Genetics of susceptibility to radiation-induced apoptosis in colon: two loci on Chromosomes 9 and 16

Apoptosis, a mechanism for removal of genetically damaged cells and for maintenance of desired size of cell populations, has been implicated in tumor development. Previously, we defined polymorphic loci for susceptibility to apoptosis of thymocytes Rapop1, Rapop2, and Rapop3 on mouse Chromosomes 16, 9, and 3, respectively, using recombinant congenic CcS/Dem strains, each of which contains a random set of 12.5% STS/A genome in the genetic background of BALB/cHeA. The STS/A alleles at these loci confer lower susceptibility to radiation-induced apoptosis of thymocytes than the BALB/cHeA. In the present study, we tested susceptibility of colon crypt cells to radiation-induced apoptosis. In contrast to apoptosis in thymus, the STS/A mice were more susceptible to apoptosis in colon than the BALB/cHeA. Among the CcS/Dem strains, CcS-4, CcS-7, and CcS-16 were more susceptible to apoptosis in colon than the BALB/cHeA; in thymus, the CcS-7 mice are less susceptible, and the CcS-4 and CcS-16 are not different from the BALB/cHeA. Thus, individual CcS/Dem strains showed different apoptosis susceptibility in the two organs. Analysis of (CcS-7 × BALB/cHeA)F₂ hybrids revealed linkage of susceptibility to radiation-induced apoptosis of colon crypt cells to two loci on Chrs 9 and 16, to which Rapop2 and Rapop1 are mapped. The STS/A allele at the locus on chromosome 9 results in high susceptibility to apoptosis of colon crypt cells in mice homozygous for the BALB/cHeA allele at the locus on Chr 16. Although these two loci may be identical to Rapop1 and Rapop2, they affect apoptosis in colon in a way different from that in thymus. Received: 9 October 1997 / Accepted: 29 December 1997     

A simple method of intestinal anastomosis (ileocolostomy) in rats.

A simple method of ileocolostomy was performed in rats. The colon near the cecum was ligated, including its arteries and veins. Main artery and vein of the cecum were ligated. Then, the cecum was cut out. A longitudinal incision was made on the anti-mesenteric side of the proximal end of the colon, approximately 7-8 mm long. A 21-G needle was inserted toward the incision 2 cm away from the proximal end of the anti-mesenteric side of the colon. A nylon suture was knotted once to the distal end of the ileum and was introduced into the tip of the needle which had previously been passed through the colon. Then, the needle was removed. The suture was pulled to introduce the distal end of the ileum into the colonic lumen. Then, the suture was knotted once on the colon again to fix the ileum to the colon. The incision in the proximal end of the colon was not closed. At the 2nd week after the operation, X-ray examinations demonstrated that the ileocolonic passages with no leakage at the anastomotic site were quite satisfactory. At the 4th week after the operation, there were no macroscopic or microscopic complications at the anastomotic site. The mucosal and serosal epithelia of the ileum and colon continued smoothly. This simple method may be very effective in preparing anastomosis in the gastrointestinal tract, especially in small laboratory animals for nutritional and surgical experiments.     

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-l-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.     

Detection of Short Protein Coding Regions within the Cyanobacterium Genome: Application of the Hidden Markov Model

The gene-finding programs developed so far have not paid muchattention to the detection of short protein coding regions (CDSs).However, the detection of short CDSs is important for the studyof photosynthesis. We utilized GeneHacker, a gene-finding programbased on the hidden Markov model (HMM), to detect short CDSs(from 90 to 300 bases) in a 1.0 mega contiguous sequence ofcyanobacterium Synechocystis sp. strain PCC6803 which carriesa complete set of genes for oxygenic photosynthesis. GeneHackerdiffers from other gene-finding programs based on the HMM inthat it utilizes di-codon statistics as well. GeneHacker successfullydetected seven out of the eight short CDSs annotated in thissequence and was clearly superior to GeneMark in this rangeof length. GeneHacker detected 94 potentially new CDSs, 9 ofwhich have counterparts in the genetic databases. Four of thenine CDSs were less than 150 bases and were photosynthesis-relatedgenes. The results show the effectiveness of GeneHacker in detectingvery short CDSs corresponding to genes.     

Protein tyrosine phosphatase receptor type Z is inactivated by ligand-induced oligomerization

Receptor-type protein tyrosine phosphatases (RPTPs) are considered to transduce extracellular signals across the membrane through changes in their PTP activity, however, our understanding of the regulatory mechanism is still limited. Here, we show that pleiotrophin (PTN), a natural ligand for protein tyrosine phosphatase receptor type Z (Ptprz) (also called PTPzeta/RPTPbeta), inactivates Ptprz through oligomerization and increases the tyrosine phosphorylation of substrates for Ptprz, G protein-coupled receptor kinase-interactor 1 (Git1) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (Magi1). Oligomerization of Ptprz by an artificial dimerizer or polyclonal antibodies against its extracellular region also leads to inactivation, indicating that Ptprz is active in the monomeric form and inactivated by ligand-induced oligomerization.     

Expression and characterization of Rab38, a new member of the Rab small G protein family

Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [alpha-32P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.     

Growth,net photosynthesis and leaf nutrient status of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings grown in brown forest soil acidified with H<Subscript>2</Subscript>SO<Subscript>4</Subscript> or HNO<Subscript>3</Subscript> solution

To obtain basic information for evaluating critical loads of acid deposition for protecting Japanese beech forests, growth, net photosynthesis and leaf nutrient status of Fagus crenata seedlings grown for two growing seasons in brown forest soil acidified with H₂SO₄ or HNO₃ solution were investigated. The whole-plant dry mass of the seedlings grown in the soil acidified by the addition of H₂SO₄ or HNO₃ solution was significantly less than that of the seedlings grown in the control soil not supplemented with H⁺ as H₂SO₄ or HNO₃ solution. However, the degrees of reduction in the whole-plant dry mass and net photosynthetic rate of the seedlings grown in the soil acidified by the addition of H⁺ as H₂SO₄ solution at 100 mg l^–1 on the basis of air-dried soil volume (S-100 treatment) were greater than those of the seedlings grown in the soil acidified by the addition of H⁺ as HNO₃ solution at 100 mg l^–1 (N-100 treatment). The concentrations of Al and Mn in the leaves of the seedlings grown in the S-100 treatment were significantly higher than those in the N-100 treatment. A positive correlation was obtained between the molar ratio of (Ca+Mg+K)/(Al+Mn) in the soil solution and the relative whole-plant dry mass of the seedlings grown in the acidified soils to that of the seedlings grown in the control soil. Based on the results, we concluded that the negative effects of soil acidification due to sulfate deposition are greater than those of soil acidification due to nitrate deposition on growth, net photosynthesis and leaf nutrient status of F. crenata, and that the molar ratio of (Ca+Mg+K)/(Al+Mn) in soil solution is a suitable soil parameter for evaluating critical loads of acid deposition in efforts to protect F. crenata forests in Japan.     

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.     

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.