Study of chaperone-like activity of human haptoglobin: conformational changes under heat shock conditions and localization of interaction sites

With respect to the mechanism of chaperone-like activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperone-like activity were not fully identical with the region that takes part in formation of the hemoglobin-haptoglobin complex. We can postulate the presence of at least two different chaperone-binding sites on each haptoglobin heavy chain.     

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.     

Focus on the research utility of intravascular ultrasound - comparison with other invasive modalities

Intravascular ultrasound (IVUS) is an invasive modality which provides cross-sectional images of a coronary artery. In these images both the lumen and outer vessel wall can be identified and accurate estimations of their dimensions and of the plaque burden can be obtained. In addition, further processing of the IVUS backscatter signal helps in the characterization of the type of the plaque and thus it has been used to study the natural history of the atherosclerotic evolution. On the other hand its indigenous limitations do not allow IVUS to assess accurately stent struts coverage, existence of thrombus or exact site of plaque rupture and to identify some of the features associated with increased plaque vulnerability. In order this information to be obtained, other modalities such as optical coherence tomography, angioscopy, near infrared spectroscopy and intravascular magnetic resonance imaging have either been utilized or are under evaluation. The aim of this review article is to present the current utilities of IVUS in research and to discuss its advantages and disadvantages over the other imaging techniques.     

Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies

The high‐throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually <1%) across samples sequenced on the same lane. Alarmingly high rates of read misassignment of up to 10% were reported for lllumina sequencing machines with exclusion amplification chemistry. This may make use of these platforms prohibitive, particularly in studies that rely on low‐quantity and low‐quality samples, such as historical and archaeological specimens. Here, we use barcodes, short sequences that are ligated to both ends of the DNA insert, to directly quantify the rate of index hopping in 100‐year old museum‐preserved gorilla (Gorilla beringei) samples. Correcting for multiple sources of noise, we identify on average 0.470% of reads containing a hopped index. We show that sample‐specific quantity of misassigned reads depends on the number of reads that any given sample contributes to the total sequencing pool, so that samples with few sequenced reads receive the greatest proportion of misassigned reads. This particularly affects ancient DNA samples, as these frequently differ in their DNA quantity and endogenous content. Through simulations we show that even low rates of index hopping, as reported here, can lead to biases in ancient DNA studies when multiplexing samples with vastly different quantities of endogenous material.     

Effect of liquid pulses with 6-benzyladenine on the induction of somatic embryogenesis from coffee (Coffea arabica L.) callus cultures

We investigated the effect of the physical state of the nutrient medium on the induction of somatic embryogenesis on cell cultures derived from coffee (Coffea arabica L.). Non-embryogenic callus tissues were pulsed initially with 50 μM 6-benzyladenine (BA) for 6, 24 or 48 h in half-strength liquid Murashige and Skoog (MS) medium. After pretreatment, calli were transferred to agar-solidified half-strength MS medium supplemented with 50 μM BA (‘standard induction medium’). Control callus tissues were incubated directly on the solid standard induction medium. Callus growth was promoted by longer pretreatment periods. Formation of globular somatic embryos was observed on callus tissues pretreated with BA for 24 or 48 h, which developed fully to cotyledonary-stage within only 2 weeks after transfer to agar-solidified medium supplemented with BA. No embryo formation occurred in control cultures. Pretreatment with BA in liquid medium was associated with changes in the redox status of cultured cells, such as alterations of the ascorbate–glutathione redox systems and the accumulation of free radicals and oxidized lipids, as well as the possible reduction of cytochrome c-mediated apoptotic pathways. In particular, the induction of somatic embryogenesis was highly positively correlated (r ² = 0.822) with the accumulation of protein carbonyls. The physiological role of BA as an inducer of both embryonic differentiation and cellular death is discussed.     

Predictors of new atherosclerotic carotid plaque development in patients with rheumatoid arthritis: a longitudinal study

Introduction

Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality attributed to both classical risk factors and chronic inflammation. We assessed longitudinally the factors associated with new carotid plaques in nondiabetic RA patients and apparently healthy individuals.

Methods

In our present prospective observational study, carotid plaques were identified by ultrasonography at baseline and follow-up end, separated by an average of 3.6 ± 0.2 years, in 64 patients (mean age 59.2 ± 12.0 and disease duration at baseline 7.8 ± 6.2 years, 83% women, clinical and laboratory evaluation every 3 to 6 months). In a substudy, 35 of the patients were matched 1:1 for traditional cardiovascular risk factors with 'healthy' controls and were studied in parallel.

Results

New atherosclerotic plaques formed in 30% of patients (first plaque in 9%) who were significantly older than the remaining patients. Tobacco use, blood pressure, body mass index, average cumulative low-density lipoprotein, high-sensitivity C-reactive protein, erythrocyte sedimentation rate level, RA stage, functional class, disease duration and treatment modalities during follow-up did not differ significantly between subgroups after application of the Bonferroni correction. RA was in clinical remission, on average, for approximately 70% of the follow-up time and was not different between subgroups. Multivariate analysis including all the above parameters revealed that age (P = 0.006), smoking (P = 0.009) and duration of low-dose corticosteroid use (P = 0.016) associated independently with new plaque formation. RA patients displayed similar numbers of newly formed carotid plaques to the tightly matched for traditional cardiovascular risk factors 'healthy' controls, although more patients than controls had carotid plaques at baseline.

Conclusions

Formation of new atherosclerotic plaques in this small cohort of patients with well-controlled RA depended mainly on traditional cardiovascular risk factors and corticosteroid use, whereas an adverse effect of residual systemic inflammation was not readily detectable.     

Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.