Uptake of γ-Aminobutyric Acid by Brain Tissue Preparations: A Reevaluation

The kinetic constants Km and Vmax for the uptake of gamma-aminobutyric acid (GABA) by various preparations from rat cerebral cortex were determined by means of Eadie-Hofstee plots and computer analysis. The Km values were much greater in 0.1-mm slices than in synaptosomal preparations, and the Km value increased further with the thickness of the slices. The apparent high Km values in slices were probably due to depletion of the GABA concentration in the extracellular fluid as the exogenous GABA ran the gauntlet of competing uptake sites on its way to sites deep within the slice, thereby bringing about a requirement for higher GABA concentrations in the incubation medium in order to maintain the internal GABA levels at the "Km level." Evidence was obtained for three GABA uptake systems with Km values (in synaptosomes) of 1.1 microM, 43 microM, and 3.9 mM, respectively. In contrast, only two uptake systems for D-aspartate were detected, with Km values of 1.8 microM and 1.8 mM, respectively. The implications of the findings in the study with respect to previous data in the literature are discussed.     

Identification of Multiple Binding Sites for [3H]5-Hydroxytryptamine in the Rat CNS

Recent studies indicate that there may be multiple subtypes of [3H]5-hydroxytryptamine ([3H]5-HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5-HT (2-3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high- and low-affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5-HT binding to the mianserin-sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]5-HT, one blocked by low concentrations of spiperone (5-HT1A), one blocked by low concentrations of mianserin (5-HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5-HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5-HT1A sites, whereas the striatum contained mainly 5-HT1B and 5-HT1C sites. Selective degeneration of 5-HT-containing nerve terminals induced by the neurotoxin 5,7-dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5-HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5-HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.     

Differentiation among populations of Sedum wrightii (Crassulaceae) in response to limited water availability: water relations,CO2 assimilation,growth and survivorship

Summary Sedum wrightii is one of only a few species in the Crassulaceae for which there is evidence for a high degree of variability in the ratio of daytime to nighttime CO₂ assimilation. There are both environmental and genetic components to this variability. S. wrightii grows over a wide altitudinal gradient. The purpose of this study was to compare low, intermediate, and high altitude populations with respect to the degree of CAM expression and the capability to tolerate limited water availability. We utilized clonallyreplicated genotypes of plants from each population in common environment greenhouse experiments. Genetic differences among the populations were found in long-term water use efficiency, in 24 hour CO₂ exchange patterns, in biomass ¹³C values, in carbon allocation, and in water status and ultimately survival during prolonged drought. The differences among the populations appear to be closely related to differences in the native habitats. The low altitude, desert plants had the greatest ability to grow and survive under conditions of limited water availability and appear to have the greatest shift to nighttime CO₂ uptake during periods without water, while the high altitude plants had the poorest performance under these conditions and appear to shut down net carbon uptake when severely water limited.     

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.     

Atriopeptin II relaxes and elevates cGMP in bovine pulmonary artery but not vein

Atriopeptin II, a 23-amino acid synthetic peptide fragment of atrial natriuretic factor, caused an endothelium-independent relaxation of isolated precontracted rings of bovine intrapulmonary artery that was accompanied by the concomitant accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) but not adenosine 3',5'-cyclic monophosphate. In contrast, rings of intrapulmonary vein were unaffected by atriopeptin II whether or not endothelium was present. Whereas methylene blue, an inhibitor of soluble guanylate cyclase, abolishes endothelium-dependent and independent arterial relaxation and cGMP accumulation in response to acetylcholine and glyceryl trinitrate, respectively, methylene blue failed to alter these responses to atriopeptin II. Similarly, the effects of atriopeptin II were unaltered by propranolol, indomethacin, or atropine. These results indicate that relaxation elicited by atriopeptin II may be selective for arterial smooth muscle receptors, does not require endothelial cells, and does not involve the soluble form of guanylate cyclase, although cGMP accumulation is stimulated.     

