Benzodiazepines in the brain

Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5.10⁻¹⁰–10⁻⁸ M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam andN-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.     

Fluoritisierte Radiolarien aus Kieselkalk-Bänken des Mittel-Viseums (Unterkarbon) des Rheinischen Schiefergebirges (Deutschland)

From siliceous shales (Lower Carboniferous, Rheinisches Schiefergebirge), in direct neighborhood to a bed of allodapic limestone, the following fluontized radiolarian fauna has been extracted by chemical transformation of originally calcified skeletons:Albaillella cartalla, Latentifistula turgida, Eostylodktya cf. eccentrica, Tetragregnon sycamorensis sycamorensis, Belowea variabilis, Callela? hexactinia, Entactinia tortispina and Entactinia variospina. The limestone bed has been dated by calcareous foraminifera as being mid Visean in age (V 2b-3a, Cf 5-foraminiferal zone). The diagenetic calcification took place after the selective dissolution of the skeletons and was in itself not selective.     

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

Summary An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.     

Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.     

In situ activation of syngeneic tumour-specific cytotoxic T lymphocytes: Intra-pinna immunization followed by restimulation in the peritoneal cavity

Summary Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the K^d major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice.     

Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice

Summary DBA/2 (H-2^d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA^– ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA⁺ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Length Polymorphisms in Human Proline-Rich Protein Genes Generated by Intragenic Unequal Crossing over

Southern blot hybridization analysis of genomic DNAs from 44 unrelated individuals revealed extensive insertion/deletion polymorphisms within the BstNI-type loci (PRB1, PRB2, PRB3 and PRB4) of the human proline-rich protein (PRP) multigene family. Ten length variants were cloned, including alleles at each of the four PRB loci, and in every case the region of length difference was localized to the tandemly repetitious third exon. DNA sequences covering the region of length variation were determined for seven of the alleles. The data indicate (1) that the PRB loci can be divided into two subtypes, PRB1 plus PRB2, and PRB3 plus PRB4, and (2) that the length differences result from different numbers of tandem repeats in the third exons. Variant chromosomes were also identified with different numbers of PRP loci resulting from homologous but unequal exchange between the PRB1 and PRB2 loci. The overall data are compatible with the observed length variants having been generated via homologous but unequal intragenic exchange. The results also indicate that these crossover events are sensitive to the amount of homology shared between the interacting DNA strands. Allelic length variants have arisen independently at least 20 times at the PRB loci, but only one has been detected at a PRH locus. Comparison of the detailed structures of the repetitious regions in PRB and PRH loci shows that the repeats in PRB genes are very similar to each other in sequence and in length. The PRH genes contain fewer repeats, which differ considerably in their individual lengths. These differences suggest that the larger number of length variants in PRB genes is related to their greater ease of homologous but unequal pairing compared to PRH genes.