Effect of Repeated Convulsive Seizures on Brain γ-Aminobutyric Acid Metabolism in Three Sublines of Mice Differing by Their Response to Acoustic Stimulations

The turnover rates and steady-state levels of gamma-aminobutyric acid (GABA) have been determined in 15 brain areas of three sublines of inbred mice differing in their susceptibility to audiogenic seizures: Rb3, which is seizure resistant; Rb2, which develops clonic seizures; and Rb1, which develops tonic-clonic seizures. In the Rb1 subline, GABA steady-state levels are lower than in the Rb3 subline in three of the 15 areas examined (cerebellum, anterior colliculus, and amygdala), whereas in the Rb2 subline, steady-state levels are either higher (posterior colliculus and hippocampus) or lower (amygdala) than in the Rb3 subline. GABA turnover rates differ in three brain areas in Rb1 (amygdala, raphe, and hypothalamus) and in a single area (amygdala) in Rb2 when compared with Rb3. Only one area has similar variations of GABA turnover rate and steady-state levels in the two susceptible sublines: the amygdala. After 2 weeks of repeated auditory stimulations (two times a day, 8,000 Hz, 100 dB), additional alterations in GABA metabolism are observed: mainly large increases in GABA turnover rates (from 40% to three- to fourfold). The Rb2 subline displays a greater number of alterations (increases of turnover rates in pons, cerebellum, anterior and posterior colliculus, amygdala, olfactory bulbs and tubercles, striatum, and frontal cortex) than the Rb1 subline (increases of turnover rates in cerebellum, posterior colliculus, olfactory tubercles, raphe, and frontal cortex and a decrease in hypothalamus). In the Rb3 subline, increases of the turnover rate in amygdala and olfactory tubercles and decreases in olfactory bulbs and hippocampus are observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in synaptosomal neurotransmitter amino acids in “petit-mal” rats at a daytime and a nighttime

The synaptosoma fractions of 6 brain areas-olfactory tubercles (OT), frontal cortex (FC), striatum (Sr), amygdala (A), thalamus (Th), hypothalamus (Hy)-have been analyzed for their neurotransmitter amino acids (AA) content in Wistar rats exhibiting petit-mal epilepsy (PM-E) and in controls (C). The analysis was carried out at 11 p.m. (nighttime corresponding to the acrophase for the hourly number of spike-wave complexes) and at 11 a.m. (daytime). A day versus night rhythmicity is recorded for synaptosomal inhibitory AA in control and in PM-E rats. However, day versus night variations are more frequent and more prominent in C rats than in PM-E rats. Two day versus night variations exist only in PM-E rats: increases of GABA level in Sr and of Asp in Hy. Differences between PME-and C in synaptosomal AA content are more likely to be present during the nighttime. During this period lower AA values for PM-E rats are found for one or several inhibitory AA in OT, Th, and FC. It seems that the differences between PM-E and C concerning the inhibitory AA correlate with the number of spike-wave discharges. Only in one brain area is there a similar difference for PM-E and C during daytime and nighttime: a decreased GABA content for PM-E rats in OT. The decrease is larger in nighttime than in daytime. This difference may serve as a marker for this epileptic disorder. Moreover, it is in OT that the greatest number of PM-E versus C differences in synaptosomal neurotransmitter AA are observed. In view of these and former data, the existence of different alterations in synaptosomal neurotransmitter AA for different types of epilepsy is suggested.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspartate - Glu glutamate - Gln glutamine - OT offactory tubereles - FC fronto-parietal cortex - Sr striatum - A amygdala - Th lateral thalamus - Hy lateral hypothalamus - AA neurotransmitter amino acids - I inhibitory - E excitatory - C control rats - PM-E petit-mal rats     

Examinations of cell mediated immunity in diffuse peritonitis]

Phagocytic index and rosette test E have been determined in 50 patients, including 15 with diffuse peritonitis and the noncomplicated diffuse peritonitis, 20 patients with septic complications, and 15 patients who underwent abdominal surgery. It was found, that phagocytic index is decreased in all patients after abdominal surgery. The most marked decrease of this index was found in patients with complicated peritonitis. It still decreases in the course of peritonitis and increases in the reference group. The marked decrease in the number of T-cells was observed in complicated peritonitis during the whole period of follow-up while it normalizes in case of noncomplicated peritonitis as in the reference group. Using the tests under study it was possible to assess cell-mediated immunity which is depressed in peritonitis. The tests enable also the prediction of septic complications of peritonitis.     

Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels.

A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.     

Demonstration of the correlation of the degree of synthesis and 4-aminobutyric acid levels in the central nervous system; effect of repeated convulsive seizures

GABA turnover rates have been determined in 15 brain areas in five inbred strains of Mice or sublines (DBA/2J, C57/6J, Swiss Rb1, Swiss Rb2, Swiss Rb3). GABA turnover rates and levels are correlated (2 P less than 0.05). After repeated seizures (twice a day for 15 days), induced by an acoustic stimulus in Swiss Rb1 Mice selected for audiogenic seizures, this correlation is no longer observed.     

