Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate

The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.     

Effect of granulocyte-macrophage colony-stimulating factor on chemiluminescence of human neutrophils

We investigated the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance the function of neutrophils. Neutrophil function was measured in terms of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced luminol-dependent chemiluminescence (LDCL). LDCL of fMLP-stimulated neutrophils was enhanced up to 4.5 fold following preincubation with rhGM-CSF. This enhancement depended on the length of preincubation, reaching an optimal level at 120 min. The dose-response relationship for fMLP-induced LDCL of neutrophils preincubated with rhGM-CSF revealed that half-maximum enhancement was achieved at an approximately 20-fold higher concentration than that of colony-forming units in culture-derived colony formation. These results suggest that differences in dose dependency may be explained by differences in the distribution of receptor(s) for GM-CSF. This may also enable GM-CSF to affect the hematopoietic system, which contains cells at various levels of differentiation, thus mediating the host-defense mechanism.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 °C for 15 min in borate buffer (20 mmol l^?1, pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 °C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l^?1, pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7–4.6% and 0.4–5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6–125 nmol (mg creatinine)^?1 (mean ± SD: 21.6 ± 26.6 nmol (mg creatinine)^?1, n = 20).     

Intracellular cAMP controls a physical association of V-1 with CapZ in cultured mammalian endocrine cells

V-1, an ankyrin repeat protein with the activity to control tyrosine hydroxylase (TH) gene expression and transmitter release in PC12D cells, associates with CapZ, an actin capping protein, and thereby regulates actin polymerization in vitro. In this study, immunoprecipitation and Western blot analysis showed that V-1 was physically associated with CapZ-beta in PC12D transfectants overexpressing V-1. These proteins were co-localized in the soma of Purkinje cells of rat cerebellum as assayed by immunohistochemistry. Furthermore, in the V-1 transfectants, the amount of CapZ which physically associated with V-1 was steeply reduced at 2h after treatment with forskolin, but was thereafter increased to reach its initial level at 12h after forskolin-treatment. These results suggest that the association of V-1 with CapZ is controlled by a cAMP-dependent signalling pathway probably to play a functional role in the regulatory mechanism of actin dynamics in the endocrine system and the central nervous system.     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.