Lower Cerebrospinal Fluid/Plasma Fibroblast Growth Factor 21 (FGF21) Ratios and Placental FGF21 Production in Gestational Diabetes

Objectives

Circulating Fibroblast Growth Factor 21 (FGF21) levels are increased in insulin resistant states such as obesity, type 2 diabetes mellitus and gestational diabetes mellitus (GDM). In addition, GDM is associated with serious maternal and fetal complications. We sought to study human cerebrospinal fluid (CSF) and corresponding circulating FGF21 levels in women with gestational diabetes mellitus (GDM) and in age and BMI matched control subjects. We also assessed FGF21 secretion from GDM and control human placental explants.

Design

CSF and corresponding plasma FGF21 levels of 24 women were measured by ELISA [12 GDM (age: 26–47 years, BMI: 24.3–36.3 kg/m²) and 12 controls (age: 22–40 years, BMI: 30.1–37.0 kg/m²)]. FGF21 levels in conditioned media were secretion from GDM and control human placental explants were also measured by ELISA.

Results

Glucose, HOMA-IR and circulating NEFA levels were significantly higher in women with GDM compared to control subjects. Plasma FGF21 levels were significantly higher in women with GDM compared to control subjects [234.3 (150.2–352.7) vs. 115.5 (60.5–188.7) pg/ml; P<0.05]. However, there was no significant difference in CSF FGF21 levels in women with GDM compared to control subjects. Interestingly, CSF/Plasma FGF21 ratio was significantly lower in women with GDM compared to control subjects [0.4 (0.3–0.6) vs. 0.8 (0.5–1.6); P<0.05]. FGF21 secretion into conditioned media was significantly lower in human placental explants from women with GDM compared to control subjects (P<0.05).

Conclusions

The central actions of FGF21 in GDM subjects maybe pivotal in the pathogenesis of insulin resistance in GDM subjects. The significance of FGF21 produced by the placenta remains uncharted and maybe crucial in our understanding of the patho-physiology of GDM and its associated maternal and fetal complications. Future research should seek to elucidate these points.     

Antiestrogenic and antitumor properties of the new triphenylethylene derivative toremifene in the rat

The effects of toremifene, a new triphenylethylene derivative, on the uterus and DMBA-induced mammary tumors in rats were compared to tamoxifen. The ability of toremifene to compete with [3H]estradiol for cytoplasmic estrogen receptor from rat uterus was similar to tamoxifen, the IC50 being 26 and 23 microM respectively. In immature intact rats the two compounds, administered orally for three consecutive days, had similar intrinsic partial estrogenic efficacy, at 50 mg/kg, about 40% of that of estradiol benzoate (EB). However, at doses less than or equal to 10 mg/kg, the estrogenic effect of toremifene was seen at doses about 40 times higher than that of tamoxifen. The two compounds, administered together with a standard dose of EB, expressed the same maximal antiestrogenic efficacy (about 65% inhibition) at 50 mg/kg. However, the minimal effective antiestrogenic dose of toremifene was about 10 times that of tamoxifen and the ratio between antiestrogenic/estrogenic properties was favourable to toremifene. The duration of the antiestrogenic (antiuterotrophic) effect of a single oral dose (10 mg/kg) of the two compounds proved similar: at least 4 days in intact rats and 3 days in ovariectomized rats. In DMBA-induced tumor bearing rats toremifene was administered p.o., 6 times/week for 4 weeks at 0.08, 0.4, 2, 10 and 50 mg/kg. It was effective at the doses of 2, 10 and 50 mg/kg, inducing 39, 35 and 46% tumor regressions. The activity of toremifene at the minimal effective dose of 2 mg/kg was then compared with that of tamoxifen given at the same dose level. The compounds had comparable activity (47 vs 44% tumor regressions).     

Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.     

