Differential microRNA Profiles and Their Functional Implications in Different Immunogenetic Subsets of Chronic Lymphocytic Leukemia

Critical processes of B-cell physiology, including immune signaling through the B-cell receptor (BcR) and/or Toll-like receptors (TLRs), are targeted by microRNAs. With this in mind and also given the important role of BcR and TLR signaling and microRNAs in chronic lymphocytic leukemia (CLL), we investigated whether microRNAs could be implicated in shaping the behavior of CLL clones with distinct BcR and TLR molecular and functional profiles. To this end, we examined 79 CLL cases for the expression of 33 microRNAs, selected on the following criteria: (a) deregulated in CLL versus normal B-cells; (b) differentially expressed in CLL subgroups with distinct clinicobiological features; and, (c) if meeting (a) + (b), having predicted targets in the immune signaling pathways. Significant upregulation of miR-150, miR-29c, miR-143 and miR-223 and downregulation of miR-15a was found in mutated versus unmutated CLL, with miR-15a showing the highest fold difference. Comparison of two major subsets with distinct stereotyped BcRs and signaling signatures, namely subset 1 [IGHV1/5/7-IGKV1(D)-39, unmutated, bad prognosis] versus subset 4 [IGHV4-34/IGKV2-30, mutated, good prognosis] revealed differences in the expression of miR-150, miR-29b, miR-29c and miR-101, all down-regulated in subset 1. We were also able to link these distinct microRNA profiles with cellular phenotypes, importantly showing that, in subset 1, miR-101 downregulation is associated with overexpression of the enhancer of zeste homolog 2 (EZH2) protein, which has been associated with clinical aggressiveness in other B-cell lymphomas. In conclusion, specific miRNAs differentially expressed among CLL subgroups with distinct BcR and/or TLR signaling may modulate the biological and clinical behavior of the CLL clones.     

Protein τ-Mediated Effects on Rat Hippocampal Choline Transporters CHT1 and τ-Amyloid β Interactions

It is suggested that intracellular tau protein (τ), when released extracellularly upon neuron degeneration, could evoke direct toxic effects on the cholinergic neurotransmitter system through muscarinic receptors and thus contribute to the pathogenesis of Alzheimer’s disease. In this study, we evaluated the in vitro effects of six naturally occurring monomeric τ isoforms on rat hippocampal synaptosomal choline transporters CHT1 (large transmembrane proteins associated with high-affinity choline transport and vulnerable to actions of amyloid β peptides (Aβ) applied in vitro or in vivo). Some τ isoforms at nM concentrations inhibited choline transport in a dose- and time-dependent saturable manner (352 = 441 > 410 = 383 > 381 = 412) and effects were associated with changes in the Michaelis constant rather than in maximal velocity. Moreover, the actions of τ 352/441 were not influenced by previous depolarisation of synaptosomes or by previous depletion of membrane cholesterol. Specific binding of [3H]hemicholinium-3 was not significantly altered by τ 352/441 at higher nM concentrations. Results of in vitro tests on CHT1 transporters from cholesterol-depleted synaptosomes supported interactions between Aβ 1-40 and τ 352. In addition, we developed surface plasmon resonance biosensors to monitor complexes of Aβ 1-42 and τ 352 using a sandwich detection format. It seems, therefore, that protein τ, similar to Aβ peptides, can contribute to the pathogenesis of Alzheimer’s disease through its actions on CHT1 transporters. However, the interaction mechanisms are quite different (τ probably exerts its effects through direct interactions of microtubule binding repeats with extracellular portions of the CHT1 protein without influencing the choline recognition site, Aβ rather through lipid rafts in the surrounding membranes). An N-terminal insert of τ is not necessary but the N-terminal projection domain plays a role. The developed biosensor will be used to detect Aβ–τ complexes in cerebrospinal fluid in order to evaluate them as prospective biomarkers of Alzheimer′s disease.     

The genetic basis of triple A (Allgrove) syndrome in a Greek family

Triple A (or Allgrove) syndrome is an autosomal recessive genetic disorder. Patients typically suffer from chronic adrenal insufficiency due to resistance to ACTH (Addison's disease), achalasia of the cardia, and defective tear formation (alacrima). The syndrome is caused by mutations in the AAAS gene which encodes the protein ALADIN, a constituent of eukaryotic nuclear pore complexes. The multi-systemic nature and variable manifestations of the triple A syndrome often confound its diagnosis and limit our understanding of its exact pathogenesis. We performed mutational screening of the AAAS gene in a Greek family of four individuals, including an affected propositus with typical symptoms of late-onset triple A syndrome. Our results are consistent with an autosomal recessive pattern of inheritance within the family, caused by a functional c.43C > A mutation in exon 1 of the AAAS gene. All members of the family were also homozygous for a silent c.855C > T nucleotide change within exon 9 of the AAAS gene, representing a common single nucleotide polymorphism. The compromising c.43C > A mutation is predicted to cause a p.Gln15Lys amino acid substitution in the ALADIN protein. However, it has been suggested that the functional impact of this mutation may be more severe, causing a shift in the reading frame of AAAS gene via formation of an aberrant premature donor splice site within exon 1. We propose that mutational analysis of the AAAS gene should be considered in adult patients with one or more clinical signs of the disease, as diagnosis of late-onset cases can be ambiguous.     

