Dissociation of human alphaB-crystallin aggregates by thiocyanate is structurally and functionally reversible

Conformational modifications and changes in the aggregation state of human B-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH₄SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH₄SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of B-crystallin in 2 M NH₄SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated B-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.     

Analysis of telomeric single-strand overhang length in human endometrial cancers

The 3' single-strand telomeric overhang (3'-OH) is a key component of telomere structure. Although telomere length has been well analyzed in a variety of human cancers, no information is available on the 3'-OH length in cancers. In the present study, we examined the 3'-OH length in normal and malignant endometria using telomere-oligonucleotide ligation assay. Although 3'-OH lengths varied among patients, 3'-OH length observed in endometrial cancers was significantly shorter than that found in samples derived from normal endometria (P < 0.001: Student's t-test), suggesting that erosion of 3'-OH length induces impaired telomeric integrity and genomic instability, leading to carcinogenesis. Interestingly, we found that the most aggressive subtypes of endometrial cancers harbored significantly longer 3'-OH length than those with non-aggressive subtypes (P < 0.001: Sheffe's test), suggesting that cancer cells with long 3'-OH length have growth advantage due to their stabilized telomere ends. In contrast, we failed to observe an association between overall telomere length and any clinicopathological characteristics of endometrial cancers. These findings suggest that erosion of 3'-OH length, rather than overall telomere length, play roles in endometrial carcinogenesis. Furthermore, long 3'-OH may serve as a molecular marker for aggressive phenotype of tumors.     

A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates

We have developed a homogeneous high-capacity assay format for measuring integrin- and selectin-dependent cell binding to immobilized ligand using V-well microtiter plates. 2',7'-Bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescence, acetoxymethylester-labeled cells are added to ligand-coated V-shaped microtiter wells. Bound cells are separated from free cells using centrifugal force to produce shear stress. Nonadherent cells accumulate in the nadir of the well and are measured using a fluorescence plate reader. Antibody or low-molecular-weight inhibitors of either the ligand or the cell surface receptor result in less cell binding, more cells in the pellet, and increased signal. The optimization and validation of the very late antigen-4/vascular cell adhesion molecule-1 assay is described in detail. We demonstrate that this assay can be rapidly adapted to measure other integrin- and selectin-mediated interactions. This assay format has several advantages over conventional assays. The centrifugal process is biologically relevant and eliminates the washing steps to remove nonadherent cells that can cause well-to-well and plate-to-plate variation. Because the assay is robust with a high signal-to-noise ratio and low variability, it is ideally suited for studying multiple parameters of cell adhesion and for high capacity screening.     

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.     

Dissociation of Human αB-Crystallin Aggregates by Thiocyanate Is Structurally and Functionally Reversible

Conformational modifications and changes in the aggregation state of human αB-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH₄SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH₄SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of αB-crystallin in 2 M NH₄SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated αB-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.     

Genome‐scale metabolic reconstruction and constraint‐based modelling of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The Antarctic strain Pseudoalteromonas haloplanktis TAC125 is one of the model organisms of cold‐adapted bacteria and is currently exploited as a new alternative expression host for numerous biotechnological applications. Here, we investigated several metabolic features of this strain through in silico modelling and functional integration of –omics data. A genome‐scale metabolic model of P. haloplanktis TAC125 was reconstructed, encompassing information on 721 genes, 1133 metabolites and 1322 reactions. The predictive potential of this model was validated against a set of experimentally determined growth rates and a large dataset of growth phenotypic data. Furthermore, evidence synthesis from proteomics, phenomics, physiology and metabolic modelling data revealed possible drawbacks of cold‐dependent changes in gene expression on the overall metabolic network of P. haloplanktis TAC125. These included, for example, variations in its central metabolism, amino acid degradation and fatty acid biosynthesis. The genome‐scale metabolic model described here is the first one reconstructed so far for an Antarctic microbial strain. It allowed a system‐level investigation of variations in cellular metabolic fluxes following a temperature downshift. It represents a valuable platform for further investigations on P. haloplanktis TAC125 cellular functional states and for the design of more focused strategies for its possible biotechnological exploitation.     

Survival of Patients with Hepatocellular Carcinoma (HCC) Treated by Percutaneous Radio-Frequency Ablation (RFA) Is Affected by Complete Radiological Response

Background

Radio-frequency ablation (RFA) has been employed in the treatment of Barcelona Clinic Liver Cancer (BCLC) early stage hepatocellular carcinoma (HCC) as curative treatments.

Aim

To assess the effectiveness and the safety of RFA in patients with early HCC and compensated cirrhosis.

Methods

A cohort of 151 consecutive patients with early stage HCC (122 Child-Pugh class A and 29 class B patients) treated with RFA were enrolled. Clinical, laboratory and radiological follow-up data were collected from the time of first RFA.A single lesion was observed in 113/151 (74.8%), two lesions in 32/151 (21.2%), and three lesions in 6/151 (4%) of patients.

Results

The overall survival rates were 94%, 80%, 64%, 49%, and 41% at 12, 24, 36, 48 and 60 months, respectively. Complete response (CR) at 1 month (p<0.0001) and serum albumin levels (p = 0.0004) were the only variables indipendently linked to survival by multivariate Cox model. By multivariate analysis, tumor size (p = 0.01) is the only variable associated with an increased likehood of CR.The proportion of major complications after treatment was 4%.

Conclusions

RFA is safe and effective for managing HCC with cirrhosis, especially for patients with HCC ≤3 cm and higher baseline albumin levels. Complete response after RFA significantly increases survival.     

Molecular characterization of novel melanoma cell lines

We isolated two novel cell lines from different types of sporadic human malignant melanoma: the hmel1 line was obtained from a melanoma skin metastasis and the hmel9 cell line from a primary superficial spreading melanoma. The karyotype and pigmentation parameters were assessed in these cell lines. Cytogenetic analysis in early stages of culture revealed that both cell lines had chromosome instability and simultaneous growth of heteroploid subpopulations. The molecular analysis of some genes involved in melanoma showed that both cell lines harbor BRAF mutations. The unpigmented hmel1 and the pigmented hmel9 lines were found to express the tyrosinase gene. The tyrosine hydroxylase activity was detectable only in hmel9 cells and practically absent in the hmel1 cell line. This activity was found to be correlated with the relative tyrosinase protein amount in both melanoma cell lines. The biological behaviour in the two melanoma cell lines, derived from two different types of melanoma lesions displaying distinct clinical and histopathological features, confirms the heterogeneous characteristics of sporadic melanoma. Similarities and/or differences between cell lines extracted from different melanoma cases could be useful in the future for diagnostic, prognostic and therapeutic purposes.     

Fish chorus and vessel noise in a marine protected coastal reef vary with lunar phase

Environmental Biology of Fishes - This study implemented Passive Acoustic Monitoring (PAM) to evaluate temporal acoustic patterns in a protected coastal reef area in Tamandaré beach...