Growth and fluctuating asymmetry of human newborns: Influence of inbreeding and parental education

Historically, medical concerns about the deleterious effects of closely inbred marriages have focused on the risk posed by recessive Mendelian disease, with much less attention to developmental instability. We studied the effects of inbreeding (first‐cousin marriage) on growth and fluctuating asymmetry of 200 full‐term infants (101 inbred and 99 outbred) whose parents were of similar socioeconomic status in Sivas Province, Turkey. In addition to differences in their mean inbreeding coefficients (f = 1/16 for first cousins and f < 1/1,024 for unrelated parents), the consanguineous parents were less well educated (3 years, on average for both husbands and wives). We measured weight, height, head circumference, and chest circumference of the newborns, as well as four bilateral traits (ear width, ear length, and second and fourth digit lengths). After taking education into account, none of the measures of size (weight, height, head circumference, and chest circumference) and fluctuating asymmetry differed between the inbred and outbred groups. Male children of well‐educated parents, however, were larger and had less fluctuating asymmetry. Female children of well‐educated parents weighed more than those of less well‐educated parents, but were otherwise indistinguishable for height, head circumference, chest circumference, and fluctuating asymmetry. We conclude that inbreeding depression causes neither an increase in fluctuating asymmetry of full‐term newborns, nor a decrease in body size. Unmeasured variables correlated with education appear to have an effect on fluctuating asymmetry and size of male children and only a weak effect on size (weight) of female children. Am J Phys Anthropol 153:45–51, 2014. © 2013 Wiley Periodicals, Inc.     

Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.     

Biological Responses of Human Gingival Fibroblasts (HGFs) in an Innovative Co-Culture Model with Streptococcus mitis to Thermosets Coated with a Silver Polysaccharide Antimicrobial System

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6) and prostaglandin E₂ (PGE₂) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE₂ production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE₂ secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.     

Telomerase Recruitment in Saccharomyces cerevisiae Is Not Dependent on Tel1-Mediated Phosphorylation of Cdc13

