Presentation of alpha-galactosylceramide by murine CD1d to natural killer T cells is facilitated by plasma membrane glycolipid rafts

CD1 molecules are non-polymorphic major histocompatibility complex class I-related proteins that bind and present glycolipid antigens to T-cell antigen receptors (TCR) expressed by alphabeta T cells or natural killer-like T cells (NKT). Anti-metastatic properties of NKT cells reactive to the CD1d-binding antigen alpha-galactosylceramide (alpha-GalCer) are now being explored as a contributor to tumour cell killing. In this study, we tested the hypothesis that presentation of alpha-GalCer by murine CD1d (mCD1d) to mCD1d-restricted NKT cells was facilitated by plasma membrane glycolipid rafts. Confocal microscopy of mCD1d-transfected A20 B cells (A20mCD1d) demonstrated that mCD1d was raft-localized. This observation was confirmed by immunoblotting of raft fractions isolated on sucrose density gradients. Raft disruption by the cholesterol-binding agent nystatin, or short-chain ceramides, inhibited presentation of low concentrations of alpha-GalCer to NKT cells. Inhibition of antigen presentation was reversed by treatment of A20mCD1d cells with higher alpha-GalCer concentrations, or removal of raft-disrupting agents. These data indicate that partitioning of mCD1d into membrane rafts increases the capacity of antigen-presenting cells to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD1d/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells.     

Association between mutations in the CARD15 (NOD2) gene and Crohn's disease in Israeli Jewish patients

Ulcerative colitis (UC) and Crohn's disease (CD) are heterogeneous disorders characterized by chronic intestinal inflammation. Genetic predisposition is a major risk factor in both diseases. The CARD15 (NOD2) gene has been implied as a candidate gene in the pathogenesis CD. Our aim was to delineate the frequency of three missense and one frameshift variant of CARD15 in Israeli Jewish CD and UC patients. DNA was extracted from blood samples from 238 unrelated inflammatory bowel disease (IBD) patients, 68 with UC and 170 with CD. The DNA was genotyped for two missense mutations, R675W and G881R, and one frameshift mutation, 980FS981X. Mutations in CARD15 were observed with significantly greater frequency in CD patients (46/170, 27%) than in UC patients (7/68, 10%) (P = 0.005). Homozygous and compound heterozygous carriers were restricted to seven (4%) patients with CD as compared to none of the UC patients (P = 0.01). Similar rates in Ashkenazi and non-Ashkenazi Jewish patients were observed. Age-of-onset of disease was lower in Ashkenazi mutation carriers as compared to non-carriers of Ashkenazi origin (18.7 +/- 8.6 years vs. 25.8 +/- 13.4 years, respectively, P = 0.03). No other phenotypic characteristics could distinguish mutation carriers from non-carriers. We conclude that germline mutations in the CARD15 gene are more frequently found in CD than UC patients and appear to predict an earlier age-of-onset in Ashkenazi Jewish patients. No association could be demonstrated between CARD15 mutations and specific disease course or behavior.     

Einflüsse von Nahrungsaufnahme und Gewöhnung auf die Verteilung von 8-14C-Coffein bei der Ratte

Zusammenfassung 200 g schwere Ratten erhielten Lösungen von¹⁴C-Coffein entweder nüchtern oder nach vorheriger Verabreichung von Kohlenhydrat, Eiweiß und Fett. Die Nahrungszufuhr bedingte folgende Änderungen der Coffeinverteilung im Organismus gegenüber den nüchternen Tieren: die Coffeinresorption war deutlich verzögert, so daß die¹⁴C-Aktivitätswerte im Serum und Carcass nur langsam anstiegen. Im weiteren Verlauf des Versuches erreichte der Abfall der¹⁴C-Aktivität in Serum und Carcass und ihre Ausscheidung im Harn ein geringeres Ausmaß.Die Coffeinverteilung im Organismus zeigte außerdem bestimmte Änderungen bei Ratten, die im Anschluß an eine 14tägige Gewöhnunsperiode¹⁴C-Coffeinhaltigen Kaffee-Infus erhielten. Im Vergleich zu den nicht an Kaffee gewöhnten Ratten war hierbei die Coffeinresorption deutlich beschleunigt. Die¹⁴C-Aktivitätswerte im Serum stiegen anfangs schneller und höher an, zeigten anschließend aber einen rascheren Abfall. Auf Grund dieser Effekte war die¹⁴C-Ausscheidung durch die Niere erheblich beschleunigt und vermehrt.

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Summary Rats weighing 200 g obtained¹⁴C-caffeine solution either after starvation or after feeding carbohydrate, proteine or fat. The following differences in caffeine distribution in the fed rats compared with the starved ones were confirmed: caffeine absorption was clearly decreased, resulting in slow increase in¹⁴C-activity in the serum and the carcass. During the course of the experiment it was observed that the decline of¹⁴C-activity in serum and carcass was delayed and its excretion in the urine was decreased. Additionally caffeine distribution in the rat body showed certain changes which were accompanied by caffeine habituation in the animals after administration of¹⁴C-caffeine contained in coffee infusion for 14 days. In the coffee habituated animals the caffeine absorption was noticeably accelerated.¹⁴C-activity in serum increased initially quicker but finally showed a quicker decline. Governed by these effects the¹⁴C-excretion via kidney was increased and its rate was accelerated.

