Effect of alkylation on the secondary structure of DNA

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.     

Multidisciplinary approach to diagnosis and management of osteosarcoma – a review of the St Vincent's Hospital experience

Background

Osteosarcoma is the most common primary malignant bone tumour in children and young adults. Despite advances in the diagnosis and management of osteosarcoma, there have been few recent studies describing the experiences of tertiary referral centres. This paper aims to describe and discuss the clinical features, pre-operative work-up, management and outcomes of these patients at St Vincent's Hospital (Melbourne, Australia).

Methods

Retrospective study of fifty-nine consecutive patients managed for osteosarcoma at St Vincent's Hospital between 1995 and 2005.

Results

Median age at diagnosis was 21 (range, 11–84) years. Gender distribution was similar, with thirty-one male and twenty-eight female patients.Twenty-five patients had osteosarcoma in the femur, eleven each were located in the humerus and tibia, six were identified in the pelvis, and one each in the clavicle, maxilla, fibula, sacrum, ulna and radius.Pre-operative tissue diagnosis of osteosarcoma was obtained through computed tomography-guided percutaneous biopsy in over ninety percent of patients.Following initial therapy, over fifty percent of patients remained relapse-free during the follow-up period, with twelve percent and twenty-seven percent of patients documented as having local and distant disease recurrence, respectively. Of patients with recurrent disease, sixty-two percent remained disease-free following subsequent surgical intervention (most commonly, pulmonary metastatectomy).

Conclusion

Patient outcomes can be optimised through a multidisciplinary approach in a tertiary referral centre. At St Vincent's Hospital, survival and relapse rates of patients managed for osteosarcoma compare favourably with the published literature.

     

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation,migration and malignant signalling events in hepatoma cells

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.     

Immunohistochemical localization of irisin in skin,eye, and thyroid and pineal glands of the crested porcupine (Hystrix cristata)

Irisin was first identified in muscle cells. We detected irisin immunoreactivity in various organs of the crested porcupine (Hystrix cristata). In the epidermis, irisin immunoreactivity was localized mainly in stratum basale, stratum spinosum and stratum granulosum layers; immunoreactivity was not observed in the stratum corneum. In the dermis, irisin was found in the external and internal root sheath, cortex and medulla of hair follicles, and in sebaceous glands. Irisin immunoreactivity was found in the neural retina and skeletal muscle fibers associated with the eye. The pineal and thyroid glands also exhibited irisin immunoreactivity.     

Evidence on the presence of two distinct alkaline phosphatases in Serratia marcescens

Certain strains of Serratia marcescens synthesized two different types of alkaline phosphatase (APase), constitutive (CAPase) and inducible (IAPase) APases, in low phosphate medium. Synthesis of the IAPase was repressed in the presence of high phosphate. Purification and separation of these electrophoretically distinct APases was achieved by using fractional (NH(4))(2)SO(4) precipitation, adsorption on a DEAE-cellulose column and elution of enzymes by a linear sodium chloride gradient. Starch gel electrophoresis of certain fractions revealed the separation of not only IAPase from CAPase but its separation into four distinct isozymes. CAPase gave maximum enzyme activity around pH 9.5, whereas for IAPase a broad range of enzyme activity was found between pH 8.5 and 10.5. Reversible inactivation at low pH occurred for IAPase but very little with CAPase. CAPase was more thermolabile than IAPase at 95 degrees C. The two APases were found to be distinct in their kinetic as well as immunological properties, suggesting two distinct enzyme species.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Evidence for cadherin-11 cleavage in the synovium and partial characterization of its mechanism

IntroductionEngagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments.MethodsCadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA.ResultsSoluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts.ConclusionsCadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0647-9) contains supplementary material, which is available to authorized users.