Immunocytochemistry of collagenase expression in transitional cell carcinoma of the bladder

OBJECTIVE: To evaluate the usefulness of collagenase immunocytochemistry as well as its immunohistochemistry in assessing the correlation with prognostic factors in transitional cell carcinoma (TCC) of the urinary bladder. STUDY DESIGN: We investigated the expression of collagenase in catheterized urine and histologic specimens from 38 patients with TCC and 20 cases with benign lesions of the urinary tract. RESULTS: Thirteen (34.2%) and 17 (44.7%) patients with TCC showed positive expression of collagenase on cytologic and histologic specimens, respectively, whereas in no cases with benign lesions was such expression found (P < .01). Invasive and nonpapillary TCC had higher positive rates than noninvasive and papillary TCC. Grade 3 TCC was positive at a higher rate than was grade 2, whereas there were no positive cases with grade 1. Collagenase expression did not correlate significantly with stage. CONCLUSION: Collagenase expression in urinary TCC correlated well with tumor growth pattern, pathologic grade and invasiveness of the carcinoma; all are known to be prognostic factors. The application of collagenase immunostaining to urinary cytology is very useful for assessing prognosis in TCC.     

In vivo gene gun-mediated DNA delivery into rodent brain tissue

Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain.     

Chat, a Cas/HEF1-associated adaptor protein that integrates multiple signaling pathways

Cas (Crk-associated substrate) and HEF1 (human enhancer of filamentation) are related adaptor proteins that function in integrin-mediated cell adhesion and antigen receptor signaling pathways. We report here a molecular cloning of Chat (Cas/HEF1-associated signal transducer) that associates with Cas and HEF1. Chat is a 78-kDa signaling molecule with an N-terminal SH2 domain and is expressed in a wide range of tissues. In hematopoietic cells, a 115-kDa isoform of Chat (Chat-H) was specifically expressed. Chat is associated with Cas in brain, and Chat-H is associated with HEF1 in splenocytes. Deletion analyses revealed that Chat and Cas are associated with each other by their C-terminal domains. Treatment of PC12 cells with epidermal growth factor or nerve growth factor increased the phosphorylation level of Chat. This increase was suppressed by an inhibitor of mitogen-activated protein (MAP) kinase kinase, PD98059, suggesting the phosphorylation of Chat by MAP kinase. In Chat-overexpressed COS7 cells, the activity of c-Jun N-terminal kinase was up-regulated. After the epidermal growth factor stimulation, Chat and Cas were colocalized with actin filaments at ruffling membranes. These findings suggest that Chat transduces signals of tyrosine kinases and MAP kinase to Cas signaling pathway.     

Phenolic constituents from Grevillea robusta

Seven phenolic compounds were isolated from a MeOH extract of the leaves of Grevillea robusta. Their structures were determined by various spectral methods including 2D NMR spectroscopy.     

L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis

L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.     

Dynamic function of the spacer region of acetogenins in the inhibition of bovine mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Studies of the action mechanism of acetogenins, the most potent and structurally unique inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), are valuable in characterizing the inhibitor binding site in this enzyme. Our previous study deepened our understanding of the dynamic function of the spacer region of bis-THF acetogenins [Abe, M., et al. (2005) Biochemistry 44, 14898-14906] but, at the same time, posed new important questions. First, while the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings) span a distance shorter than that of the extended 13 carbon atoms [-(CH 2) 13-], what is the apparent optimal length of the spacer for the inhibition of 13 carbon atoms? In other words, what is the functional role of the additional methylene groups? Second, why was the inhibitory potency of the mono-THF derivative, but not the bis-THF derivative, drastically reduced by hardening the spacer covering 10 carbon atoms into a rodlike shape [-CH 2-(C identical withC) 4-CH 2-]? This study was designed not only to answer these questions but also to further disclose the dynamic functions of the spacer. We here synthesized systematically designed acetogenins, including mono- and bis-THF derivatives, and evaluated their inhibitory effects on bovine complex I. With regard to the first question, we demonstrated that the additional methylenes enhance the hydrophobicity of the spacer region, which may be thermodynamically advantageous for bringing the polar gamma-lactone ring into the membrane-embedded segment of complex I. With regard to the second question, we observed that a decrease in the flexibility of the spacer region is more adverse to the action of the mono-THF series than that of the bis-THF series. As a cause of this difference, we suggest that for bis-THF derivatives, one of the two THF rings, being adjacent to the spacer, is capable of working as a pseudospacer to overcome the remarkable decrease in the conformational freedom and/or the length of the spacer. Moreover, using photoresponsive acetogenins that undergo drastic and reversible conformational changes with alternating UV-vis irradiation, we provided further evidence that the spacer region is free from steric congestion arising from the putative binding site probably because there is no receptor wall for the spacer region.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.