The primary structure and oxygen-binding properties of the single haemoglobin of the high-Antarctic fish Aethotaxis mitopteryx DeWitt

Summary The complete amino acid sequence of the single haemoglobin of the Antarctic fish Aethotaxis mitopteryx DeWitt has been established by automated repetitive Edman degradation on the intact and cleaved (enzymatically and chemically) and chains. A very high sequence identity with other Antarctic fish haemoglobins has been detected. The haemoglobin has a moderate Bohr effect and no Root effect. Organic phosphates and chloride also regulate oxygen binding only to a moderate extent. The lack of Root effect is consistent with the substitution His — Val at the HC3 C-terminal position of the chain. The low overall heat of oxygenation suggests that in this species oxygen transport is an energy-saving process, presumably related to cold adaptation. The comparative analysis of the haemoglobins of Antarctic fishes emphasises some unique features of the oxygen-transport system of A. mitopteryx, which are likely to be related to its also rather unique mode of life.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Species-specific diversity among simian immunodeficiency viruses from African green monkeys

The prevalence, natural history, and genetic characteristics of simian immunodeficiency virus (SIV) infections in most feral African monkey species are presently unknown, yet this information is essential to elucidate their origin and relationship to other simian and human immunodeficiency viruses. In this study, a combination of classical and molecular approaches were used to identify and characterize SIV isolates from West African green monkeys (Cercopithecus sabaeus) (SIVagm isolates). Four SIVagm viruses from wild-caught West African green monkeys were isolated and analyzed biologically and molecularly. Amplification, cloning, and sequencing of a 279-bp polymerase fragment directly from uncultured peripheral blood mononuclear cells was facilitated by the use of nested polymerase chain reaction. The results indicated that West African green monkeys are naturally infected with SIVs which are closely related to East African SIVagm isolates. However, structural, antigenic, and genetic differences were observed which strongly suggest that the West African green monkey viruses comprise a phylogenetically distinct subgroup of SIVagm. These findings support our previous hypothesis that SIVagm viruses may have evolved and diverged coincident with the evolution and divergence of their African green monkey host. In addition, this study describes a polymerase chain reaction-based approach that allows the identification and molecular analysis of divergent SIV strains directly from primary monkey tissue. This approach, which does not depend on virus isolation methods, should facilitate future studies aimed at elucidating the origins and natural history of SIVs in feral African green monkey populations.     

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.     

Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings

Low-temperature stress was shown to cause a rapid increase in steady-state levels of alcohol dehydrogenase-1 message (Adh1) and protein activity (ADH1) in maize (Zea mays) (B37N, A188) and rice (Oryza sativa) (Taipei 309, Calmochi 101) seedlings. Maize roots and rice shoots and roots from 7-day seedlings shifted to low temperature (10°C) contained as much as 15-fold more Adh1 mRNA and 8-fold more ADH1 protein activity than the corresponding tissues from untreated seedlings. Time-course studies showed that these tissues accumulated Adh1 mRNA and ADH1 activity severalfold within 4- to 8-hour, levels plateaued within 20 to 24 hours, and remained elevated at 4 days of cold treatment. Within 24 hours of returning cold-stressed seedlings to ambient temperature, Adh1 mRNA and ADH1 activity decreased to pretreatment levels. Histochemical staining of maize and rice tissue imprints showed that ADH activity was enhanced along the lengths of cold-stressed maize primary roots and rice roots, and along the stems and leaves of rice shoots. Our observations suggest that short-term cold stress induces Adh1 gene expression in certain plant tissues, which, reminiscient of the anaerobic response, may reflect a fundamental shift in energy metabolism to ensure tissue survival during the stress period.     

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.     

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.