyal2, human Hyal1, or no protein. In two independent experiments, the first effects were observed at 1.2 ng/ml MSP and the maximum effect was observed at 12 ng/ml. Identical responses were also observed for 208F/Stk c13 cells that expressed mouse, rat, or human Hyal2, human Hyal1, or no protein. In this case, the responses were first noticeable at 4 ng/ml MSP and were maximal at 40 ng/ml MSP. Untreated 208F/Stk c12 cells are more refractile and express more Stk than the 208F/Stk c13 cells (data not shown), and the higher level and basal activity of Stk in these cells likely explains their higher sensitivity to MSP.The ability of JSRV Env to transform cells in culture, the identification of Hyal2 as the cell-surface receptor for the Env protein of JSRV, and the localization of Hyal2 to the chromosome 3p21.3 lung cancer tumor suppressor locus suggested the hypothesis that JSRV might cause cancer by inhibiting a tumor suppressor activity of Hyal2 . The facUnfortunately and despite numerous attempts we have been unable to reliably measure transformation in BEAS-2B cells using the originally described expression plasmids and transfection techniques . OccasioBased on earlier findings that Stk could transform NIH 3T3 mouse fibroblasts ,24, we aIn contrast, the 208F cells showed no background transformation and allowed us to detect a low frequency of transformation by RON and Stk. This low rate of transformation could be due to a requirement for a very high level of RON/Stk expression that occurs only in a small number of cells, or a requirement for mutations that activate the RON/Stk proteins and occur at low frequency. We isolated several foci of Stk-transformed 208F cells and found that these cells expressed Stk protein of the expected size by western blotting and that the cells responded to MSP, indicating that the Stk protein was not constitutively activated by mutations or deletions, and suggesting that the protein is simply overexpressed in these cells. In contrast, cells from the one focus of cells transformed by transfection of the RON gene were unresponsive to MSP and thus might have been the result of activating mutations or deletions in RON. To generate 208F cells expressing RON we transfected RON with a selectable marker and isolated cells showing a weakly transformed phenotype.We presume that the low level phenotypic alteration observed in 208F/RON and 208F/Stk cells in the absence of MSP addition is due either to low-level constitutive activity of RON/Stk or to RON/Stk activation by low levels of MSP made by the cells or present in the culture medium. Given that the parental 208F cells show no response to MSP, we conclude that the cell scattering phenotype observed after addition of MSP is a direct measure of RON/Stk activation. The transformed phenotype of these cells in the absence of added MSP and the scattering phenotype observed after treatment of the cells with MSP seem to represent variations of the same transformation phenotype. Indeed, 208F cells transformed by other oncogenes, including JSRV Env, exhibit a scattered phenotype similar to MSP treated 208F cells expressing RON or Stk. Furthermore, 208F cells expressing RON or Stk can exhibit a scattered phenotype in the absence of added MSP, especially if the cells are selected for a highly transformed phenotype by passaging substrate-independent cells that float in the culture medium. Given this interpretation, the response of the RON/Stk-expressing cells to MSP can be considered a form of ligand-dependent transformation, similar to what has been observed previously for cells expressing the receptors for human colony-stimulating factor 1 , insulinAlternatively, it may be that the basal and MSP-induced phenotypes of the RON/Stk-expressing cells are mediated by separate signaling pathways, as has been found for the related receptor tyrosine kinase MET . These iWe did not find any effect of human Hyal2 on the basal phenotype or the response to MSP of the 208F/RON cells, nor did we find an effect of human, rat, or mouse Hyal2 on the basal phenotype or the MSP response of 208F/Stk cells. These results argue against direct regulation of RON activity by Hyal2 as was indicated by prior experiments using BEAS-2B cells . It is pRecently we have found that Env proteins from JSRV and from the related retrovirus enzootic nasal tumor virus (ENTV) can transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism different from that observed in BEAS-2B cells and similar to that observed in rodent fibroblast cell lines . In partWe have shown that expression of the mouse or human orthologs of the receptor tyrosine kinase Mst1r  can induce phenotypic changes indicative of transformation in a rat fibroblast cell line. These