Isolation, tissue distribution, and chromosomal localization of a novel testis-specific human four-transmembrane gene related to CD20 and FcepsilonRI-beta.

CD20 and the beta subunit of the high affinity receptor for IgE (FcepsilonRIbeta) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcepsilonRIbeta. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcepsilonRIbeta respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcepsilonRIbeta have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcepsilonRIbeta family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex.

Identification of active-site residues of the pro-metastatic endoglycosidase heparanase.

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).

DNA adducts of 9-anilinoacridine mustards: characterization by NMR.

Two types of 9-anilinoacridine-linked mustards, containing aniline mustard side chains linked either at the 4-position of the intercalating acridine chromophore (type A) or at the 1'-position of the 9-anilino group (type B), were reacted with calf thymus DNA. Adducts were isolated by preparative TLC, and their structures were determined by a variety of one-dimensional and two-dimensional NMR experiments. The only isolated product from the reaction of the 4-linked mustard (type A) was a guanine N7 adduct (55% yield), arising from alkylation in the major groove. In contrast, the major product (57% yield) from reaction of the 1'-linked mustard (type B) was an adenine N3 adduct from alkylation in the minor groove, with a smaller proportion (20%) of guanine N7 adduct. Computer modeling studies of drug-DNA alkylation complexes resulted in minimum-energy structures and averaged molecular dynamics structures that agreed with the adduct studies. The models suggest the aniline ring of the carrier is located in the DNA minor groove, with the acridine ring intercalated between two base pairs with the 4-position pointing into the major groove.

An experimental and mathematical model for the extravascular transport of a DNA intercalator in tumours.

A new in vitro model has been developed for investigating extravascular diffusion of therapeutic agents in tumour tissue. V79-171b or EMT6/Ak cells are grown on porous Teflon support membranes and submerged in a large reservoir of medium, to give diffusion-limited 'multicellular membranes' (MMs) c. 200 microm in thickness. MMs are histologically similar to multicellular spheroids, but their planar rather than spherical geometry facilitates direct measurement of the flux of radiolabelled agents through the multicellular structure. For [14C]urea, flux kinetics through V79-171b MMs was modelled as simple diffusion, yielding a diffusion coefficient in the MM (DMM) of 1.45 x 10(-6) cm2 s(-1), 11-fold lower than in culture medium. Flux of the 3H-labelled DNA intercalator 9-[3-(N,N-dimethylamino)propylamino]acridine (DAPA) was dramatically slower than urea. Modelling this over the first 5 h gave a DMM of 1.3 x 10(-8) cm2 s(-1), but over longer times the kinetics was not consistent with simple diffusion. Flux of DAPA was markedly increased in the presence of 50 mM ammonium chloride, indicating that sequestration in acidic endosomes is a major impediment to flux. Accumulation in cytoplasmic vesicles was confirmed by fluorescence microscopy. The DAPA flux kinetics, with and without ammonium chloride, was well fitted by a reaction-diffusion model with reversible cellular uptake (modelled as binding), using uptake parameters determined in separate experiments with V79-171b single-cell suspensions. This study demonstrates the utility of the MM model for determining extravascular transport parameters, and indicates that much of the impediment to diffusion of basic DNA intercalators in tumour tissue may arise from lysosomal sequestration rather than DNA binding.