Immunogenic gangliosides in human ovarian carcinoma

Ganglioside signatures of four poorly and three moderately differentiated ovarian epithelial cancer (OEC) cell lines reveal the presence of GM3, GM2, GD2, O-AcGD2, GD1a and GM1b. The expression of GM3, presence of GD1a and GM1b in the ascitic fluid and plasma, together with a positive correlation in the total-gangliosides levels between ascitic fluid and plasma of OEC patients support the earlier contention that the tumor-gangliosides may be released (or shed) into the tumor-microenvironment. The immunogenicity of OEC-gangliosides is determined by comparing anti-ganglioside-IgM titers in ascitic fluid (n = 14) and plasma (n = 23) of OEC-patients and age-matched healthy (n = 14). The titers were measured by ELISA. Strikingly, the level of anti-GD1a-IgM is significantly higher in ascitic fluid and plasma of patients than in the plasma of healthy volunteers. Paired sample analysis of ascitic fluid and plasma from the same patients confirmed the significant expression of anti-GD1a IgM in OEC patients, while no such difference was observed with other anti-ganglioside IgMs among different groups. The significance of the endogenous IgM response to GD1a may be to eliminate this immunosuppressive-ganglioside from the tumor-microenvironment.     

Less calcemic Vitamin D analogs enhance creatine kinase specific activity and modulate responsiveness to gonadal steroids in the vasculature

Vitamin D receptors are widely expressed in the cardiovascular system, in which Vitamin D and its metabolites exert a variety of biological activities such as regulation of cellular proliferation and differentiation, cell calcium transients and cell energy metabolism in vitro. The latter is mediated through the control of the brain type creatine kinase specific activity (CK), which serves to provide a readily available reservoir for ATP generation under increased work-load. In the present study we undertook to assess the role of Vitamin D on energy metabolism in the rat heart and aorta in vivo by using CK, which is a key energy metabolizing enzyme and compare Vitamin D depleted and repleted animals. Vascular tissues from female or male Vitamin D-depleted rats showed 61-80% lower CK activity in the aorta (Ao) and left ventricle of the heart (Lv) than control, Vitamin D-replete rats. Moreover, neither estradiol-17beta (E2) nor dihydrotestosterone (DHT), which increases CK specific activity in Ao and Lv of intact female or male rats, respectively, were able to stimulate CK in Vitamin D-depleted rats. Treatment of intact female rats for 2 weeks or 2 months with the less-calcemic Vitamin D analogs JKF 1624F2-2 (JKF) or QW 1624F2-2 (QW) (Fig. 1), did not significantly affect CK specific activity. However, after pretreatment with these analogs, there was an up regulation of the E2-induced CK response in Ao and Lv. In intact female rats, all Vitamin D analogs also potentiated the in vivo CK response to the SERMs raloxifene (Ral) and tamoxifen (TAM) in Ao and Lv. However the inhibitory effect of Ral or TAM on E2-induced CK activity was lost after pretreatment with Vitamin D analogs. The non-calcemic analog CB 1093 (CB) induced a significant increase in estradiol receptor alpha (ERalpha) protein in both myocardial and aortic tissue from intact and from ovariectomized female rats. Collectively, these results indicate that Vitamin D analogs modulate cell energy homeostasis in vascular tissues through induction of CK and up regulation of the response and sensitivity of CK in vascular tissues to E2 and to SERMs, possibly through via an increase in ERalpha protein in female derived organs. These results corroborate our previous in vitro studies in human vascular cells and further suggest that the Vitamin D system plays an important physiological role in maintaining normal cell energy reservoir in the vasculature.     

Pluripotency genes overexpressed in primate embryonic stem cells are localized on homologues of human chromosomes 16, 17, 19, and X

While human embryonic stem cells (hESCs) are predisposed toward chromosomal aneploidities on 12, 17, 20, and X, rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genes overexpressed in pluripotent rhesus ESCs (nhpESCs) and comparing them both to their genetically identical differentiated progeny (teratoma fibroblasts) and to genetically related differentiated parental cells (parental skin fibroblasts from whom gametes were used for ESC derivation), we find that some of those overexpressed genes in nhpESCs cluster preferentially on rhesus chromosomes 16, 19, 20, and X, homologues of human chromosomes 17, 19, 16, and X, respectively. Differentiated parental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are genetically identical to the pluripotent nhpESCs. Twenty over- and underexpressed pluripotency modulators, some implicated in neurogenesis, have been identified. The overexpression of some of these genes discovered using pedigreed nhpESCs derived from prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinically discarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions in pluripotency and suggests specific candidates for targeting differentiation and transformation decisions.     