changes are enhanced by treatment of the cells with MSP, the natural ligand for RON/Stk, which induces a pronounced scattering/transformation response. Transformation of the BEAS-2B immortalized human epithelial cell line by JSRV appears to require RON activation, as a result of JSRV Env binding to and degradation of the virus receptor Hyal2, which appeared to negatively regulate RON activity. However, we find no evidence for direct regulation of the basal or MSP-induced RON/Stk activity by Hyal2 in rat fibroblasts.None declared.ADM designed the study, performed most of the experiments, and drafted the manuscript. NSVH and SLL performed the western blot and FACS experiments and provided intellectual input, and SLL performed the preliminary studies on BEAS-2B cells. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta.Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, in vivo.Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions  Drosophila melanogaster. They are evolutionarily conserved, functioning similarly in animals from worms to humans [Caenorhabditis elegans, however, the differential accumulation of the Da homolog HLH-2 is the earliest detectable difference between the cells taking up alternate fates during lateral inhibition [Notch (N) and Delta (Dl) are cell surface proteins that are required for differentiation of almost all tissues in the fruit fly o humans ,2. The bo humans ,4. Theseo humans -7. Da iso humans  and up ro humans . Whetherhibition . As N anhibition , it is vintra) from the plasma membrane. Nintra translocates to the nucleus and, in association with the transcription factor Suppressor of Hairless (SuH), activates transcription of the Enhancer of split Complex (E(spl)C) genes. Cells that express a high level of E(spl)C RNA suppress their neuronal predisposition, become the epidermal precursor cells (EPCs), and differentiate the epidermis. Cells that express a low level of E(spl)C RNA and a high level of Da protein become the Neuronal Precursor Cells (NPCs) and differentiate the nervous system [E(spl)C RNA as SuH/Nintra signaling. A 1.5 to 2-fold difference in the level of SuH/Nintra signaling is sufficient to initiate specification of the EPCs and the NPCs [intra signaling. The lateral inhibition process described above is repeatedly used during development for differentiation of various tissues with minor variations or changes in target genes.When N expressed on one proneural cell binds Dl expressed on the neighboring proneural cell, N is proteolytically cleaved to release the Notch intracellular domain  is a secreted factor that is produced at high levels in the NPCs and functions non-autonomously to promote specification of the EPCs during differentiation of the compound eye and the bristle organ ,13. In iin vivo and N receptor activities in response to Dl binding are widely used during development. In developmental instances where Sca is present, the expression data suggest that both N and Dl will have access to Sca. Thus, it is very difficult to separate in vivo the activities of N alone, Dl alone, N on Dl, Dl on N, Sca on N, Sca on Dl, and Sca on N and Dl together. Therefore, we addressed the questions posed above in an in vitro model system based on Drosophila Schneider (S2) cells. S2 cells do not express the endogenous N, Dl, or Sca [N and Dl are expressed in almost all cells , or Sca ,16. S2 c, or Sca . Using t, or Sca , we showAlthough N and Sca complexes could be immuno-precipitated , we, andLive S2-Dl cells showed the strongest GFP signals, followed by live S2-N cells, and then live S2 cells Fig. . The sigWe have previously shown that Sca and N form complexes . To deteE(spl)C m3 gene in response to Dl [E(spl)C m3 in S-N cells or S2-Dl cells and found that it was indeed the case with S2-N cells, but not with S2-Dl cells C m3 expression when S2-Dl or S2-DlΔI cells were replaced with S2 cells, in the absence of Sca C m3 RNA expression in S2-N cells in the absence of ligands is due to the low level of Nintra produced upon induction of N expression in S2 cells [E(spl)C m3 RNA expression C m3 expression (data not shown).N promotes expression of se to Dl ,20. We eS2 cells . This exS2 cells , resultiE(spl)C m3 expression appeared to be solely dependent on N activation and the Notch intracellular domain as it was promoted in S2-N cells treated with either S2-Dl cells or S2-DlΔI cells  is required for SuH/Nintra signaling, possibly for promoting Dl internalization that results in exerting a 'pull' on N and increased production of Nintra [intra signaling in S2-N cells [intra signaling in S2 cells. In any case, in our S2 cell system, the S2-N and S2-Dl cells require shaking for formation of cell aggregates. As a consequence, we shake