Comprehensive DNA microarray expression profiles of tumors in tenascin-C-knockout mice

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays a pivotal role in tumor growth. However, the mechanism whereby TNC affects tumor biology remains unclear. To investigate the exact role of TNC in primary tumor growth, a mouse mammary tumor cell line, GLMT1, was first developed. Subsequently, global gene expression in GLMT1-derived tumors was compared between wild-type (WT) and TNC-knockout (TNKO) mice. Tumors in WT mice were significantly larger than those in TNKO mice. DNA microarray analysis revealed 447 up and 667 downregulated in the tumors inoculated into TNKO mice as compared to tumors in WT mice. Validation by quantitative gene expression analysis showed that Tnc, Cxcl1, Cxcl2, and Cxcr2 were significantly upregulated in WT mice. We hypothesize that TNC stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation.     

Effects of base damages on DNA replication--mechanism of preferential purine nucleotide insertion opposite abasic site in template DNA.

DNA polymerase preferentially inserts purine nucleotides opposite non-instructive lesions such as abasic sites during DNA replication. In order to elucidate the mechanism of the preferential insertion, a DNA template containing a model abasic site and primers containing 4 different nucleotides (A,G,C,T) at primer terminus were synthesized. The stability of the primer terminus nucleotide placed opposite the abasic site was evaluated on the basis of its sensitivity to 3'-5' exonuclease associated with DNA polymerase.     

Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Scale-dependent effects of windthrow disturbance on forest arthropod communities

The effect of disturbance on local communities may operate within the context of the spatial landscape. We examined the scale-dependent effects of windthrow disturbance caused by a large typhoon on three arthropod communities in a temperate forest of Japan. Canopy arthropods were collected by beating foliage, forest-floor arthropods were collected by sweeping the vegetation, and flying arthropods were collected in Malaise traps. To assess the “functional spatial scale” at which arthropods responded to tree-fall disturbance, the gap rate was quantified at different spatial scales by sequentially enlarging the radius of a circular landscape sector in 10-m increments from 10 to 500 m. We then analyzed the responses of order richness and abundance to the gap rate for each arthropod community. The spatial scale of the significant best-fitting model, which was selected from the models fitted to the gap rate at stepwise spatial scales, was regarded as the arthropod-specific functional spatial scale. Arthropod order richness was not dependent on the gap rate. In contrast, arthropod order abundance depended significantly on the gap rate in many orders, but varied in the response direction and functional spatial scale. These order-specific, scale-dependent responses to tree-fall gaps could complicate interactions among organisms, leading to complex community organization. An understanding of the spatial processes that link the use of space by organisms with the spatial scale at which ecological processes are experienced is required to elucidate the responses of populations, communities, and biotic interactions to disturbances in a spatial landscape context.     

DCCD-sensitive Na+-transport in the membrane vesicles of Halobacterium halobium

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

The fate of prorenin during granulopoiesis in epithelioid cells. Immunocytochemical experiments with antisera against renin and different portions of the renin prosegment

Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH2 terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex. After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme. The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a somewhat earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

A novel purification method of human renin

Human renal renin was isolated from a partially purified preparation, Haas' preparation step 5 material, with a remarkably high yield of 28% by a newly developed method. This method consisted of only four steps including affinity chromatography on pepstatin-aminohexyl-agarose, chromatofocusing, gel filtration and DEAE-chromatography after the initial extraction and concentration. This method enabled us to obtain pure human renin without another affinity column used by other investigators. Pure human renin had a molecular weight of 40,000 daltons as estimated by gel filtration and sodiumdodecylsulfate-polyacrylamide gel electrophoresis. The pI of purified renin was 5.6.