Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

Characterization of Microtubule-Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex

Microtubule-associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A-5m gel filtration. The purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using both a gradient gel (3.75-12.5%) and a low-percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to those of porcine brain MAP2. Immunoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.     

CAM Photosynthesis under Drought Conditions in Kalanchoe blossfeldiana Grown with Nitrate or Ammonium as the Sole Nitrogen Source

K. blossfeldiana Poelln. cv. Hikan was grown in vermiculite,supplied daily with nutrient solution containing 1 mM (or 10mM) nitrate or ammonium as the sole nitrogen source. The nitrate-grownplants had more activity of CAM (Crassulacean acid metabolism)photosynthesis (nocturnal CO₂ uptake in the shoot and nocturnalincreases of titratable acidity and malate content in the leaves)than the ammonium-grown plants. Interruption of the solutionsupply for 5 or more days (drought conditions) increased theactivity of CAM photosynthesis in nitrate- or ammonium-grownplants, and the diurnal CO₂ uptake pattern in the nitrate-grownplants shifted from ‘weak-CAM’ to ‘full-CAM’.The difference in the activity of CAM photosynthesis betweennitrate- and ammonium-grown plants increased under the droughtconditions. When the solution was resupplied, the activity ofCAM photosynthesis rapidly decreased to the levels before theinterruption. The physiological mechanism and ecological significanceof the effect of the nitrogen source on CAM photosynthesis arediscussed (Received January 5, 1988; Accepted April 13, 1988)     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Radiometric assays for mammalian epoxide hydrolases and glutathione S-transferase

A number of epoxides, including cis- and trans-stilbene oxides, were assayed as substrates for epoxide hydrolases (EHs) by gas-liquid chromatography. Radiolabeled stilbene oxides were prepared by sodium borotritide reduction of desyl chloride followed by ring closure with base treatment. Rapid radiometric assays for EHs were performed by differential partitioning of the epoxide into dodecane, while the product diol remained in the aqueous phase. Glutathione (GSH) transferase was similarly assayed by partitioning the epoxide and diol, if formed metabolically, into 1-hexanol, while the GSH conjugate was retained in the aqueous phase. The cytosolic EH rapidly hydrates the trans isomer while the cis is very poorly hydrated. In contrast, the cis is a better substrate for the microsomal EH than the trans. GSH transferase utilized both epoxides as substrates, but conjugation is faster with the cis isomer. Cytosolic EH activity is high in mouse but very low in rat and guinea pig. Microsomal EH activity, in contrast, is highest in guinea pig, intermediate in rat, and the lowest in mouse. GSH transferase activity, which is high in all three species, can be inhibited by chalcone, with an I₅₀ of 3.1 × 10^?5m. These assays facilitate the rapid evaluation and direct comparison of epoxide-metabolizing systems in cell homogenates used in short-term mutagenicity assays, cell or organ culture, and possibly in vivo.     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)