Effect of age on surface molecules and cytokine expression in human dendritic cells

Dendritic cells (DCs) are central in regulating both innate and acquired immunity, but their possible age-related functional modifications are still unclear. Here we have analyzed the effect of age on LPS-treated monocyte-derived DCs (MDDCs). A negative correlation between age and cell expression of ICAM-1, CD25 and IL-10 was observed in a group of healthy donors. This has been confirmed by a significantly reduced expression of the same molecules in cells of subgrouped elderly versus younger individuals. On the contrary, a positive correlation between age and cell expression of IL-6 and IL-18 has been reported in all the subjects and further supported by a significant increase of the two pro-inflammatory cytokines in cells of elderly versus young subjects.Our data indicate that aging can impair the expression of ICAM-1 and CD25 and skew the production of cytokines, including IL-18, which concur to make a pro-inflammatory milieu. Altogether, the present results point to additional molecules whose role might be relevant in immunosenescence of human DCs, confirming that these cells undergo functional changes during aging.     

Stabilization of a supplemental digestive enzyme by post-translational engineering using chemically-activated polyethylene glycol

Many enzymes used as digestive aids exhibit, at best, moderate stability when incubated under gastrointestinal conditions. A supplemental β-galactosidase administered orally to treat lactose intolerance was conjugated to 40 kDa, branched polyethylene glycol (PEG). PEGylation increased the enzyme's relative activity at lower pH values (2.5-4.5) and doubled enzyme stability at pH 2.5. The PEGylated enzyme retained significantly more residual activity after exposure to simulated gastric conditions (52% versus 31%), a consequence of protection from both pepsin and low pH mediated inactivation. Conjugation also provided significant protection against the proteolytic component of pancreatin. Overall, the PEGylated enzyme retained over twice the levels of residual activity recorded for non-PEGylated enzyme after exposure to complete simulated gastrointestinal conditions. PEGylation also marginally improved the enzyme's kinetic characteristics. When using its physiological substrate (lactose), K(m) values recorded were slightly decreased (from 83 to 60 μM) and k(cat)/K(m) values (M(-1) s(-1)) were increased from 100 to 147. This appears to be the first report of the use of a conjugated PEG to stabilize a digestive enzyme and the first report of the ability of conjugated PEG to stabilize a protein at low pH.     

Dual nature of translational control by regulatory BC RNAs

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3' stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.     

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.     

Cloning and sequencing of four new metallothionein genes from Tetrahymena thermophila and T. pigmentosa: evolutionary relationships in Tetrahymena MT family

The structure of four new MT (metallothionein) genes of Tetrahymena thermophila and T. pigmentosa were characterized. The MT-2 genes from the two species are very similar, differing by 10 out of 2259 sequenced nucleotides, and the deduced amino acid sequences are identical. The MT-1 genes from T. pigmentosa and T. thermophila are also very similar, differing only by 3 nucleotides in the 5′-UT region. The promoter regions contain a TATA box and many stretches partially matching some regulatory elements such as metal-responsive (MREs), antioxidant-responsive (AREs), a CAAT box, a G-box, and AP1 and ACE-1 binding sites. The related coding and amino acid sequences were compared with those previously sequenced in Tetrahymena. This analysis revealed two independent events of duplication occurring in Cd- (MT-1 and MTT1) and Cu- (MT-2) induced MTs. This evolutionary pathway also explains the unusual length of these proteins, which are much longer than many MTs studied so far. Additionally, the orthology and paralogy relationships of the various MTs are presented. Finally, on the basis of phylogenetic analyses of Tetrahymena MTs, two evolutionary hypotheses are proposed.     

Insight into genetic determinants of resting heart rate

Background

Recent studies suggested that resting heart rate (RHR) might be an independent predictor of cardiovascular mortality and morbidity. Nonetheless, the interrelation between RHR and cardiovascular diseases is not clear. In order to resolve this puzzle, the importance of genetic determinants of RHR has been recently suggested, but it needs to be further investigated.

Objective

The aim of this study was to estimate the contribution of common genetic variations on RHR using Genome Wide Association Study.

Methods

We performed a Genome Wide Association Study in an isolated population cohort of 1737 individuals, the Italian Network on Genetic Isolates — Friuli Venezia Giulia (INGI-FVG). Moreover, a haplotype analysis was performed. A regression tree analysis was run to highlight the effect of each haplotype combination on the phenotype.

Results

A significant level of association (p < 5 × 10^− 8) was detected for Single Nucleotide Polymorphisms (SNPs) in two genes expressed in the heart: MAML1 and CANX. Founding that the three different variants of the haplotype, which encompass both genes, yielded a phenotypic correlation. Indeed, a haplotype in homozygosity is significantly associated with the lower quartile of RHR (RHR ≤ 58 bpm). Moreover no significant association was found between cardiovascular risk factors and the different haplotype combinations.

Conclusion

Mastermind-like 1 and Calnexin were found to be associated with RHR. We demonstrated a relation between a haplotype and the lower quartile of RHR in our populations. Our findings highlight that genetic determinants of RHR may be implicated in determining cardiovascular diseases and could allow a better risk stratification.     

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available.     

Clinical and Biochemical Correlates of Serum L-Ergothioneine Concentrations in Community-Dwelling Middle-Aged and Older Adults

Background

Despite the increasing interest towards the biological role of L-ergothioneine, little is known about the serum concentrations of this unusual aminothiol in older adults. We addressed this issue in a representative sample of community-dwelling middle-aged and older adults.

Methods

Body mass index, estimated glomerular filtration rate, serum concentrations of L-ergothioneine, taurine, homocysteine, cysteine, glutathione, cysteinylglycine, and glutamylcysteine were evaluated in 439 subjects (age 55–85 years) randomly selected from the Hunter Community Study.

Results

Median L-ergothioneine concentration in the entire cohort was 1.01 IQR 0.78–1.33 µmol/L. Concentrations were not affected by gender (P = 0.41) or by presence of chronic medical conditions (P = 0.15). By considering only healthy subjects, we defined a reference interval for L-ergothioneine serum concentrations from 0.36 (90% CI 0.31–0.44) to 3.08 (90% CI 2.45–3.76) µmol/L. Using stepwise multiple linear regression analysis L-ergothioneine was negatively correlated with age (rpartial = −0.15; P = 0.0018) and with glutamylcysteine concentrations (rpartial = −0.13; P = 0.0063).

Conclusions

A thorough analysis of serum L-ergothioneine concentrations was performed in a large group of community-dwelling middle-aged and older adults. Reference intervals were established. Age and glutamylcysteine were independently negatively associated with L-ergothioneine serum concentration.     

Sequential Cross-Species Chromosome Painting among River Buffalo,Cattle, Sheep and Goat: A Useful Tool for Chromosome Abnormalities Diagnosis within the Family Bovidae

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards.