Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus

Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor in establishing the vasculature in renal cell carcinomas (RCCs). Since little is known about VEGF signalling in RCCs, the profile of phosphorylated KDR (pKDR) has been investigated and the intracellular location of the receptor has been examined in the present study. Using two monoclonal antibodies raised against the phosphorylated KDR epitopes (Y1059 and Y1214) known to mediate different VEGF functions, together with a commercial anti-KDR antibody and immunohistochemistry, the expression of pKDR was investigated in a series of normal (n = 25) and neoplastic kidneys (n = 54; clear cell n = 35; papillary n = 10; oncocytomas n = 8). pKDR was present in many tissue elements of both normal and neoplastic renal tissues, with strong expression in the cell membrane, cytoplasm, and nuclei of normal kidney and tumour cells, as well as endothelial cells in tumours of all histological types. Patterns and intensity were similar using both anti-pKDR antibodies. There was no significant correlation in clear cell carcinomas between pKDR expression and age (p = 0.57), tumour size (p = 0.2), gender (p = 0.59), grade (p = 0.2) or histological type (p = 0.36). To delineate further the intracellular processing that might account for the cellular distribution, confocal microscopy was also performed. Antibodies to the different phosphorylated epitopes demonstrated different intracellular staining patterns. This study shows that pKDR is present in a wide variety of renal tumours, suggesting that anti-VEGF therapy might have direct effects on tumour cells. It further suggests that cells traffic pKDR depending on the precise KDR tyrosines that are autophosphorylated in a manner that enables receptor activation to result in different functions.     

Protection of Chinese hamster cells against the cytotoxic and mutagenic effects of alkylating agents by transfection of the Escherichia coli alkyltransferase gene and a truncated derivative

The cytotoxic and mutagenic effects of various monofunctionaland bifunctional alkylating agents have been assessed in V79Chinese hamster cells that express either the entire O⁶-alkylguanine(O⁶AG) and alkylphosphotriester alkyltransferase (ATase) gene(clone 8 cells) or a truncated form that codes only for O⁶AGATase activity (clone SB cells). Protection ratios, as determinedby D₃₇ values, were greater for clone 8 cells than for SB cells.Significant protection against the mutagenic effects of N-methyl-N-nitrosoureaand ethylmethanesulphonate at the hypoxanthine phosphoribosyltransferase(HPRT) locus was observed in clone 8 and SB cells. Streptozotocinand the haloethyl nitrosoureas, chlorozotocin and bis-chloroethylnitrosoureawere less efficient in inducing HPRT-deficient mutants and asmaller degree of protection was afforded by the transfectedgenes. This is possibly due to the propensity of these compoundsto induce multi-locus deletions. Southern analysis of DNA fromclone 8 and SB cells indicated the presence of multiple copiesof the plasmid integrated into clone 8 cells but few copiesin clone SB cells. The copy number did not change but ATaselevels fell when cells were grown in the absence of G418. ²Present address: Corporate Biosciences Laboratory, ICI plc,The Heath, Runcorn, Cheshire, UK      

Laser induced thermal injury of rabbit cornea and treatment with anti-inflammatory agents

A moderately severe thermal injury of the central cornea of 48 Dutch-belted rabbit eyes was produced with a carbon (CO₂) laser. The lesions were photographed with a slit lamp (SL) camera immediately following the injury and at 1, 2, 4, 7, 14, 21, 30 and 60 days after the exposure. Lesion size, opaqueness, and depth were graded clinically by SL biomicroscopy at the same intervals. No significant differences were found (p 0.05) between groups of eyes treated with flurbiprofen (0.03%), prednisolone acetate (1%), and vehicle control four-times-a-day for three weeks following injury. Additionally, eyes were studied histopathologically at 3 and 60 days following injury by light and transmission electron microscopy, and clinically at 30 and 60 days by endothelial specular microscopy. Important clinical and histopathological findings included coagulative necrosis of the corneal epithelium, epithelial sloughing, fusion of stromal collagen, stromal edema and inflammatory cell infiltration, stromal scar formation, corneal thinning, endothelial hyperplasia and metaplasia, fibrinous anterior chamber reaction with hypopyon, and retrocorneal fibrous membrane formation.     

Direct membrane method for the study of interface-controlled transport of cholesterol in aqueous media

Studies were designed to demonstrate the use of a silicone rubber membrane diffusion cell in the mechanistic study of cholesterol mass transfer in aqueous media. The method is shown to be simple, precise, and well suited for delineating conditions which facilitate cholesterol transport. Traditional membrane diffusion resistance was determined with cholesterol solubilized in the nonionic surfactant, polyoxyethylene(10)-nonylphenol ether. The use of a charged surfactant additive, either sodium oleate or benzyldimethyltetradecylammonium chloride, reduced cholesterol membrane flux in a manner consistent with a transport barrier residing in the membrane and micelle interfacial regions. Quantitative determination of total transport resistance was good (CV of greater than 95%) for cases more than 99% interface controlled. Interfacial resistance imparted by the charged surfactant additive was essentially abolished by strong electrolyte (sodium chloride). Electrolyte was utilized in either the upstream or the downstream aqueous compartment to enhance cholesterol transport by a mechanism which is consistent with a marked increase in the frequency of micelle collision with the corresponding membrane surface. When the downstream interfacial component of total transport resistance was "short circuited" by electrolyte in sequential transport runs using the same membrane, a "dumping" of cholesterol by the membrane compartment was observed. Limited studies with a second nonionic surfactant, polyoxyethylene(15)-tridecyl ether, suggest that the structure of separate micelle components may also be related to cholesterol mass transfer which occurs via a micelle collision in the interfacial region.     

Standardized mean regional method for calculating global positron emission tomographic measurements

A new "mean regional" method for calculating global hemispheric values of blood flow, blood volume, and metabolism with positron emission tomography is presented. It is based on a standardized set of regions defined according to coordinates in a stereotactic atlas of the brain. Region locations in each individual scan were determined by a localization technique that is independent of the appearance of the physiological images. Measurements obtained with this mean regional method minimize contributions from nonbrain structures such as ventricles or venous sinuses and provide the necessary basis for comparisons among different subjects and laboratories.     

Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.

Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane.     

CT metrizamide myelography in syringomyelia: sensitivity and specificity

In a retrospective study of 32 patients with "proven" syringomyelia and 15 patients with an alternate proven diagnosis, a change in the caliber of the spinal cord with different positions ("collapsing cord sign or cord collapse") had a sensitivity of 38% and a specificity of 87%. Central cord enhancement ("bull's-eye") on delayed CT had a sensitivity and specificity of 91% and 87%, respectively. The positive predictive value of cord collapse was 87%, while the positive predictive value of central cord enhancement was 94%.