all cell mixtures, including those containing the secreted ligand Sca. This shaking might have simulated the pulling effect and overcome any deficiency DlΔI might have in this regard thereby resulting in a level of SuH/Nintra signaling that is comparable to that produced by the full length Dl.f Nintra ,25-29. H-N cells . Thus, iWe examined the expression of various proteins known to be involved in lateral inhibition to find out if Dl expression affected them. They were Numb, Dishevelled, Suppressor of Hairless, Wingless, Hairless, Hairy, Achaete, Da, and Armadillo. We found a relatively high level of Da protein in S2-Dl cells compared with the level in S2-DlΔI cells C m3 RNA were very variable (data not shown). This was possibly due to the varying combinations of Sca effect on S2-Dl cells, Sca effect on S2-N cells, Dl effect on S2-N cells, and N effect on S2-Dl cells.When S2-N and S2-Dl cells were together in the presence of Sca, the levels of Da protein and da gene [E(spl)C m3 RNA expression in S2-N cells even in the absence of Dl.Dl is processed to produce Dl intracellular domain (DlIC), constitutively, and the levels of DlIC increase upon N treatment -33. Therda gene . We examtransformer, was also up (1.76X) and it is significant in the light of our observation that Dl promotes expression of Da: both Da and transformer are involved in sex determination. Expression of da RNA was not significantly increased in S2-Dl cells, possibly due to the negative part of the da gene autoregulation system [fringe (fng) and pangolin (pan) as responding to Dl expression. fng is a glycosyl transferase that regulates the affinity of N for Dl [pan is a transcription factor functioning in the Wingless (Wg) pathway [fng and pan for further investigation.To gather additional evidence for Dl activity independent of its activity as a ligand of Notch, we performed microarray experiments using the Affymetrix Drosophila GeneChip Arrays to compare gene expression in S2 cells and S2-Dl cells. Many genes relevant to known Dl functions responded in S2-Dl cells (at p < 0.05, n = 3 × 2 pooled samples): axonal path finding genes , actin-based cell motility and kinases , N signaling pathway genes , and oogenesis genes . Expression of n system . The detN for Dl -37, and  pathway . Notch a pathway ,41-44. Tfng and pan was higher in S2-Dl cells compared with S2-N or S2-DlΔI cells  strongly suggest that the Dl activities we have described in S2 cells represent the in vivo Dl activities during development.Results described in Figures in vivo.The N independent Dl activity we have described is dependent on the full length Dl, not just on its intracellular domain or the extracellular domain Figs. ; 3; 5A-Bin vivo dissection of Dl response to N, as Sca and N appear to have a similar effect on Dl. It is quite likely that our experiments did not pick up Dl receptor activity in response to N or Sca. In any case, the potential developmental significance of our findings is briefly discussed below.Dl activity that is independent of its N ligand activity has been speculated for some time. Efforts to identify it have intensified since the discovery that Dl gets proteolytically processed in the same manner as N -33. Howeintra/SuH signaling, small changes in Da levels might be sufficient for initiating or augmenting NPC specification and promoting neuronal differentiation. Small changes in Da levels might also be imposed by the built-in autoregulation of the da gene [fng, or pan might not be the direct target of the full length Dl activity. Our microarray data indicate that many other genes  are strongly up regulated in Dl expressing cells. It is possible that one of these genes is the primary target. It is also possible that Da, fng, or pan accumulation is significant only in the context of these other genes. We will have to await validation of other putative targets of Dl activity, and evaluation of their role in lateral inhibition or other activities involving N and/or Dl, to determine if Da, fng, or pan are typical or atypical targets of Dl activity.Da is a widely expressed protein and cells requiring its function show only a modest increase in its levels ,6,8 indi da gene . Accordi da gene . Accordi da gene ,51. Our  da gene . It is p da gene , promoteintra signaling are strongly affected by the functions of glycosyl transferases such as fng. The possibility that Dl, and not N, regulates fng RNA expression might explain some of the very complex functions of these glycosyl transferases and the complex interactions between N and Dl during lateral inhibition. As N and Dl activities are known to strongly interact with the Wg signaling pathway, it is interesting that Dl promotes pan expression. It is possible that Dl activity