Evaluating developmental shape changes in Homo antecessor subadult facial morphology

The fossil ATD6-69 from Atapuerca, Spain, dated to ca. 900 ka (thousands of years ago) has been suggested to mark the earliest appearance of modern human facial features. However, this specimen is a subadult and the interpretation of its morphology remains controversial, because it is unclear how developmental shape changes would affect the features that link ATD6-69 to modern humans. Here we analyze ATD6-69 in an evolutionary and developmental context. Our modern human sample comprises cross-sectional growth series from four populations. The fossil sample covers human specimens from the Pleistocene to the Upper Paleolithic, and includes several subadult Early Pleistocene humans and Neanderthals. We digitized landmarks and semilandmarks on surface and CT scans and analyzed the Procrustes shape coordinates using multivariate statistics. Ontogenetic allometric trajectories and developmental simulations were employed in order to identify growth patterns and to visualize potential adult shapes of ATD6-69. We show that facial differences between modern and archaic humans are not exclusively allometric. We find that while postnatal growth further accentuates the differences in facial features between Neanderthals and modern humans, those features that have been suggested to link ATD6-69's morphology to modern humans would not have been significantly altered in the course of subsequent development. In particular, the infraorbital depression on this specimen would have persisted into adulthood. However, many of the facial features that ATD6-69 shares with modern humans can be considered to be part of a generalized pattern of facial architecture. Our results present a complex picture regarding the polarity of facial features and demonstrate that some modern human-like facial morphology is intermittently present in Middle Pleistocene humans. We suggest that some of the facial features that characterize recent modern humans may have developed multiple times in human evolution.     

Synthesis and evaluation of carbamoylmethylene linked prodrugs of BMS-582949, a clinical p38α inhibitor

A series of carbamoylmethylene linked prodrugs of 1 (BMS-582949), a clinical p38α inhibitor, were synthesized and evaluated. Though the phosphoryloxymethylene carbamates (3, 4, and 5) and α-aminoacyloxymethylene carbamates (22, 23, and 26) were found unstable at neutral pH values, fumaric acid derived acyloxymethylene carbamates (2, 28, and 31) were highly stable under both acidic and neutral conditions. Prodrugs 2 and 31 were also highly soluble at both acidic and neutral pH values. At a solution dose of 14.2 mpk (equivalent to 10 mpk of 1), 2 gave essentially the same exposure of 1 compared to dosing 10 mpk of 1 itself. At a suspension dose of 142 mpk (equivalent to 100 mpk of 1), 2 demonstrated that it could overcome the solubility issue associated with 1 and provide a much higher exposure of 1. To our knowledge, the unique type of prodrugs like 2, 28, and 31 was not reported in the past and could represent a novel prodrug approach for secondary amides, a class of molecules frequently identified as drug candidates.     

Aminoimidazole Carboxamide Ribonucleotide (AICAR) Inhibits the Growth of Retinoblastoma In Vivo by Decreasing Angiogenesis and Inducing Apoptosis

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.     

Elevated p16ink4a Expression in Human Labial Salivary Glands as a Potential Correlate of Cognitive Aging in Late Midlife

Background

The cell-cycle inhibitor and tumor suppressor cyclin dependent kinase inhibitor, p16^ink4a, is one of the two gene products of the ink4a/ARF (cdkn2a) locus on chromosome 9q21. Up-regulation of p16^ink4a has been linked to cellular senescence, and findings from studies on different mammalian tissues suggest that p16^ink4a may be a biomarker of organismal versus chronological age.

Objective

The aim of this study was to examine the immunolocalization pattern of p16^ink4a in human labial salivary gland (LSG) tissue, and to analyze whether its expression level in LSGs is a peripheral correlate of cognitive decline in late midlife.

Methods

The present study was a part of a study of causes and predictors of cognitive decline in middle-aged men in a Danish birth cohort. It is based on data from 181 male participants from the Danish Metropolit birth cohort, born in 1953, who were examined for age-associated alterations in cognition, dental health, and morphological and autonomic innervation characteristics of the LSGs. The participants were allocated to two groups based on the relative change in cognitive performance from young adulthood to late midlife. LSG biopsies were analyzed by qRT-PCR for the expression level of p16^ink4a. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of LSGs.

Results

p16^ink4a immunoreactivity was observed in LSG ductal, myoepithelial, and stromal cells, but not in acinar cells. The mean relative expression of p16^ink4a in LSGs was higher in the group of participants with decline in cognitive performance. A logistic regression analysis revealed that the relative p16 expression was predictive of the participant’s group assignment. A negative correlation was found between relative p16^ink4a expression and the participant’s standardized regression residuals from early adulthood to late midlife cognitive performance scores.

Conclusions

p16^ink4a expression in human LSGs may constitute a potential peripheral correlate of cognitive decline. Human labial salivary glands seem suitable for studies on organismal as opposed to chronological age.