Stimulatory effect of 1 alpha, 25-dihydroxyvitamin D3 on the formation of skin tumors in mice treated chronically with 7,12-dimethylbenz[a]anthracene

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) and its 24,24-difluoro analog on the formation of skin tumors in mice was evaluated in a complete carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as the carcinogen. Twice weekly topical application of 0.25-0.50 nmol of 1 alpha, 25-(OH)2D3 or 0.05-0.10 nmol of the difluoro analog of 1 alpha, 25-(OH)2D3 1 hour prior to treatment with 50 nmol DMBA stimulated tumor formation several fold compared to animals receiving DMBA alone. Topical application of 0.50 nmol of 1 alpha, 25-(OH)2D3 24 hours after treatment with DMBA, or half of this dose of the vitamin D3 metabolite, applied 1 hour before and 24 hours after treatment with DMBA, also stimulated tumor formation several fold. These results are in marked contrast to the potent inhibitory effect of 1 alpha, 25-(OH)2D3 and its difluoro analog on the formation of skin tumors in mice promoted by 12-O-tetradecanoylphorbol-13-acetate.     

Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum

We proposed that Dictyostelium discoideum contains two linked pools of mature alpha-mannosidase (Wood, L., R. N. Pannell, and A. Kaplan, 1983, J. Biol. Chem., 258:9426-9430). To obtain physical evidence for these pools, cells were pulse-labeled with [35S]methionine, homogenized, and subjected to Percoll gradient centrifugation. After immune precipitation of alpha-mannosidase, its polypeptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by fluorography. After a 30-min pulse with [35S]methionine, the precursor and small amounts of cleaved enzyme were detected in a low density fraction (1.04 g/ml). Subsequently, cleaved enzyme was transferred to higher density fractions (1.05 and 1.07 g/ml) that were enriched in lysosomal enzymes. The half time for formation of the 1.07 g/ml pool was approximately 45 min, whereas formation of the 1.05 g/ml pool was not detected until 1.5 h after the pulse. The transfer of mature forms out of the 1.04 g/ml pool was inhibited by monensin (3.5 microM). Thus, alpha-mannosidase precursor appears to be cleaved in a prelysosomal organelle. The data also indicate that starving cells secrete precursor directly from this organelle to the extracellular space, whereas cleaved forms are first transferred into lysosomes before they are secreted. Furthermore, 2 h after starvation, the secretion of mature forms ceases even though both transit of mature forms between the two pools and secretion of precursor continues. From this we inferred that the cessation of secretion of mature forms is due to a halt in fusion of lysosomes with the plasma membrane and that precursor follows a different route to the plasma membrane.     

The four biochemically distinct species of human interleukin 1 all exhibit similar biologic activities

The supernatants of human monocytes incubated with endotoxin are able to stimulate the proliferation of murine thymocytes in the presence of PHA. This is known as LAF (lymphocyte activating factor) activity and is a characteristic activity of interleukin 1 (IL 1). The LAF activity can be resolved into four major fractions: a 15,000 dalton (pI 7), a 15,000 (pI 5.5), a 35,000 (pI 7), and a 35,000 (pI 5.5) fraction. To determine whether these four fractions shared the other biologic activities ascribed to IL 1, they were compared in a series of bioassays. When standardized with respect to their LAF activities, the four fractions did not differ significantly as mitogens for murine thymocytes, inducers of IL 2, murine or human B cell activators, human chondrocyte or synoviocyte stimulants, or inducers of acute phase proteins in vivo. On the other hand, the samples differed markedly as stimulators of porcine synoviocytes, with the 15,000 dalton (pI 5.5) fraction being the only strongly active fraction. These results are consistent with the hypothesis that all four LAF could be products of a single gene, although the porcine receptor may be able to distinguish between them. If this is the case, all four fractions can properly be termed IL 1.     

Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-leu-8

Previous studies have shown that monoclonal anti-Leu-8 antibody identifies functionally distinct subpopulations within both the Leu-2 (T8+) and Leu-3 (T4+) lineages of human T lymphocytes. We now report in detail on the tissue distribution of the Leu-8 antigen and on extensive functional studies of T cells subsets distinguished by their expression or lack of expression of this marker. Leu-8 is present on a wide variety of hematologic cells, including granulocytes, T and B lymphocytes, monocytes, and null or NK cells. Within lymph nodes and tonsils, Leu-8 is absent from both B and T cells within germinal centers but is present on nearly all paracortical lymphocytes. Leu-8 is present on most but not all EBV-transformed B cell lines, reflecting its presence on a subset of normal peripheral blood B cells. None of six malignant T cell lines tested were Leu-8+, whereas most circulating T cells are Leu-8+. Although standard immunoprecipitation techniques failed to demonstrate any specific bands on SDS polyacrylamide gels, the antigenic determinant recognized by anti-Leu-8 is protein or protein-associated, because brief treatment of target cells with pronase abrogated binding of anti-Leu-8. Both Leu-3+8+ and Leu-3+8- cells proliferated in response to several soluble antigens and to autologous and allogeneic non-T cells. Nonetheless, nearly all of the helper T cells for PWM- and AMLR-induced PFC were contained within the Leu3+8- subset. Optimal suppression of the PWM-induced PFC response required both Leu-2+8+ and Leu-2+8- cells, and irradiation of either subset with 3000 R abrogated the capacity of the recombined subsets to effect suppression. In contrast to help for B cell differentiation, both Leu-3+8+ and Leu-3+8- cells were capable of amplifying the development of allospecific T killer cells; precursor and effector T killer cells could be found within both Leu-2+8+ and Leu-2+8- subpopulations. The correlation between Leu-8 phenotype and selected immune functions of T cells (and B cells; see companion paper) indicates that anti-Leu-8 distinguishes important immunoregulatory T and B lymphocyte subsets in man.     

Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature.

In April 1983, an influenza virus of low virulence appeared in chickens in Pennsylvania. Subsequently, in October 1983, the virus became virulent and caused high mortality in poultry. The causative agent has been identified as an influenza virus of the H5N2 serotype. The hemagglutinin is antigenically closely related to tern/South Africa/61 (H5N3) and the neuraminidase is similar to that from human H2N2 strains (e.g., A/Japan/305/57) and from some avian influenza virus strains (e.g., A/turkey/Mass/66 [H6N2]). Comparison of the genome RNAs of chicken/Penn with other influenza virus isolates by RNA-RNA hybridization indicated that all of the genes of this virus were closely related to those of various other influenza virus isolates from wild birds. Chickens infected with the virulent strain shed high concentrations of virus in their feces (10(7) 50% egg infective dose per g), and the virus was isolated from the albumin and yolk of eggs layed just before death. Virus was also isolated from house flies in chicken houses. Serological and virological studies showed that humans are not susceptible to infection with the virus, but can serve as short-term mechanical carriers. Analysis of the RNA of the viruses isolated in April and October by gel migration and RNA-RNA hybridization suggested that these strains were very closely related. Oligonucleotide mapping of the individual genes of virulent and avirulent strains showed a limited number of changes in the genome RNAs, but no consistent differences between the virulent and avirulent strains that could be correlated with pathogenicity were found. Polyacrylamide gel analysis of the early (avirulent) isolates demonstrated the presence of low-molecular-weight RNA bands which is indicative of defective-interfering particles. These RNAs were not present in the virulent isolates. Experimental infection of chickens with mixtures of the avirulent and virulent strains demonstrated that the avirulent virus interferes with the pathogenicity of the virulent virus. The results suggest that the original avirulent virus was probably derived from influenza viruses from wild birds and that the virulent strain was derived from the avirulent strain by selective adaptation rather than by recombination or the introduction of a new virus into the population. This adaptation may have involved the loss of defective RNAs, as well as mutations, and thus provides a possible model for a role of defective-interfering particles in nature.