independent of N accounts for some of the interactions between the N and the Wg pathways. So far, these interactions have been considered only from the perspective of N receptor activity.N/Dl binding and SuH/NE(spl)C RNA and processed Dl through suppression of Da expression. Consequently, lateral inhibition might initiate with symmetrical actions of N and Dl promoting EPC specification in all proneural cells. Sca might boost N and Dl processing in the incipient EPCs while suppressing them or not affecting them in the incipient NPCs. Thus, it is possible that Sca or Sca-like molecule have a role in breaking the symmetrical actions of N and Dl during certain lateral inhibition instances. It is also possible that Sca mediates long range N signaling during differentiation of some other tissues, either alone or in association with Dl, as proposed by Renaud and Simpson [in vivo, cleanly sort the different activities of N, Dl, and Sca, and understand the fascinating in vivo developmental mechanisms involving N, Dl, and Sca.Lastly, our data suggest interesting interactions among Dl, N, and Sca in instances of lateral inhibition and tissue differentiation when their functions overlap. The full length Dl promotes Da accumulation, not any of its parts that might result from processing in response to N or Sca binding. Thus, both the processed N and Dl might promote EPC specification- processed N through  Simpson . By extefng RNA, and pan RNA responds positively to the N independent Dl activity we have discovered. These could be direct or indirect targets. Our microarray analysis has identified many more putative targets of N independent Dl activity that can be explored for a better understanding of the complex interactions among Dl, Sca, and N during Drosophila development.Sca binds Dl and suppresses a Dl activity that is independent of Dl's activity as a ligand of N. This Dl activity requires the full length Dl and is not enhanced by expression of just the Dl intracellular domain, which is different from the mechanism underlying Notch activity. Da protein, sca was replaced with a glycine codon and fused in-frame with GFP to obtain Sca-GFP. A Bam HI-KpnI fragment containing this sca sequence was cloned into pEGFP vector (Clontech). The XbaI fragment containing Sca-GFP coding fragment was cloned into the pCaSpeR-hs vector. DlΔI: A stop codon and a XbaI restriction site was introduced after the transmembrane domain using PCR. The PCR product was checked for mutations and used to replace the BstEII-BcgI fragment in the Dl cDNA. An Eco RI-XbaI fragment from this construct (Dl amino acid 1 to 620) was cloned into the pCaSpeR-hs vector. DlIC: The Dl intracellular region (codon 619 to the stop codon 881) was PCR amplified, checked for errors, and cloned into the BglII-XbaI sites in the pCaSpeR-hs vector.Sca-Gfp: The stop codon of S2-N, S2-Dl, and S2-Sca cells have been previously described ,20,49. Ofringe cDNA (from Dr. Ken Irvine), rp49 cDNA, and rt-PCR amplified pangolin cDNA were used to prepare probes for northern blots. Incubation times with ligands were two hours for western blots and 45 minutes for all northern blots; it was three hours for fng and pan northern blot showing the effect of Sca  and αDlEC (C594.9B) were obtained from the Developmental Studies Hybridoma Bank; αGFP (G-6539) and αHsp70 (H-5147) from Sigma; αDlIC (GPC2) from Dr. Marc Muskavitch, αDlIC (dC-19) from Santa Cruz Biotechnology, αDa (DAM 109-10) from Dr. Claire Cronmiller; and αNI from Dr. Toby Lieber.Heat shocked S2 and S2-Dl cells were treated with Sca or non-Sca medium for 45 minutes before extracting RNA. GeneChip Drosophila Genome Arrays from Affymetrix were used. RNAs were extracted, checked, and processed for hybridization according to procedures suggested by Affymetrix. We pooled RNA from two independent experiments and used three such pooled samples as replicates for each treatment. The MicroArray Core Facility at the University of Vermont prepared the probes, hybridized the chips, and statistically analyzed the data (using the GeneSifter program). We used the Microarray Suite program to examine the data.LPM and TQ designed and carried out many of the experiments in cultured cells and flies; BB carried out the immuno-precipitation experiments with cultured cells and helped in interpretation of data; MLC made the Sca GFP construct and assisted in many experiments; AH performed some experiments in cultured cells and prepared and maintained cell lines; FA assisted in statistical analyses and interpretation of data; and CSW conceived the study, designed experiments, and performed or participated in many of experiments in cultured cells and flies. LPM, BB, and MLC helped CSW in drafting the manuscript. All authors read and approved the final manuscript.


