Frog (Pelophylax bergeri, Günther 1986) endocrine disruption assessment: characterization and role of skin poly(ADP-ribose) polymerases.

Model of the our research was the adult male amphibian anura, Pelophylax bergeri, poikilotherm species not considered threatened by the IUCN, sampled in representative sites at different degree. In the first phase, a biochemical characterization of the ADP-ribosylation on the skin of barcoded amphibian anura collected from Matese Lake (clean reference site in CE, Italy) was carried out. Two PARP isoforms were evidence: the first of 66 kDa is localized into nucleus and activated by DNA damage; the second of 150 kDa is in cytoplasm, as demonstrated by biochemical and immunohistochemical analysis. Subsequently, the PARP activity, the quantitative expression of androgen receptor gene, and the levels of arsenic and chromium in skin and testis of frog and soil, water, and sediment collected from sites at different degrees of pollution were measured. A significant variation of PARP activity and androgen receptor expression levels was detected in both tissues of barcoded frogs from Sarno and Scafati, along Sarno River (SA, Italy), suggesting that a PARP activation is correlated to pollution and to steroid-regulated physiology disruption.

Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis.

BACKGROUND: To objectively measure changes in nuclear morphology and cell distribution following induction of apoptosis. METHODS: A spontaneously immortalized retinal pigment epithelial cell line (ARPE-19) was cultured for three days in DMEM/F12 with 10% fetal bovine serum followed by 24 hours incubation in staurosporine to induce apoptosis. Cells that were not incubated in staurosporine served as control. Caspase-3 expression in apoptotic cells was demonstrated by quantitative immunofluorescence. Nuclei were counterstained with DAPI. Assessments of nuclear morphology and cell distribution were performed using ImageJ software. Statistical analyses included Student's t-test and Pearson's correlation coefficient. Nearest neighbor analysis was used to assess cell nuclei distribution. RESULTS: Caspase-3 expression in staurosporine-incubated cells increased by 471% ± 182% compared to control (P=0.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68% ± 5%; P<0.001) and nuclear circumference (78 ± 3%; P<0.001), while nuclear form factor was larger (110% ± 1%; P<0.001). Cell nuclei from the staurosporine-group (R=1.12 ± 0.04; P<0.01) and the control (R=1.28 ± 0.03; P<0.01) were evenly spaced throughout the cultures, thereby demonstrating a non-clustered and non-random cell distribution. However, the staurosporine-incubated group had a significantly lower R-value compared to the control (P=0.002), which indicated a move towards cell clustering following induction of apoptosis. Caspase-3 expression of each individual cell correlated significantly with the following morphological indicators: circumference of the nucleus divided by form factor (r=-0.475; P<0.001), nuclear area divided by form factor (r=-0.470; P<0.001), nuclear circumference (r=-0.469; P<0.001), nuclear area (r=-0.445; P<0.001), nuclear form factor (r=0.410; P<0.001) and the nuclear area multiplied by form factor) (r=-0.377; P<0.001). CONCLUSIONS: Caspase-3 positive apoptotic cells demonstrate morphological features that can be objectively quantified using freely available ImageJ software. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3271993311662947.

Chromogenic in situ hybridisation (CISH) is a powerful method to detect ALK-positive non-small cell lung carcinomas.

AIM: We assessed the potential of a chromogenic in situ hybridisation (CISH) assay in comparison with quantitative reverse transcription (RT)-PCR (qPCR) to detect anaplastic lymphoma kinase (ALK) break apart-positive lung carcinomas. METHODS: Dual-colour CISH using a break apart probe for the ALK gene on 2p23 was performed with 181 formalin-fixed, paraffin-embedded tissue and agar block sections from 175 cases of non-small cell lung carcinomas (NSCLC). Stained slides were analysed with a standard bright-field microscope at 1000× magnification by counting signals from 60 non-overlapping nuclei from three different tumour areas. Samples with ≥15% of positive nuclei were judged as ALK break apart-positive. All samples were simultaneously analysed by qPCR for EML4-ALK to validate CISH results, and positive samples were subject to Sanger sequencing. RESULTS: CISH was successful with 173 of 181 hybridised samples (96%), and seven ALK break apart-positive cases were detected. CISH signals were specific and distinct for both colours. All positive cases were confirmed by qPCR and Sanger sequencing, and concordance between CISH and qPCR was 100%. Nearly all samples (9/10) which failed by qPCR were accessible to CISH analysis. CONCLUSIONS: CISH is a very reliable, convenient and inexpensive method to detect ALK-positive NSCLC. CISH success rate is comparably high as with qPCR, and it detects all ALK break apart events in a single assay. It is of special value when RNA quality is poor, or when small biopsies with a very limited amount of tumour cells have to be analysed.

Computing the length of the shortest telomere in the nucleus.

The telomere length can either be shortened or elongated by an enzyme called telomerase after each cell division. Interestingly, the shortest telomere is involved in controlling the ability of a cell to divide. Yet, its dynamics remains elusive. We present here a stochastic approach where we model this dynamics using a Markov jump process. We solve the forward Fokker-Planck equation to obtain the steady state distribution and the statistical moments of telomere lengths. We focus specifically on the shortest one and we estimate its length difference with the second shortest telomere. After extracting key parameters such as elongation and shortening dynamics from experimental data, we compute the length of telomeres in yeast and obtain as a possible prediction the minimum concentration of telomerase required to ensure a proper cell division.

