Sequence and Properties of Cagein,a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as “basal-body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are “gold standard” because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the “mix & measure” assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca⁺² measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compard the IC₅₀ values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca⁺² mobilization assays can be an alternate to radioligand binding assays.     

Prognostic impact of coronary microcirculation abnormalities in systemic sclerosis: a prospective study to evaluate the role of non-invasive tests

Introduction

Microcirculation dysfunction is a typical feature of systemic sclerosis (SSc) and represents the earliest abnormality of primary myocardial involvement. We assessed coronary microcirculation status by combining two functional tests in SSc patients and estimating its impact on disease outcome.

Methods

Forty-one SSc patients, asymptomatic for coronary artery disease, were tested for coronary flow velocity reserve (CFR) by transthoracic-echo-Doppler with adenosine infusion (A-TTE) and for left ventricular wall motion abnormalities (WMA) by dobutamine stress echocardiography (DSE). Myocardial multi-detector computed tomography (MDCT) enabled the presence of epicardial stenosis, which could interfere with the accuracy of the tests, to be excluded. Patient survival rate was assessed over a 6.7- ± 3.5-year follow-up.

Results

Nineteen out of 41 (46%) SSc patients had a reduced CFR (≤2.5) and in 16/41 (39%) a WMA was observed during DSE. Furthermore, 13/41 (32%) patients showed pathological CFR and WMA. An inverse correlation between wall motion score index (WMSI) during DSE and CFR value (r = -0.57, P <0.0001) was observed; in addition, CFR was significantly reduced (2.21 ± 0.38) in patients with WMA as compared to those without (2.94 ± 0.60) (P <0.0001). In 12 patients with abnormal DSE, MDCT was used to exclude macrovasculopathy. During a 6.7- ± 3.5-year follow-up seven patients with abnormal coronary functional tests died of disease-related causes, compared to only one patient with normal tests.

Conclusions

A-TTE and DSE tests are useful tools to detect non-invasively pre-clinical microcirculation abnormalities in SSc patients; moreover, abnormal CFR and WMA might be related to a worse disease outcome suggesting a prognostic value of these tests, similar to other myocardial diseases.     

Anti-cyclic citrullinated peptide antibody titer predicts time to rheumatoid arthritis onset in patients with undifferentiated arthritis: results from a 2-year prospective study

Introduction

The diagnostic, predictive and prognostic role of anti-cyclic citrullinated peptide (CCP) antibodies in rheumatoid arthritis (RA) patients is widely accepted. Moreover, detection of these antibodies in subjects presenting with undifferentiated arthritis (UA) is associated with a significant risk to develop the disease. On the other hand, clinical and prognostic significance of evaluating anti-CCP levels in subjects with inflammatory arthritis at disease onset has not been fully clarified. The goal of this prospective study is to analyze the value and prognostic significance of anti-CCP titer quantification in UA subjects.

Methods

Serial anti-CCP assays were measured in 192 consecutive patients presenting with UA lasting less than 12 weeks. Clinical and serological data and arthritis outcome were evaluated every 6 months until two years of follow-up.

Results

Anti-CCP positivity, at both low and high titer, and arthritis of hand joints significantly predicted RA at two years, risk increasing in subjects with high anti-CCP titers at baseline. Moreover, time to RA diagnosis was shorter in patients with high anti-CCP2 titers at enrollment with respect to those with low antibody concentration.

Conclusions

Presence of anti-CCP antibodies, at both low and high concentration, is significantly associated with RA development in subjects with recent onset UA. However, time interval from the onset of the first symptoms to the fulfilment of the classification criteria appears to be directly related to the initial anti-CCP level.     

Synthesis of Novel 5′-Uridine-Head Amphiphiles as Model for DNA Molecular Recognition

Here we describe uridine functionalization in the 5′ position, which provides new classes of cationic and nonionic amphiphiles specifically designed as DNA transfection agents. The synthetic procedures developed to obtain the cationic uridine-head surfactants prevented intramolecular cyclization that occurs when uridine is functionalized in this position without using protecting groups in the uracil.     

Ghrelin induces apoptosis in colon adenocarcinoma cells via proteasome inhibition and autophagy induction

Ghrelin is a metabolism-regulating hormone recently investigated for its role in cancer survival and progression. Controversially, ghrelin may act as either anti-apoptotic or pro-apoptotic factor in different cancer cells, suggesting that the effects are cell type dependent. Limited data are currently available on the effects exerted by ghrelin on intracellular proteolytic pathways in cancer. Both the lysosomal and the proteasomal systems are fundamental in cellular proliferation and apoptosis regulation. With the aim of exploring if the proteasome and autophagy may be possible targets of ghrelin in cancer, we exposed human colorectal adenocarcinoma cells to ghrelin. Preliminary in vitro fluorimetric assays evidenced for the first time a direct inhibition of 20S proteasomes by ghrelin, particularly evident for the trypsin-like activity. Moreover, 1 μM ghrelin induced apoptosis in colorectal adenocarcinoma cells by inhibiting the ubiquitin–proteasome system and by activating autophagy, with p53 having an “interactive” role.     

Grazing history influences biodiversity: a case study on ground-dwelling arachnids (Arachnida: Araneae, Opiliones) in the Natural Park of Alpi Marittime (NW Italy)

Alpine pastures are typical examples of “high nature value farmlands”, representing important habitats harbouring unique biological communities. Disturbance induced by overgrazing influences significantly ecosystem processes, in which invertebrates play a major role. To develop new models of sustainable management of pastures, more knowledge is needed of the animal communities, essential to the ecological functioning of pasture ecosystems. The apparent poor ecological state of several pastures in the Natural Regional Park of Alpi Marittime (NW-Italy) lead us to evaluate the influence of grazing history on biodiversity, using spider and harvestmen assemblages as key groups. Four different pastoral types characterized by four different grazing histories were identified using the Daget-Poissonet method. Spiders and harvestmen were collected by means of pitfall traps. Arachnid assemblages were characterized in terms of composition, abundance, species richness, richness of endemic species and taxonomic relatedness. Generalized linear mixed models (GLMM) were used to test differences among assemblages occurring in each pastoral type. Furthermore, we tested differences in terms of plant communities (species richness and percentage of zoogenic species). Specificity and fidelity of every spider and harvestmen species within pastoral types were explored by the IndVal (Indicator Value) procedure. Fifty-eight species of spiders and seven species of harvestmen were collected (2,304 individuals). Pastoral types related to intensive grazing were characterized by the dominance of diurnal wanderer spiders (namely Lycosidae) while, conversely, nocturnal wanderers (mainly Gnaphosidae) were more abundant in extensive pastoral types. Results show that both species richness and spider abundance were higher in abandoned areas of extensive grazing, while endemic assemblages were richer in extensive grazed areas, which also hosted the most diverse plant community. Furthermore, most of the indicator species of spiders of this type were endemic, characterized by more demanding ecological requirements.     

Development and set-up of a portable device to monitor airway exhalation and deposition of particulate matter

The aim of this study was to assess and monitor airway exhalation and deposition of particulate matter (PM). After standardizing inspiratory/expiratory flow and volumes, a novel device was tested on a group of 20 volunteers and in a field study on workers exposed to cristobalite. Both male and female subjects showed a higher percentage of deposition in the 0.5?μm channel than in the 0.3?μm channel on a laser particle counter, but it was higher in the males because of their higher exhaled lung volumes. The device was tested on a wider range of particles (0.3–0.5–1.0–2.5?μm) in the cristobalite productive division. The device has low intrasubject variability and good reproducibility, with geometric mean of %CV?相似文献