Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1.

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats.

The E4 region of human adenovirus type 9 (Ad9) transforms established rat embryo fibroblasts and encodes an essential determinant for the production of estrogen-dependent mammary tumors in rats. Testing of the seven Ad9 E4 open reading frames (ORFs) individually for transformation of the established rat embryo fibroblast cell line CREF indicated that only Ad9 E4 ORF1 possessed a significant ability to generate transformed foci on these cells. In contrast, the E4 ORF1 sequences from human Ad5 and Ad12 lacked the transforming potential exhibited by Ad9 E4 ORF1. Cell lines derived from Ad9 E4 ORF1-transformed foci expressed the 14-kDa Ad9 E4 ORF1 protein and formed colonies in soft agar. In addition, the Ad9 E4 ORF1 protein was required for initiation of mammary oncogenesis in vivo, as E4 ORF1 mutant viruses failed while E4 ORF2 and ORF3 mutant viruses succeeded in eliciting mammary tumors in animals. A role for Ad9 E4 ORF1 in tumor maintenance was suggested by the fact that 100% of virus-induced mammary tumors expressed the E4 ORF1 protein. Taken together, the facts that the Ad9 E4 ORF1 protein exhibits transforming potential in culture and is required by Ad9 to produce mammary tumors in animals suggest that Ad9 E4 ORF1 is a new viral oncoprotein.     

Overcoming seed dormancy in ex situ plant germplasm conservation programmes; an example in the endemic Argyranthemum (Asteraceae: Anthemideae) species from the Canary Islands

Germplasm of 21 diverse Argyranthemum taxa was collected from contrasting ecological zones in the Canary Islands. Seed dormancy was considerable in the majority of taxa. Extensive investigations, based on a germination test procedure algorithm for Asteraceae, with achenes from ray and disc florets of five contrasting taxa identified a procedure to promote full (85%) germination of the seeds from both ray and disc florets of all five taxa; viz, excision of the seeds from the achenes, followed by testing at 15°C with 2.6×10^-3 m GA₃ co-applied. Subsequent tests showed that this regime was effective in promoting full germination in seeds from both ray and disc florets of the remaining 16 taxa. The results are discussed in the context of ex situ plant germplasm conservation.     

Bats of the Gulf of Guinea islands: faunal composition and origins

The present study compares the bat faunas of the islands of the Gulf of Guinea. Species composition. endemism and hypothetical origins are discussed. All families present in the mainland region are found in Bioko, a typical landbridge island. Foliage gleaning guild species (Nycteridae) show limited colonization abilities. This is also true of the family Rhinolophidae, but not for the closely related family Hipposideridae. The majority of the oceanic island species are African bats which show a widespread distribution and, therefore, have a high ecological plasticity. The continental relatives of the two endemic species Myonycteris brachycephala and Chaerephon tomensis are restricted to relatively small forested areas. Bioko's bat fauna is the result of the recent isolation from a formerly land-connected community. The oceanic bat faunas originated from the establishment of incomers from other areas. Nevertheless, extinction appears in both vicariant and dispersal processes, as an important factor in modelling the current bat communities of the Gulf of Guinea islands.     

Trinucleotide (CAG) repeat expansion in chromosomes of Spanish patients with Huntington's disease

Huntington's disease (HD) is a neurodegenerative and hereditary disease characterized by progressive movement disorders and mental and behavioral abnormalities. The HD gene is an expanding and unstable trinucleotide repeat (CAG repeat sequences). We studied 77 individuals from 38 families with HD in an attempt to obtain information for genetic counselling and differential diagnosis. Our results indicate that individuals with more than 40 repeats will be affected by the disease, whereas those with fewer than 30 will be healthy. There can be some overlap between 30 and 40 repeats, and one should be careful when interpreting these results.     

Adenylate Uptake by Proplastids from Cultured Cells of Tobacco {Nicotiana tabacum L. cv. BY2) Indicates That an Adenylate Translocator is Present in All Types of Plastid

The presence of an adenylate translocator in the envelope membranesof proplastids isolated from the cultured cells of tobacco (Nicotianatabacum L. cv. BY2) was examined by means of transport experimentsusing the silicone oil filtering centrifugation technique. Itwas observed that proplastids can import [³H]ATP, [³H]ADP, [³H]AMPand less specifically ADP-[¹⁴C]Glc which can eventually be usedfor starch biosynthesis. The effects of specific inhibitorsof the mitochondrial adenylate translocator, i.e. atractyloside,bongkrekic acid and carboxyatractyloside were tested. Similarto the case of amyloplasts isolated from the cultured cellsof sycamore and chloroplasts isolated from spinach leaves, onlyATP and ADP-Glc uptake were shown to be partially inhibitedby carboxyatractyloside. On the other hand, neither atractylosidenor bongkrekic acid exerted a significant inhibitory effecton adenylate uptake. (Received August 8, 1992; Accepted November 26, 1992)     

City refuse compost as a phosphorus source to overcome the P-fixation capacity of sesquioxide-rich soils

In sesquioxide-rich soils of tropical and subtropical areas and volcanic-ash soils with high levels of active Al(Fe), large amounts of phosphate fertilizers are needed to overcome their high P-fixation capacity (quenching strategy). A greenhouse pot experiment has been used to evaluate the effectiveness of city refuse compost (CRC) as a P-source for these variable-charge soils, compared to inorganic P. Mature CRC and K₂HPO₄ were applied at rates equivalent to 125, 250, 375, 500 and 625 kg P ha^–1 to a ferrallitic soils from Tenerife Island (Andeptic Paleudult) with a high content in active Al+Fe (4.82%) and a high P-fixation capacity (87%). Perennial ryegrass (Lolium perenne L.) was grown in pots and plants were harvested at regular intervals after seedling emergence. CRC increases plant P concentration and soil labile-P proportional to the applied rate. The best results were obtained from a compost application of 30 t ha^–1 equivalent-rate, after a residence time of at least three months. An important residual effect in the supply capacity of P in relation to the phosphate fertilizer was also observed. The relative agronomic effectiveness (RAE) in comparison to K₂HPO₄ was 66% after 6 months, considering P uptake + soil labile-P. The soil P-fixation capacity was significantly reduced from a compost application of 40 t ha^–1 equivalent-rate. Competition in adsorption between organic ligands and phosphate, in combination with net mineralization of organic P in compost, might account for the high RAE value obtained. The main conclusion is that the city refuse compost could be a suitable P-amendment for resquioxic soils due to its high RAE, and the residual effect on P-supply. ei]H. Lambers     

Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Induction of apoptosis and c-myc in L1210 lymphocytic leukemia cells by adenosine

High concentrations of adenosine (Ado), when added to L1210 lymphocytic leukemia cells, resulted in apoptosis or programmed cell death. The apoptotic process was accompanied by distinct morphological changes including chromatin condensation and blebbing of plasma membranes. Extensive DNA fragmentation was correlated with Ado concentrations. Furthermore, apoptosis in these cells was preceded by an early but transient expression of c-myc proto-oncogene, and was not influenced by homocysteine thiolactone added to the cells. Since severe combined immunodeficiency (SCID) is associated with a deficiency of adenosine deaminase, leading to defects in both cellular and humoral immunity, Ado-induced apoptosis may thus be a contributing factor in the pathology of SCID.