Glioblastoma is the deadliest and most prevalent brain tumor. Dexamethasone (DXM) is a commonly used steroid for treating glioblastoma patients for alleviation of vasogenic edema and pain prior to treatment with chemotherapeutic drugs. Temozolomide (TMZ), an alkylating agent, has recently been introduced in clinical trials for treating glioblastoma. Here, we evaluated the modulatory effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells.2+], as determined by fura-2 assay. Western blot analyses showed alternations in the levels of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) proteins resulting in increased Bax:Bcl-2 ratio in TMZ treated cells. Western blot analyses also detected overexpression of calpain and caspase-3, which cleaved 270 kD α-spectrin at specific sites for generation of 145 and 120 kD spectrin break down products (SBDPs), respectively. However, 1-h pretreatment of cells with 40 μM DXM dramatically decreased TMZ induced apoptosis, decreasing Bax:Bcl-2 ratio and SBDPs.Freshly grown cells were treated with different doses of DXM or TMZ for 6 h followed by incubation in a drug-free medium for 48 h. Wright staining and ApopTag assay showed no apoptosis in cells treated with 40 μM DXM but considerable amounts of apoptosis in cells treated with 100 μM TMZ. Apoptosis in TMZ treated cells was associated with an increase in intracellular free [CaOur results revealed an antagonistic effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells, implying that treatment of glioblastoma patients with DXM prior to chemotherapy with TMZ might result in an undesirable clinical outcome. Steroids, however, may modulate the sensitivity of tumor cells to chemotherapeutic drugs. Dexamethasone (DXM), a synthetic glucocorticoid, is commonly used to reduce inflammation and pain associated with glioblastoma . However calpain . Activat calpain ,9. Mitocchondria ,11, and chondria ,13, whicchondria . The pro cytosol .WAF1/CIP1 protein accumulation has been reported [WAF1/CIP1 expression in glioblastoma cells but this effect appears to be cell-type specific [Glucocorticoids are steroid hormones, which are secreted in response to stress and can modulate the ability of cells to undergo apoptosis . For exareported , the exareported . In addireported . It has reported -5. DXM areported -5. It haspecific . Thus, t6 position of guanine, with an additional methylation at its N7 position [6-methylguanine (O6-meG) may be removed by O6-methylguanine methyl transferase (MGMT) [6-meG. Replication of DNA introduces a T instead of C opposite to O6-meG, resulting in GT mismatches [TMZ is an alkylating chemotherapeutic drug that readily crosses the blood-brain-barrier in glioblastoma patients . It is cposition ,24. Howee (MGMT) . Cells dsmatches . Activatsmatches . These s2+-dependent cysteine protease, which plays a role in the mechanism of cell death in human malignant brain tumors including glioblastoma [We found that TMZ caused apoptosis in U87MG cells as detected by morphological and biochemical assays. Alterations in the levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins are known to regulate the commitment to apoptosis ,15. Therblastoma . Besidesblastoma .P < 0.001). A pretreatment of cells with 40 μM DXM decreased TMZ induced apoptosis by three-fold, compared to treatment of cells with TMZ only.Exclusion of trypan blue dye by viable U87MG cells was evaluated under a light microscope using a hemocytometer after all treatments. A pretreatment with DXM prevented decrease in cell viability  in TMZ treated cells, compared to CTL cells. A pretreatment of cells with DXM considerably attenuated apoptotic DNA fragmentation in TMZ treated cells.Results obtained from Wright staining were further supported by the ApopTag assay  was seen between CTL cells and cells treated with DXM alone. Cells treated with TMZ showed a significant increase (P = 0.007) in intracellular free [Ca2+], compared to CTL cells. This increase was attenuated almost 100% by a pretreatment of the cells with DXM. There was no significant difference (P = 0.999) between intracellular free [Ca2+] in CTL cells and those treated with DXM plus TMZ.Using fura-2 assay, intracellular free [Caups Fig. . No signP = 0.983) in Bax:Bcl-2 ratio between CTL and DXM treated cells  in the Bax:Bcl-2 ratio  in Bax:Bcl-2 ratio between cells treated with TMZ alone and those treated with DXM plus TMZ, indicating a loss of commitment to apoptosis due to a pretreatment with DXM. There was no significant difference (P = 0.871) in Bax:Bcl-2 ratio between CTL cells and cells pretreated with DXM and then treated with TMZ.A commitment to apoptosis was measured by examining any increase