     

Cerebellar damage impairs automaticity of a recently practiced movement

It has been suggested that the cerebellum plays a critical role in learning to make movements more "automatic" (i.e., requiring less attention to the details of a movement). We hypothesized that cerebellar damage compromises learning of movement automaticity, resulting in increased attentional demands for movement control. The purpose of our study was to determine whether cerebellar damage disrupts the ability to make a practiced movement more automatic. We developed a dual task paradigm using two tasks that did not have overlapping sensory or motor requirements for execution. Our motor task required subjects to maintain an upright posture while performing a figure-8 movement using their arm. This motor task was chosen to simulate requirements of everyday movements (e.g., standing while reaching for objects), but it was novel enough to require practice for improvement. Our secondary task was an auditory vigilance task where subjects listened to letter sequences and were asked to identify the number of times a target letter was heard. We tested controls and people with cerebellar damage as they practiced the movement task alone and then performed it with the auditory task. We recorded 3D position data from the arm, trunk, and leg during the movement task. Errors were recorded for both the movement and the letter tasks. Our results show that cerebellar subjects can improve the movement to a very limited extent with practice. Unlike controls, the motor performance of cerebellar subjects deteriorates to prepractice levels when attention is focused away from the movement during dual task trials. Control subjects' insensitivity to dual task interference after practice was due to learned movement automaticity and was not a reflection of better dual task performance generally. Overall, our findings suggest that the cerebellum may be important for shifting movement performance from an attentionally demanding (unpracticed) state to a more automatic (practiced) state.     

Localization of corticotropin-releasing activity in the rat hypothalamus

Hypothalamic nuclei were removed from frozen sections of rat brain and examined for their corticotropin-releasing activity. The highest concentration was measured in the median eminence. In addition there was significantly more activity detected in the nuclei paraventricularis, supraopticus, suprachiasmaticus and arcuatus than in the other nuclei.     

Further analysis of ATP-mediated activation of K+ channels in renal epitheloid Madin Darby canine kidney (MDCK) cells

ATP activates K⁺ channels by increasing intracellular calcium activity in Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for the involvement of G-proteins and of protein kinase C in the intracellular transmission of these effects. To this end, the effect of ATP on intracellular calcium and K⁺ channel activity has been studied in cells pretreated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or pertussis toxin. The ATP-induced increase of intracellular calcium is not significantly affected by pretreatment with pertussis toxin, is significantly blunted by pretreatment with TPA and is abolished by pretreatment with both pertussis toxin and the phorbol ester. The ATP activation of K⁺ channels is similarly blunted by pretreatment with TPA, but is not abolished by pretreatment with both the phorbol ester and pertussis toxin. Furthermore, the ATP-induced hyperpolarization is not abolished in cells pretreated with both pertussis toxin and TPA. In those cells, ATP may activate K⁺ channels by calcium-independent mechanisms or lead to localized increases of intracellular calcium sufficient to activate the K⁺ channels but escaping detection with fura-2 fluorescence.     

蛙类肠系膜内微小动脉的压力检测

根据Wiederhielm的阻抗平衡压力检测原理,设计了一个改进的伺服零微血管测压系统。利用该系统检测了蟾蜍及蛙肠系膜内微小动脉的压力、获得了各管径级的压力和脉压的参数,并观察了药物的作用,探讨了微动脉内压力的波动特性以及应激情况下压力的骤变式与脉动式的交替,为微循环研究提供一些有价值的现象。     

结核分枝杆菌联合DNA疫苗初免-BCG加强的免疫效果观察

目的:研究并比较结核分枝杆菌免疫保护性抗原DNA(Ag85A和ESAT-6)疫苗联合免疫,BCG免疫以及联合DNA疫苗初免-BCG加强免疫等不同的免疫策略,诱导免疫应答效果观察.方法:健康雌性BALB/c小鼠24只,随机分成PBS 阴性对照组,DNA初免-BCG异源加强组,DNA(Ag85A和ESAT-6)初免DNA同源加强组和BCG阳性对照组,共进行3次免疫,初免2次,最后1次加强,间隔2周1次.PBS组3次均注射PBS 溶液;DNA/BCG组以质粒DNA免疫2次,最后1次以BCG加强免疫;DNA/DNA组3次均以质粒DNA进行免疫;BCG组则注射PBS溶液2次后以BCG免疫.末次免疫后4、6、8周分别分离血清测定总IgG水平,同时分离小鼠脾细胞,体外经TB-PPD刺激后进行淋巴细胞增殖实验(XTT法)并测定脾细胞培养上清中IFN-γ和IL-4水平.结果:DNA/BCG、DNA/DNA、BCG组体外经TB-PPD刺激后均检测到特异性IgG抗体产生,3组平均效价为1:120、1:160、1:80,DNA/DNA组的抗体效价高于另外2组;小鼠脾细胞体外经TB-PPD刺激后,均能产生特异性淋巴细胞增殖并诱生较强的IFN-γ反应,其中DNA/BCG组IFN-γ的分泌水平高于DNA/DNA组和BCG组(P<0.05).结论:联合DNA疫苗初免-BCG加强的免疫策略能在小鼠体内诱导较强的特异性细胞免疫反应,产生高水平的IFN-γ.