Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

In Vitro Cellular &; Developmental Biology - Plant -     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.     

Identification of de novo copy number variants associated with human disorders of sexual development

Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10⁻¹²). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development.     

Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration

In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.     

Grazing of two toxic Planktothrix species by Daphnia pulicaria: potential for bloom control and transfer of microcystins

The role of zooplankton in the control of cyanobacterial bloomsand the transfer of cyanotoxins to higher trophic levels areof great importance to the management of water resources. Manystudies have focused on the cyanobacterium Microcystis, butfew have examined the interactions between zooplankton and filamentouscyanobacteria. In this study, we provide experimental evidencefor the potential grazing of two toxic strains of filamentouscyanobacteria, Planktothrix rubescens and P. agardhii, by Daphniapulicaria, and for transfer of toxins in the planktonic foodchain. We determined clearance rates (CRs) by adult and juvenileD. pulicaria of the two Planktothrix strains, Scenedesmus acutusand a mixture of S. acutus cells with P. rubescens culture filtrate.Filament lengths were analyzed, and microcystin (MCY) presencein Daphnia was assessed using the Protein Phosphatase-2A (PP-2A)Inhibition Assay. The two Planktothrix strains were equallygrazed by D. pulicaria, but at lower CRs than S. acutus. Potentialanti-grazer toxins in P. rubescens filtrate did not inhibitDaphnia grazing. Small P. rubescens (<100 µm) filamentswere preferentially grazed by adult D. pulicaria, suggestingtheir limited ability to control a Planktothrix population duringa bloom. Large quantities of MCYs were found in unstarved Daphniapreviously exposed to Planktothrix, whereas quantities weresignificantly smaller in individuals starved for 24 h beforepreservation. This indicated a potential for transfer of toxinsin the food chain by Daphnia, especially immediately after ingestionof toxic cyanobacteria.     

Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species

Chronic inflammation is associated with a variety of human diseases, including cancer, with one possible mechanistic link involving over-production of nitric oxide (NO*) by activated macrophages. Subsequent reaction of NO* with superoxide in the presence of carbon dioxide yields nitrosoperoxycarbonate (ONOOCO2-), a strong oxidant that reacts with guanine in DNA to form a variety of oxidation and nitration products, such 2'-deoxy-8-oxoguanosine. Alternatively, the reaction of NO and O2 leads to the formation of N2O3, a nitrosating agent that causes nucleobase deamination to form 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from dG; 2'-deoxyinosine (dI) from dA; and 2'-deoxyuridine (dU) from dC, in addition to abasic sites and dG-dG cross-links. The presence of both ONOOCO2- and N2O3 at sites of inflammation necessitates definition of the relative roles of oxidative and nitrosative DNA damage in the genetic toxicology of inflammation. To this end, we sought to develop enzymatic probes for oxidative and nitrosative DNA lesions as a means to quantify the two types of DNA damage in in vitro DNA damage assays, such as the comet assay and as a means to differentially map the lesions in genomic DNA by the technique of ligation-mediated PCR. On the basis of fragmentary reports in the literature, we first systematically assessed the recognition of dX and dI by a battery of DNA repair enzymes. Members of the alkylpurine DNA glycosylase family (E. coli AlkA, murine Aag, and human MPG) all showed repair activity with dX (k(cat)/Km 29 x 10(-6), 21 x 10(-6), and 7.8 x 10(-6) nM(-1) min(-1), respectively), though the activity was considerably lower than that of EndoV (8 x 10(-3) nM(-1) min(-1)). Based on these results and other published studies, we focused the development of enzymatic probes on two groups of enzymes, one with activity against oxidative damage (formamidopyrimidine-DNA glycosylase (Fpg); endonuclease III (EndoIII)) and the other with activity against nucleobase deamination products (uracil DNA glycosylase (Udg); AlkA). These combinations were assessed for recognition of DNA damage caused by N2O3 (generated with a NO*/O2 delivery system) or ONOOCO2- using a plasmid nicking assay and by LC-MS analysis. Collectively, the results indicate that a combination of AlkA and Udg react selectively with DNA containing only nitrosative damage, while Fpg and EndoIII react selectively with DNA containing oxidative base lesions caused by ONOOCO2-. The results suggest that these enzyme combinations can be used as probes to define the location and quantity of the oxidative and nitrosative DNA lesions produced by chemical mediators of inflammation in systems, such as the comet assay, ligation-mediated polymerase chain reaction, and other assays of DNA damage and repair.     

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥10(17)M(-1) at 23°C, pH 7.0).     

Long time to diagnosis of medulloblastoma in children is not associated with decreased survival or with worse neurological outcome

Background

The long time to diagnosis of medulloblastoma, one of the most frequent brain tumors in children, is the source of painful remorse and sometimes lawsuits. We analyzed its consequences for tumor stage, survival, and sequelae.

Patients and Methods

This retrospective population-based cohort study included all cases of pediatric medulloblastoma from a region of France between 1990 and 2005. We collected the demographic, clinical, and tumor data and analyzed the relations between the interval from symptom onset until diagnosis, initial disease stage, survival, and neuropsychological and neurological outcome.

Results

The median interval from symptom onset until diagnosis for the 166 cases was 65 days (interquartile range 31–121, range 3–457). A long interval (defined as longer than the median) was associated with a lower frequency of metastasis in the univariate and multivariate analyses and with a larger tumor volume, desmoplastic histology, and longer survival in the univariate analysis, but not after adjustment for confounding factors. The time to diagnosis was significantly associated with IQ score among survivors. No significant relation was found between the time to diagnosis and neurological disability. In the 62 patients with metastases, a long prediagnosis interval was associated with a higher T stage, infiltration of the fourth ventricle floor, and incomplete surgical resection; it nonetheless did not influence survival significantly in this subgroup.

Conclusions

We found complex and often inverse relations between time to diagnosis of medulloblastoma in children and initial severity factors, survival, and neuropsychological and neurological outcome. This interval appears due more to the nature of the tumor and its progression than to parental or medical factors. These conclusions should be taken into account in the information provided to parents and in expert assessments produced for malpractice claims.