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.     

Transition Metal Complexes of 5-Amino-1-β-D-Ribofuranosylimidazole-4-Carboxylic Acid 5′-Phosphate,an Intermediate in the de novo Biosynthesis of Purine Nucleotides: Synthesis and Effects on Enzyme Activity

Abstract

Transition metal complexes [Cu(II), Co(I1) and Ni(II)] of 5-amino-l-β-D-ribofuranosylimidazole-4-carboxylic acid have been prepared and shown to form a series of stoichiometry ML₂, nM(OH₂) (n = 0,1,2) and structures have been assigned. Analogous complexes of 5-amino-l-β-D-ribofuranosylimidazole-4-carboxy1ic acid 5′-phosphate (CAIR), a central intermediate in the de novo pathway to purine nucleotides, produced in aqueous solution have been found to affect the activity of the enzyme AIR- carboxylase (E.C. 4.1.1.21).     

Configuration and Conformation Studies of Nucleoside Analogues Based on the Benzo[c]furan Core

Abstract

The glycone 1,3-dihydro-1-hydroxy-3-hydroxymethylbenzo[c]furan (1, R =H, B =OH) has been coupled to the regular nucleoside bases to a series of novel nucleoside analogues (1, B = thymine, adenine). Both cis and trans forms of these compounds have been obtained and the configuration is unequivocally established by NMR. The assignment of stereochemistry for each isomer of the compounds was initially based on the magnitude of the coupling between the dihydrohran ring protons. The NMR spectra of the 1,3-dihydrobenzo[c]fran system have been investigated for several compounds with one or no substituent in the dihydrohran ring. The observed coupling between H-1 and H-3 in a cis arrangement is in the range 0–2 Hz and the corresponding trans coupling is in the range 2.0–3.4 Hz. The data in Table 1 indicate that there are several spectral features which taken together strongly support the assignment of a common configuration to the compounds with a measurable cross-ring coupling. Further support is found in the NOESY spectrum of the mixed isomers of 1 (R = Bn, B = T). This spectrum showed a strong contact between the thymine proton, H-6, and H-3′ in the trans isomer (protons on the same side of the fixan ring) but no analogous contact in the cis isomer (protons on the opposite side of the furan ring).     

Novel Synthesis of a 2, 5'-O-Cycloimidazole Nucleoside

Abstract

A novel conversion of ethyl 5-amino-1 -(2, 3, 0-isopropylidene-β-D-ribofuranosyl)imidazole-4-carboxylate (2) to the corresponding 2, 5'-cyclo derivative (4) occurs with alkaline hypobromite or N-chlorosuccinimide and alkali.     

The Australian giant fleas Macropsylla Rothschild, 1905 (Siphonaptera: Macropsyllidae: Macropsyllinae), their identification,evolution, ecology,and conservation biology

Systematic Parasitology - Macropsylla Rothschild, 1905 is an endemic Australian flea genus represented by two species: M. hercules Rothschild, 1905 and M. novaehollandiae Hastriter, 2002. However,...     

Expression of Betapapillomavirus Oncogenes Increases the Number of Keratinocytes with Stem Cell-Like Properties

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44^high EpCAM^high cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.     

TMEM106B p.T185S regulates TMEM106B protein levels: implications for frontotemporal dementia

Frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in individuals under age 65. In many patients, the predominant pathology includes neuronal cytoplasmic or intranuclear inclusions of ubiquitinated TAR DNA binding protein 43 (FTLD‐TDP). Recently, a genome‐wide association study identified the first FTLD‐TDP genetic risk factor, in which variants in and around the TMEM106B gene (top SNP rs1990622) were significantly associated with FTLD‐TDP risk. Intriguingly, the most significant association was in FTLD‐TDP patients carrying progranulin (GRN) mutations. Here, we investigated to what extent the coding variant, rs3173615 (p.T185S) in linkage disequilibrium with rs1990622, affects progranulin protein (PGRN) biology and transmembrane protein 106 B (TMEM106B) regulation. First, we confirmed the association of TMEM106B variants with FTLD‐TDP in a new cohort of GRN mutation carriers. We next generated and characterized a TMEM106B‐specific antibody for investigation of this protein. Enzyme‐linked immunoassay analysis of progranulin protein levels showed similar effects upon T185 and S185 TMEM106B over‐expression. However, over‐expression of T185 consistently led to higher TMEM106B protein levels than S185. Cycloheximide treatment experiments revealed that S185 degrades faster than T185 TMEM106B, potentially due to differences in N‐glycosylation at residue N183. Together, our results provide a potential mechanism by which TMEM106B variants lead to differences in FTLD‐TDP risk.

     

RCAN1 regulates vesicle recycling and quantal release kinetics via effects on calcineurin activity

We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over‐expressing RCAN1 (RCAN1^ox), but not in wild‐type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK‐506, replicates this effect in WT cells but has no additional effect in RCAN1^ox cells. When we chronically expose WT cells to cyclosporine A and FK‐506 we find that catecholamine release per vesicle and pre‐spike foot (PSF) signal parameters are decreased, similar to that in RCAN1^ox cells. Inhibiting calcineurin activity in RCAN1^ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1^ox cells, the smaller vesicle and dense core size we observe in RCAN1^ox cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.     

Linking phospholipase C isoforms with differentiation function in human vascular smooth muscle cells

The phosphoinositol-phospholipase C (PLC) family of enzymes consists of a number of isoforms, each of which has different cellular functions. PLCγ₁ is primarily linked to tyrosine kinase transduction pathways, whereas PLCδ₁ has been associated with a number of regulatory proteins, including those controlling the cell cycle. Recent studies have shown a central role of PLC in cell organisation and in regulating a wide array of cellular responses. It is of importance to define the precise role of each isoform, and how this changes the functional outcome of the cell. Here we investigated differences in PLC isoform levels and activity in relation to differentiation of human and rat vascular smooth muscle cells. Using Western blotting and PLC activity assay, we show that PLCδ₁ and PLCγ₁ are the predominant isoforms in randomly cycling human vascular smooth muscle cells (HVSMCs). Growth arrest of HVSMCs for seven days of serum deprivation was consistently associated with increases in PLCδ₁ and SM α-actin, whereas there were no changes in PLCγ₁ immuno-reactivity. Organ culture of rat mesenteric arteries in serum free media (SFM), a model of de-differentiation, led to a loss of contractility as well as a loss of contractile proteins (SM α-actin and calponin) and PLCδ₁, and no change in PLCγ₁ immuno-reactivity. Taken together, these data indicate that PLCδ₁ is the predominant PLC isoform in vascular smooth muscle, and confirm that PLCδ₁ expression is affected by conditions that affect the cell cycle, differentiation status and contractile function.