in the ratio of Bax (pro-apoptotic protein) expression to Bcl-2 (anti-apoptotic protein) expression. The bax gene encodes different isoforms. The antibody we used in this investigation could recognize 21 kD Baxα and 24 kD Baxβ bands  between CTL cells and DXM treated cells in generation of 145 kD SBDP, indicating similar levels of calpain activity in these two cases  than CTL cells, indicating that the level of calpain activity was increased in cells due to treatment with TMZ. Cells pretreated with DXM and then treated with TMZ showed a significant decrease in the generation of 145 kD SBDP, indicating an inhibitory effect of DXM on TMZ mediated increase in calpain activity  in caspase-3 activity. Caspase-3 activity in cells treated with TMZ was almost 1.5 times more (P = 0.001) than CTL cells  between CTL cells and cells that were pretreated with DXM and then treated with TMZ  in caspase-3 activation over CTL cells. But there was a significant increase (P = 0.001) in production of active 20 kD caspase-3 fragment in cells treated with TMZ, compared to CTL cells. Treatment of cells with DXM prior to TMZ appeared to significantly decrease the activation of caspase-3  and 1% penicillin and streptomycin in a fully-humidified incubator containing 5% CO2 at 37°C. Prior to drug treatments, the cells were starved in 1 × RPMI 1640 supplemented with 0.5% FBS for 24 h. Dose-response studies were conducted to determine the suitable doses of the drugs for using in the experiments. Cells were pretreated with 40 μM DXM for 1 h. The DXM pretreated or untreated cells were subsequently treated with 100 μM TMZ for 6 h. Cells were washed with drug-free medium and allowed to grow for 48 h. Then, cells were collected for determination of viability, apoptosis, or Western blot analysis. DXM and TMZ were obtained from Sigma Chemical (St. Louis, MO) and Schering Corporation (Kenilworth, NJ), respectively. The drugs were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions, which were then stored at -20°C until used for treating cells.Human glioblastoma U87MG cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in 75-cmFollowing all treatments the viability of attached and detached cell populations was evaluated by trypan blue dye exclusion test . Viable The cells from each treatment were washed with PBS, pH 7.4, and sedimented onto the microscopic slides using Cytobucket and Centra CL2 centrifuge (IEC) at 1200 rpm for 5 min. Cells were fixed in 95% (v/v) ethanol before examination of morphology with Wright staining . The morApopTag Peroxidase kit (Intergen, Purchase, NY) was used to assess the extent of cell death following drug treatments. Briefly, cells for each treatment were grown on six-well cell culture plates (Corning Corporation., Corning, NY) and were treated as described above. Following treatments, cells were washed with PBS and then centrifuged to sediment onto the microscopic slides. Residual PBS was then removed and cells were fixed using 95% (v/v) ethanol and allowed to dry overnight. Slides were pretreated with a protein-digesting enzyme for 15 min and then washed with distilled water for 2 min. Cells were quenched with 3% (v/v) hydrogen peroxide for 5 min followed by washing with PBS. Terminal deoxynucleotidyl transferase (TdT) enzyme was added to the pre-equilibrated cells and incubated for 1 h at 37°C. Stop-buffer was added to the slide and agitated for 15 sec followed by 10 min incubation at room temperature. After washing three times with PBS for 1 min each, anti-digoxigenin peroxidase conjugate was added to the slides and incubated for 30 min. After slides were washed twice with PBS, freshly prepared peroxidase substrate 3,3'-diaminobenzidine was added to the slides and kept for 6 min and then slides were washed with water two times. Slides were counterstained with 0.5% (w/v) methyl green for 10 min followed by washing with water and then 100% n-butanol. After 10 min, cells were dehydrated in xylene for 2 min and then mounted with glass coverslip. Experiments were conducted in triplicates and the percentage of ApopTag-positive cells was determined by counting cells under light microscopy.2+] in U87MG cells. Briefly, cells were grown to 80% confluency in phenol red-free medium for 72 h, suspended in the culture medium, centrifuged at 2000 rpm for 5 min to obtain pellet, and washed twice in phosphate buffered saline (PBS, pH 7.4). Cells were resuspended in culture medium, and incubated at 37°C for 2 h with gentle shaking. Following incubation, cells wee washed twice in Ca2+-free Locke's buffer [7 cells/ml) were dispersed in Locke's buffer with 10% FBS. Fura-2 (Molecular Probes, Eugene, OR) was dissolved in DMSO and diluted in Ca2+-free Locke's buffer containing 10% FBS. Cells