The surface charge of NKLy ascitic tumor cells differing in their proliferative activity, in their place in the cell cycle and in their ploidy

The NKLy ascitic tumor cells in the stationary phase of growth were fractionated by velocity sedimentation method. Cells from the obtained fractions were characterized by measurements of DNA contents and 3H-thymidine incorporation. The surface properties of the cells from five fractions, differing in proliferative capacity, stage of the cell cycle and ploidy were considered using cell electrophoresis, two polymer aqueous phase system and Alcian blue sorption. A correlation between electrophoretic mobility and cell partition constant for these fractions has been obtained. No correlation was found between these parameters and dye absorption. The surface charge of the cells from G0/G1 and S fractions was higher than that of other cells. The polyploid NKLy cells demonstrated a lower surface charge. The surface properties of the tumor cells differing in proliferative capacity, stage of the cell cycle and ploidy are discussed.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Comparative study of the effect of nonionic detergents as inhibitors of respiration of tumor ascites cells]

The action of derivatives of polyoxyethylene oxide (triton X-100, triton X-305, tween 21, tween 40, tween 60, and tween 80) on respiration of Ehrlich's tumor and rats ovarium tumor cells involved inhibition of the cells respiration due to alteration of commonly uncorrelated with the distribution of detergents between the cell and the extracellular milieu.     

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye)

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Vitamin D in a Northern Canadian First Nation Population: Dietary Intake,Serum Concentrations and Functional Gene Polymorphisms

The wide spectrum of vitamin D activity has focused attention on its potential role in the elevated burden of disease in a northern Canadian First Nations (Dené) cohort. Vitamin D insufficiency, and gene polymorphisms in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) have been implicated in susceptibility to infectious and chronic diseases. The objectives of this study were to determine the contribution of vitamin D from food, and measure the serum concentrations of 25-hydroxyvitamin D₃ (25-OHD₃) and VDBP in Dené participants. Single nucleotide polymorphisms (SNPs) associated with the dysregulation of the innate immune response were typed and counted. Potential correlations between the SNPs and serum concentrations of 25-OHD₃ and VDBP were evaluated. Venous blood was collected in summer and winter over a one-year period and analyzed for 25-OHD₃ and VDBP concentrations (N = 46). A questionnaire was administered to determine the amount of dietary vitamin D consumed. Sixty-one percent and 30% of the participants had 25-OHD₃ serum concentrations <75 nmol/L in the winter and summer respectively. Mean vitamin D binding protein concentrations were within the normal range in the winter but below normal in the summer. VDBP and VDR gene polymorphisms affect the bioavailability and regulation of 25-OHD₃. The Dené had a high frequency of the VDBP D432E-G allele (71%) and the Gc1 genotype (90%), associated with high concentrations of VDBP and a high binding affinity to 25-OHD₃. The Dené had a high frequency of VDR Fok1-f allele (82%), which has been associated with a down-regulated Th1 immune response. VDBP and VDR polymorphisms, and low winter 25-OHD₃ serum concentrations may be risk factors for infectious diseases and chronic conditions related to the dysregulation of the vitamin D pathway.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.