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.     

Selection for reduced translation costs at the intronic 5′ end in fungi

It is generally believed that introns are not translated; therefore, the potential intronic features that may be related to the translation step (occurring after splicing) have yet to be thoroughly studied. Here, focusing on four fungi, we performed for the first time a comprehensive study aimed at characterizing how translation efficiency is encoded in introns and affects their evolution. By analysing their intronome we provide evidence of selection for STOP codons close to the intronic 5′ end, and show that the beginning of introns are selected for significantly high translation, presumably to reduce translation and metabolic costs in cases of non-spliced introns. Ribosomal profiling data analysis in Saccharomyces cerevisiae supports the conjecture that in this organism intron retention frequently occurs, introns are partially translated, and their translation efficiency affects organismal fitness. We show that the reported results are more significant in highly translated and highly spliced genes, but are not associated only with genes with a specific function. We also discuss the potential relation of the reported signals to efficient nonsense-mediated decay due to splicing errors. These new discoveries are supported by population-genetics considerations. In addition, they are contributory steps towards a broader understanding of intron evolution and the effect of silent mutations on gene expression and organismal fitness.     

Fate of adenovirus type 12 genomes in nonpermissive cells

The fate of ³H-thymidine-labeled adenovirus type 12 deoxyribonucleic acid (DNA) was studied in Nil-2 cells of Syrian hamster origin. It was found that a substantial fraction of ³H-adenovirus type 12 DNA became degraded within 24 hr after infection and was released into the culture fluid. After infection of 5-bromodeoxyuridine (BUdR)-prelabeled cells with ³H-adenovirus type 12, viral DNA became readily separable from cellular DNA by equilibrium centrifugation in CsCl. Part of the viral radioactivity was found to shift gradually to the position of cellular DNA as time progressed after infection. When exponentially growing cells were exposed simultaneously to BUdR, 5-fluorodeoxyuridine, and ³H-adenovirus type 12, up to 50% of the viral radioactivity shifted within 24 hr from the density of viral DNA to that of cellular DNA after equilibrium centrifugation in CsCl. Upon denaturation of the cellular DNA, the isotope was preferentially found to be associated with the “heavy” strand which was synthesized after infection. Upon hybridization of the “heavy” and the “light” strands with sonically treated, denatured ³H-adenovirus type 12 DNA, small and nearly equal amounts of counts hybridized with both strands. The number of counts annealed was in a range similar to that of those annealed with the same amount of DNA derived from adenovirus type 12-transformed hamster cells. These results demonstrate that (i) a substantial proportion of the adsorbed virus becomes degraded within 24 hr; (ii) part of the degradation products is reutilized for cellular DNA synthesis; (iii) only a small fraction, mainly fragments, of viral DNA becomes integrated into both the newly synthesized and the parental strands of cellular DNA.     

The convergence in distribution of some simple epidemics

A group of n susceptible individuals exposed to a contagious disease isconsidered. It is assumed that at each point in time one or more susceptible individuals can contract the disease. The progress of this simple batch epidemic is modeled by a stochastic process X_n(t), t[0, ∞), representing the number of infectiveindividuals at time t. In this paper our analysis is restricted to simple batch epidemics with transition rates given by [α²X_n(t){n −X_n(t) +X_n(0)}]^1/2, t[0, ∞), α(0, ∞). This class of simple batch epidemics generalizes a model used and motivated by McNeil (1972) to describe simple epidemic situations. It is shown for this class of simple batch epidemics, that X_n(t), with suitable standardization, converges in distribution as n→∞ to a normal random variable for all t(0, t₀), and t₀ is evaluated.