In Saccharomyces cerevisiae, association between the Est1 telomerase subunit and the telomere-binding protein Cdc13 is essential for telomerase to be recruited to its site of action. A current model proposes that Tel1 binding to telomeres marks them for elongation, as the result of phosphorylation of a proposed S/TQ cluster in the telomerase recruitment domain of Cdc13. However, three observations presented here argue against one key aspect of this model. First, the pattern of Cdc13 phosphatase-sensitive isoforms is not altered by loss of Tel1 function or by mutations introduced into two conserved serines (S249 and S255) in the Cdc13 recruitment domain. Second, an interaction between Cdc13 and Est1, as monitored by a two-hybrid assay, is dependent on S255 but Tel1-independent. Finally, a derivative of Cdc13, cdc13–(S/TQ)₁₁→(S/TA)₁₁, in which every potential consensus phosphorylation site for Tel1 has been eliminated, confers nearly wild-type telomere length. These results are inconsistent with a model in which the Cdc13–Est1 interaction is regulated by Tel1-mediated phosphorylation of the Cdc13 telomerase recruitment domain. We propose an alternative model for the role of Tel1 in telomere homeostasis, which is based on the assumption that Tel1 performs the same molecular task at double-strand breaks (DSBs) and chromosome termini.TELOMERE length homeostasis is a highly regulated process that must balance telomere loss (as the result of incomplete replication and/or nucleolytic degradation) with telomeric repeat addition (through the action of telomerase and/or recombination). In the budding yeast Saccharomyces cerevisiae, a key regulatory event is recruitment of telomerase to chromosome ends by the telomere end-binding protein Cdc13 (Nugent et al. 1996; Evans and Lundblad 1999; Pennock et al. 2001; Bianchi et al. 2004; Chan et al. 2008). Recruitment relies on a direct interaction between Cdc13 and the Est1 subunit of telomerase (Pennock et al. 2001), which brings the catalytic core of the enzyme to its site of action. Disruption of this interaction, due to mutations in either CDC13 (cdc13-2) or EST1 (est1-60), results in an Est (ever-shorter-telomere) phenotype, as manifested by progressive telomere shortening and an eventual senescence phenotype. The recruitment activity of Cdc13, which resides in a 15-kDa N-terminal domain (Pennock et al. 2001), is sufficient to direct telomerase even to nontelomeric sites (Bianchi et al. 2004). As predicted by the recruitment model, association of telomerase with telomeres is greatly reduced in strains expressing the recruitment-defective cdc13-2 allele (Chan et al. 2008).Telomerase action at individual telomeres is highly regulated. Using an assay that monitors telomere addition at single nucleotide resolution (single telomere extension, STEX), Lingner and colleagues showed that only ∼7% of telomeres with wild-type (i.e., 300 bp) length are elongated by telomerase during a single cell cycle (Teixeira et al. 2004). However, as telomere length declines, the extension frequency increases: ∼20% of telomeres 200 bp in length and >40% of 100-bp-long telomeres are elongated (Teixeira et al. 2004; Arneric and Lingner 2007). The mechanism by which telomerase distinguishes short from long telomeres has been the subject of intense investigation. Work from a number of laboratories has led to the proposal that Tel1-dependent phosphorylation of Cdc13 at underelongated telomeres mediates the interaction between Cdc13 and the telomerase-associated Est1 protein, thus ensuring that telomerase is directed to the shortest telomeres in a population. In support of this model, the Est1 and Est2 telomerase subunits exhibit enhanced association with telomeres that have been artificially shortened, whereas Cdc13 displays length-independent association with telomeres (Bianchi and Shore 2007; Sabourin et al. 2007). This suggests that the preferential elongation of shorter telomeres is controlled at the level of recruitment of the telomerase holoenzyme by Cdc13. Furthermore, efficient association of Est1 and Est2 with chromosome ends requires Tel1 and Mre11 (which acts in the same pathway as Tel1 for telomere length regulation; Nugent et al. 1998; Ritchie and Petes 2000) but not Mec1 (Takata et al. 2005; Goudsouzian et al. 2006). Tel1 itself is also telomere bound (Takata et al. 2004), with enhanced binding to shorter telomeres (Bianchi and Shore 2007; Hector et al. 2007; Sabourin et al. 2007; Abdallah et al. 2009), although there is considerable controversy over the degree and timing of Tel1 association with chromosome termini during the cell cycle. As expected for a key regulator of the interaction between Cdc13 and a telomerase subunit, a tel1-Δ strain has short telomeres (Lustig and Petes 1986), although telomere length is not impaired enough to confer the Est phenotype displayed by cdc13-2 and est1-60 strains.Implicit in the above proposal is that Cdc13 must be a direct substrate of Tel1. In support of this, Teng and colleagues reported several years ago that the recruitment domain of Cdc13 has a cluster of potential Tel1 (and/or Mec1) phosphorylation sites (Tseng et al. 2006). Substrates of the DNA damage kinases often contain several closely spaced phosphorylation sites, termed S/TQ cluster domains (SCDs), which are considered a structural hallmark of many DNA damage-response proteins (Traven and Heierhorst 2005). On the basis of in vitro kinase assays with GST fusions to 75- to 90-amino-acid portions of the Cdc13 recruitment domain, Tseng et al. 2006 concluded that four SQ sites in the recruitment domain of Cdc13 are overlapping substrates for both Tel1 and Mec1, leading to the proposal that telomerase recruitment in S. cerevisiae is regulated by Tel1-dependent phosphorylation of Cdc13.The above model makes a key prediction: in a tel1-Δ strain, telomerase should no longer exhibit a length-dependent pattern of elongation. However, preferential elongation of short telomeres still occurs at native chromosome ends in the absence of Tel1 (Arneric and Lingner 2007). In addition, Petes and colleagues have argued, on the basis of epistasis data, that Tel1 performs an indirect role in the telomerase pathway, rather than directly targeting a telomerase regulator (Ritchie et al. 1999; Ritchie and Petes 2000). These observations are not easily explained, if preferential recognition of short telomeres by telomerase is mediated by Tel1-dependent phosphorylation of Cdc13. In this current study, we have re-examined the evidence for phosphorylation of Cdc13 as a regulatory mechanism for telomere length homeostasis. We report on a series of observations that indicate that Tel1 contributes to telomere length control through a mechanism other than phosphorylation of the Cdc13 S/TQ cluster.