A serotonergic system in the brain of the Antarctic fish, Trematomus bernacchii

The immunohistochemical distribution of serotonin-containing nerve fibres and cells has been described in the brain of the Antarctic fish, Trematomus bernacchii. The largest serotonergic system was associated with the diencephalic and rhombencephalic ventricles. In particular, serotonin-positive cells have been found in the lateral recess and neuropile zone of the diencephalic ventricle, where we have identified the serotonergic portion of the paraventricular organ. Numerous serotonin cells were localized in the dorsal nucleus of the raphe, the dorsal tegmental nucleus and the central gray. Two large cell groups, arranged in a pair of well-defined columns and connecting the central gray with the dorsal reticular formation, were immunostained in the region of the trigeminal nuclei. In addition, few positive cells have been found in the preoptic area and the cerebellar valvula, and few serotonergic nerve fibres, probably belonging to the lateral lemniscus, have been identified. The distribution of serotonin elements in the brain of T. bernacchii has been compared with that described in other fish, where it showed some modifications in the immunoreactive pattern. Finally, the lack of a serotonergic system at the level of the reticular superior formation has been reported; however, it was not possible to rule out a phylogenetic or environmental explanation.     

Neuronal Basis of Innate Olfactory Attraction to Ethanol in Drosophila

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine – the invertebrate analogue of noradrenaline – in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.     

Carbohydrate moieties of rat MHC class I antigens

The rat major histocompatibility complex class I antigens RT1.A^u and RT1.E^u from the u haplotype and RT1.Aⁿ from the n haplotype were labeled with ¹⁴C-asparagine or with ³H-fucose, mannose, galactose, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed complete removal of radioactivity from the sugar-labeled antigen heavy chains by digestion with glycopeptidase F, an enzyme that removes N-linked glycans completely. High performance liquid chromatography analysis of the tryptic digests of the mixed sugar-labeled and asparagine-labeled antigens demonstrated that all the sugar-labeled peptides were coincident with asparagine-labeled peptides. The Aⁿ antigen showed three glycopeptides, each of which had different amounts of sugar radioactivity. The antigens A^u and E^u showed two glycopeptides with different amounts of radioactivity but at identical positions in the two antigens. Antigen E^u had an additional glycopeptide with a lower amount of radioactivity. The positions of the glycopeptides from the A^u and E^u antigens were different from those of the Aⁿ antigen. The peptide profiles of the ¹⁴C-asparagine-labeled A^u and E^u antigens demonstrated distinct differences between the molecules. The results of this study show that: (a) all the glycans on rat class I antigens are N-linked, as they are on H-2 and HLA class I antigens; (b) there are compositional differences among the glycans in each of the three antigens; (c) the glycosylation pattern of the rat class I antigens is similar to that of the mouse class I antigens, which contain two or three glycans, in contrast to that of the human class I antigens, which contain only one glycan; and (d) the antigens A^u and E^u from the same haplotype are more closely related to each other than they are to the Aⁿ antigen.     

Development of a yeast system to assay mutational specificity

We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing. The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity. Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain. Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing. We have used this system to examine mutations arising spontaneously in the SUP4-o gene. The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division. Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid. Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation. Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size. These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.     

Gas exchange studies in two Portuguese grapevine cultivars

Gas exchange characteristics of leaves of Vitis vinifera L. cvs Tinta Amarela and Periquita, two grapevine cultivars grown in distinct climatic regions of Portugal, were studied under natural and controlled conditions. Daily time courses of gas exchange were measured on both a hot, sunny day and a cooler, partly cloudy day. Responses of net photosynthesis to irradiance and internal partial pressure of CO₂, were also obtained. A strong correlation between net photosynthesis (P_N) and leaf conductance (g_s) was found during the diurnal time courses of gas exchange, as well as a relatively constant internal partial pressure of CO₂ (P_i), even under non-steady-state conditions. On the cloudless day, both P_N and g_s were lower in the afternoon than in the morning, despite similar conditions of leaf temperature, air to leaf water vapor deficit and irradiance. The response curves of net photosynthesis to internal CO₂ showed linearity up to p_i values of 50 Pa, possibly indicating a substantial excess of photosynthetic capacity. When measured at low partial pressures of O₂ (1 kPa), P_N became inhibited at high CO₂ levels. Inhibition of P_N at high CO₂ was absent under normal levels of O₂ (21 kPa). Significant differences in gas exchange characteristics were found between the two cultivars, with T. Amarela having higher rates under similar measurement conditions. In particular, the superior performance of T. Amarela at high temperatures may represent adaptation to the warmer conditions at its place of origin.     

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.     

TheRT1.G locus in the rat encodes a Qa/TL-like antigen

A new antigenic system in the rat homologous to theQa/TL antigen system in the mouse has been characterized. It was detected by antibodies raised in donor-recipient combinations that were matched for theRT1. A, B, D, E loci in the major histocompatibility complex (MHC): (R11×BN)F₁ anti-BN.1L(LEW), (R18×BN)F₁ anti-BN.1L, and BN.1LV1(F344) anti-BN.1L. Absorption analyses using these antisera and a variety of inbred, congenic and recombinant strains identified three alleles,RT1.G ^a ,G ^b ,G ^c , of whichG ^c is a null allele. The strain distribution of these alleles was determined, using 37 strains of rats representative of all of the prototypic haplotypes and a number of congenic and recombinant strains. The use of the congenic and recombinant strains showed that theRT1.G locus was linked to the MHC and that the most probable gene order wasA-E-G. Testcross analysis showed that the map distance betweenA andG was 1.4 cM(4/285 recombinants). The RT1.G antigen has a heavy chain ofM _r 46 000 and is present on both T and B cells.     

Transport of l(-)malic acid and other dicarboxylic acids in the yeast Candida sphaerica

Summary dl-Malic acid grown cells of Candida sphaerica (anamorph of Kluyveromyces marxianus) formed a saturable transport system that mediated accumulative transport of l(-)malic acid with the following kinetic parameters at pH 5.0: V _max, 0.44 nmol l(-)malate·s^-1 per milligram dry weight; K _m ,0.1 mM l(-)malate. Initial uptake of the acid was accompanied by disappearance of extracellular protons, the rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. Variation with extracellular pH of the K _m values, calculated either as the concentrations of anions or of undissociated acid, pointed to anions as the transported form. Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonylcyanide-M-chlorophenyl-hydrazone (CCCP). These results suggested that the transport system was a dicarboxylate-proton symporter. The system was inducible and was subject to glucose repression. Succinic, fumaric, -ketoglutaric, oxaloacetic and d-malic acid, but not maleic, malonic, oxalic nor l(+)-tartaric acid, apparently used the same transport system since they acted as competitive inhibitors of l(-)malic acid transport and induced proton movements that followed Michaelis-Menten kinetics. Experiments with glucose-repressed cells showed that undissociated dicarboxylic acid (measured with labelled succinic acid) entered the cells slowly by simple diffusion. The permeability of the cells for undissociated acid increased exponentially with pH, the diffusion constant increasing 100-fold between pH 3.5 and 6.0.