contributed equally to this workWe have cloned and characterized a new member of the voltage-dependent Ca 2+ channel g subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential g subunits identi®ed by their homology to the stargazin gene, CACNG7 is a ®ve-, and not four-exon gene whose mRNA encodes a protein we have designated g 7 . Expression of human g 7 has been localized speci®cally to brain. N-type current through Ca V 2.2 channels was almost abolished when co-expressed transiently with g 7 in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that g 7 has this effect by causing a large reduction in expression of Ca V 2.2 rather than by interfering with traf®ck-ing or biophysical properties of the channel. No effect of transiently expressed g 7 was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like g subunits, different gene structure and the unique functional properties of g 7 imply that it represents a distinct subdivision of the family of proteins identi®ed by their structural and sequence homology to stargazin.

Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable to bind beyond the outer region of the beads. A harsher CIP treatment of 1 M NaCl dissolved in 1 M NaOH was also ineffective in removing all the foulant protein but did remove PicoGreen-conjugated dsDNA within 15 min of incubation. Cy5.5-labelled BSA was able to bind throughout the bead interior after this more aggressive CIP treatment but at a lower capacity than in the case of fresh beads. The competitive adsorption of BacLight Red-labelled whole cells or cell debris and PicoGreen-conjugated dsDNA was also visualised using CSLM.

Potential biomarkers were identifi ed for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefi tinib in head and neck cancer. Gefi tinib sensitivity was determined in cell lines, followed by transcript profi ling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefi tinib (inhibitor concentration required to give 50% growth inhibition [GI 50 ] < 1 µM), three had intermediate sensitivity (GI 50 1-7 µM), and six were resistant (GI 50 > 7 µM); an exploratory principal component analysis revealed a separation between the genomic profi les of sensitive and resistant cell lines. Subsequently, a hypothesis-driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E-cadherin (CDH1); transforming growth factor, alpha (TGF-α); amphiregulin (AREG); FLJ22662; EGFR; p21-activated kinase 6 (PAK6); glutathione S-transferase Pi (GSTP1); and ATP-binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis-free analysis identifi ed 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF-α, CDH1, EGFR, keratin 16 [KRT16], fi broblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme-linked immunoabsorbant assay analysis confi rmed Affymetrix data, and EGFR gene mutation, amplifi cation, and genomic gain correlated strongly with gefi tinib sensitivity. We identifi ed biomarkers that predict for in vitro responsiveness to gefi tinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

SUMMARYThe ionophore monensin inhibited the formation of herpes simplex virus type 2 (HSV-2) particles by about 30~ but the yields of infectious particles were reduced to 5~ and 1 ~ for cell-associated and extracellular virus, respectively. The presence of monensin did not affect the processing of the two viral glycoproteins gB-2 and gG-2. However, two other glycoproteins, gC-2 and gD-2, were not processed to their fully mature forms in the monensin-treated cells and only the faster moving pgC-2 and pgD-2 were detected. The cell-associated virus particles contained the glycoproteins gB-2, gC-2, gD-2 and gG-2, whereas the extracellular virus particles contained only gG-2 glycoprotein. These results suggest that HSV

Human tumour xenografts have commonly been used to explore the mechanisms of tumour angiogenesis and the interaction of tumour cells with their microenvironment, as well as predict potential utility of anti-angiogenic inhibitors across different tumour types. To investigate how well human tumour xenografts can be used to differentiate the effects of stromal targeting agents we performed a comparative assessment of the murine angiogenic response across a panel of pre-clinical tumour xenografts. By analysing a panel of 22 tumour xenografts with a range of vascular morphologies, micro-vessel densities and levels of fibroblast and inflammatory infiltrate, we have examined the relationship between angiogenic stroma and human tumour models. These models were studied using a combination of immunohistochemistry and species specific mRNA profiling to differentiate the tumour and stromal transcript mRNA profiles. Principal Component Analysis (PCA) and regression analysis was used to investigate the transcriptional relationships between the individual models and the correlation with the stromal architecture. We found the human tumour cell expressed factors to be independent of the murine host responses such as microvessel density, and fibroblast or macrophage cellular infiltrate. Moreover mRNA profiling of the mouse stroma suggested that the host response to the different tumours was relatively uniform despite differences in stromal structures within the tumour. Supporting this, models with different stromal compositions responded similarly to cediranib, a small molecule inhibitor of VEGF signalling. The data indicate that although the angiogenic response to the tumour results in reproducible stromal architectures, these responses are not differentiated at the level of gene expression.