were mixed with 5 μM fura-2, incubated at 37°C for 30 min, washed twice and diluted to 1 × 106 cells/ml in Ca2+-free Locke's buffer. The intracellular free [Ca2+] was calculated spectrofluorometrically using the equation [Ca2+] = dβ(R-Rmin)/(Rmax-R)K, where β is the ratio of F380max, fluorescence intensity exciting at 380 nM for zero free Ca2+ to F380min, and fluorescence intensity at saturating free [Ca2+] as reported previously [(R) was performed using an SLM 8000 fluorometer (Thermospectronic, Shelton, CT) at 340 and 380 nm wavelengths. The maximal max)  (Rratios were determined using 200 μl of 250 μM digitonin (Sigma) and 500 mM EGTA (Sigma, St. Louis, MO), respectively. The value of Kd, a cell-specific constant, was determined experimentally to be 0.476 μM using standards of the Calcium Calibration Buffer Kit with Magnesium (Molecular Probes, Eugene, OR).We recently reported this method , which ws buffer  and theneviously . The detMonoclonal antibody against α-spectrin (Affiniti, Exeter, UK) was used to measure calpain activity as well as caspase-3 activity. Caspase-3 polyclonal antibody  was used to determine caspase-3 activation. Bax and Bcl-2 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used to assess apoptotic threshold by determining the Bax:Bcl-2 ratio. Antibody to β-actin  was used to standardize protein loading in Western blot experiments. The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG , except in case of calpain and α-spectrin where HRP-conjugated goat anti-rabbit IgG was used .2+ (GIBCO, Grand Island, NY). The cells were then washed twice in phosphate buffered saline (PBS, pH 7.4) and centrifuged in Eppendorf 5804R (Brinkmann Instruments, Westbury, NY) at 106 × g for 10 min. Cells were resuspended in a homogenizing buffer composed of 50 mM Tris-HCl (pH 7.4), 1 mM PMSF (Bethesda Research Laboratories, Gaithersburg, MD), and 5 mM EGTA (Sigma). A polypropylene pestle (Kontes Glass, Vineland, NJ) was placed inside the microcentrifuge tube (1.5 ml) containing cells and the tube was placed inside an ice bucket for 2 min to let the pestle cool to 4°C. Cells were completely homogenized for 30 sec (the homogenization was performed while holding the microcentrifuge tube in ice with one hand). Following homogenization, protein concentration was determined using Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL) and spectrophotometric measurement at 595 nm (Spectronic, Rochester, NY). Samples were then diluted (1:1) in sample buffer (62.5 mM, Tris pH 6.8, 2% SDS, 5 mM β-mercaptoethanol, 10% glycerol) and boiled for 5 min. Samples were then loaded onto the 4–20% gradient gels for electrophoresis at 200 V for 30 min (Bio-Rad, Hercules, CA). For detection of α-spectrin bands, a 5% gel was used for electrophoresis at 100 V for 2 h. Following electrophoresis, gels with the resolved proteins were electroblotted to nylon membranes (Millipore, Bedford, MA) in an electroblotting Genie apparatus (Idea Scientific, Minneapolis, MN). The membranes were blocked for 1 h in blocking buffer . Primary antibody was diluted  in blocking solution and then added to the blots for 1 h. The blots were washed three times with a wash buffer (Tris/Tween solution) and covered with secondary antibody  at a 1:2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia, Buckinghamshire, UK) and exposed to X-OMAT AR films (Eastman Kodak, Rochester, NY). The films were scanned on a UMAX PowerLook Scanner (UMAX Technologies, Fremont, CA) using Photoshop software (Adobe Systems, Seattle, WA), and optical density of each band was determined using Quantity One software (Bio-Rad, Hercules, CA). Estimation of the degradation products of α-spectrin indicated calpain and caspase-3 activities. The 145 kD spectrin breakdown product (SBDP) is specific for calpain activation [Cells were washed in culture flasks using Hank's balanced salt solution without Cap ≤ 0.05.Data from various experiments were analyzed using StatView software (Abacus Concepts, Berkeley, CA). Results were compared using one-way analysis of variance (ANOVA) with Fisher's protected least significant difference (PLSD) post hoc test at a 95% confidence interval. All results were presented as mean ± standard error of mean (SEM) of separate experiments (n ≥ 3). A difference between two values was considered significant at AD performed the experiments and participated in writing the manuscript. NLB contributed to the interpretation of results. SJP participated in the discussion of the study and provided comments on clinical importance of this study. SKR conceived the study, planned experimental design, supervised the study, and helped writing the manuscript. All authors read and approved the final version of the manuscript.