Expression of soluble adenylyl cyclase in lentigo maligna: use of immunohistochemistry with anti-soluble adenylyl cyclase antibody (R21) in diagnosis of lentigo maligna and assessment of margins.

CONTEXT: Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a signaling molecule involved in regulating melanocyte functions. R21, a mouse monoclonal antibody against sAC, shows a striking pan-nuclear staining in lentigo maligna, indicating possible utility for diagnosis and margin assessment. OBJECTIVE: To evaluate R21 in the diagnosis and evaluation of margins in lentigo maligna. DESIGN: Thirty one re-excision specimens for lentigo maligna were evaluated for R21 expression using previously published protocol. In addition, 153 cases including 41 lentigo malignas, 30 non-lentigo maligna-type melanomas, 38 lentigos, and 44 nevi were evaluated using a modified stringent protocol to eliminate all nonmelanocyte staining. RESULTS: The sensitivity of nuclear staining with R21 in lentigo maligna was 87.8%. Nuclear expression of sAC was observed in 40% of other melanomas and 2.3% of benign nevi. R21 did not stain nuclei of resting melanocytes but was observed in 28.9% of melanocytic hyperplasias. These cases were easily distinguished from lentigo maligna in routine sections. R21 staining facilitated extent of the lesion in resection margins. In cases examined under the less stringent conditions, interpretation was facilitated by comparing R21 and Mart1/Melan A staining. Greater than 9 pan-nuclear staining melanocytes within one high-power field along with a pan-nuclear sAC/Melan A ratio greater than 0.5 was consistent with a positive margin whereas 5 or less pan-nuclear staining melanocytes along with a sAC/Melan A ratio of less than 0.3 constituted a negative margin. CONCLUSION: R21 is a useful diagnostic adjunct in the diagnosis and evaluation of margins in re-excision specimens in lentigo maligna.

Involvement of the integrin-linked kinase pathway in hexachlorobenzene-induced gender-specific rat hepatocarcinogenesis.

Overexpression of the integrin-linked kinase (ILK) pathway disrupts cell-cell interactions, an epigenetic event leading to epithelial cell transformation. Female rats exposed to hexachlorobenzene (HCB) for 5 consecutive days and sampled 45 days later show a decrease in liver gap junctional intercellular communication. We hypothesized that HCB also alters E-cadherin expression and that this alteration is mediated by the ILK pathway. Hepatic ILK levels were markedly increased in HCB-treated female rats. Cytoplasmic/membrane levels of protein kinase B (Akt), a target of ILK, and its phosphorylated active form were decreased in treated female rats. Flow cytometric analysis showed a concomitant increase in nuclear Akt levels. Both ILK and Akt can phosphorylate glycogen synthetase kinase-3beta (GSK3beta), rendering it inactive. Phosphorylated-GSK3beta levels were higher in treated females and resulted in a twofold decrease in the activity of GSK3beta. The inactivation of GSK3beta in HCB-treated female rats resulted in the nuclear translocation of beta-catenin, as demonstrated by both immunocytochemistry and flow cytometric analyses. Western blot analysis showed an 84% decrease in E-cadherin levels in HCB-treated rats as compared to controls, and this decrease was not mediated by Snail activation. Mimicking the activation of ILK with specific GSK3beta inhibitors resulted in downregulation of E-cadherin levels but had no effect on Cx32 expression in the MH(1)C(1) cells. Overall, these results indicate that hepatic E-cadherin is downregulated as a result of an overexpression of the ILK pathway. The concomitant HCB-induced downregulation of intercellular communication does not occur as a result of either E-cadherin downregulation or GSK3beta inactivation.

Identifying chimerism in proteins using hidden Markov models of codon usage.

Protein chimerism is a phenomenon involving the combination of multiple ancestral sequences into a single, multi-domain protein through evolution. We propose a novel method for detecting chimeric proteins by analyzing their nucleotide sequence. The method tests for differences in the distributions of synonymous (isoaccepting) codons in different regions of the protein. The test involves the comparison of the ability of varying size hidden Markov models (HMMs) of codon usage to fit the natural sequence, relative to a set of randomized controls. We demonstrate the method on the families of yeast nuclear and mitochondrial amino-acyl tRNA synthetases. The method is potentially useful for the automated screening of entire genomes or large databases.

Assessment of the interaction between calmodulin and casein kinase II.

Calmodulin is an in vitro substrate for casein kinase II and is also reported to inhibit casein kinase II activity in rat liver nuclear extracts. Here we demonstrate that in the presence or absence of Ca2+, calmodulin did not significantly alter either in vitro casein kinase II activity or autophosphorylation of its beta-subunit. In contrast, Ca2+ inhibited in a dose-dependent manner both casein kinase II activity and casein kinase II-catalysed calmodulin phosphorylation. These data indicate that calmodulin does not directly inhibit casein kinase II activity, but casein kinase II is sensitive to physiologic Ca2+ concentrations.