Purification of rat brain succinate-semialdehyde dehydrogenase and study of its inhibition by branched chain fatty acids]

Rat brain succinic semialdehyde deshydrogenase has been purified 1300 fold. This enzyme is inhibited non competitively by the same branched chain fatty acids which inhibit GABA-transaminase competitively with respect to GABA. The respective activities of GABA-T and SSADH found in rat brain indicate that at anticonvulsant doses, the acids dipropylacetic and 2-methyl 2-ethyl caproic preferentially inhibit GABA-transaminase thus inducing a rise in cerebral GABA level. This increase is therefore not due to metabolism of the succinic semialdehyde by GABA-T.     

Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer

The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.     

鼠李糖乳杆菌LV108及其发酵乳免疫调节作用的比较

目的探讨鼠李糖乳杆菌LV108及其发酵乳对免疫抑制小鼠免疫功能的调节作用。方法将BALB/c小鼠随机分为5组,每组10只,即空白组(正常小鼠)、模型组(免疫抑制小鼠)、药物组(免疫抑制小鼠食物中添加左旋咪唑)、LV108菌悬液组(免疫抑制小鼠食物中添加LV108菌悬液)和LV108发酵乳组(免疫抑制小鼠食物中添加LV108发酵乳),除空白组外其余组构建免疫抑制小鼠模型。干预4周后,分别测定各组小鼠体质量和脏器指数,血清中白细胞介素2(IL2)、肿瘤坏死因子α(TNFα)和免疫球蛋白G(IgG)含量,血清溶血素含量、耳肿胀度和肝、脾巨噬细胞吞噬能力。结果相比模型组,LV108菌悬液组和LV108发酵乳组小鼠体质量增长速度、脏器指数、血清IL2与IgG水平、血清溶血值、耳肿胀度和巨噬细胞吞噬能力显著升高(均P<0.05);在脾脏指数、血清IL2与TNFα水平、血清溶血素含量和耳肿胀度免疫指标上,LV108菌悬液组与LV108发酵乳组之间比较差异具有统计学意义(均P<0.05)。结论LV108菌体及发酵乳对免疫抑制小鼠具备较全面的免疫调节作用,均可提高小鼠的自身免疫力;LV108发酵乳对小鼠的免疫调节作用强于LV108菌体。     

Site-specific conjugation of boron-containing dendrimers to anti-EGF receptor monoclonal antibody cetuximab (IMC-C225) and its evaluation as a potential delivery agent for neutron capture therapy

The gene encoding EGFR often is amplified in human gliomas, and the receptor itself has been considered as a potential target for the specific delivery of therapeutic agents to brain tumors. The purpose of the present study was to investigate the use of the chimeric MoAb cetuximab (IMC-C225), which is directed against EGFR and EGFRvIII, as a boron delivery agent for neutron capture therapy (NCT) of brain tumors. As determined by 125I-cetuximab radioligand binding assays, F98 rat glioma cells, which had been transfected with the gene encoding EGFR (F98EGFR), expressed 1.60 +/- 0.13 x 10(5) receptor sites/cell with a Ka = 1.64 +/- 0.32 x 10(8) M-1). F98 cells transfected with the gene encoding a mutant form of EGFR, designated the F98EGFRvIII glioma, expressed 1.07 +/- 0.10 x 10(5) receptor sites/cell with a Ka = 2.18 +/- 0.54 x 10(9) M-1 compared to background levels expressed on F98 wild-type cells (F98WT). A heavily boronated, fifth generation polyamidoamine (PAMAM or "starburst") dendrimer, G5-B1100, was linked to oligosaccharide moieties, which were distant from antigen binding sites of cetuximab, by means of the heterobifunctional reagents N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and N-(k-maleimidoundecanoic acid) hydrazide (KMUH). The resulting bioconjugate, designated C225-G5-B1100, was separated from the unconjugated dendrimer using a Sephacryl S-300 column. On the basis of the relative concentration ratios of boron and protein, there were approximately 1100 boron atoms per molecule of cetuximab with only a slight reduction of Ka. The localization of C225-G5-B1100 or G5-B1100 in rats bearing intracerebral implants of either F98EGFR or F98WT gliomas was determined 24 h following direct intratumoral (i.t.) injection at which time 92.3 +/- 23.3 micrograms B/g tumor was localized in F98EGFR gliomas versus 36.5 +/- 18.8 micrograms B/g tumor in F98WT gliomas and 13.4 +/- 6.1 micrograms in normal brain. In contrast, only 6.7 +/- 3.6 micrograms B/g tumor of G5-B1100 was localized in F98EGFR gliomas following i.t. injection, thereby demonstrating specific molecular targeting of EGFR. Based on these data, BNCT studies will be initiated in F98EGFR glioma bearing rats to evaluate C225-G5-B1100 for the treatment of intracerebral brain tumors.