Connectivity changes in an isolated molluscan ganglion during in vivo culture

The stability of neuronal connections in the isolated buccal ganglia of Helisoma trivolvis was examined during in vivo culture for periods up to one month. After 4--8 days the characteristic IPSP input to protractor motoneurons (PMNs) was either abolished or reduced in efficacy. This is apparently due to reduced efficacy of chemical synapses, since the input resistance and resting potential of the motoneurons is unchanged and a fraction of spike-evoked IPSPs from premotor neurons (cyberchrons) onto PMNs was absent. PMNs lacking IPSP input nevertheless exhibit vigorous cyclical bursts of action potentials driven by electrical EPSPs. The IPSP of PMNs showed partial or full restoration after 14--32 days of culture despite the lack of reinnervation of normal targets. Existing electrical synapses were apparently more stable during culture, but electrical connections between cyberchrons and PMNs were strengthened. Probably because of the reinforcement of these electrical synapses, regenerative cycles of activity in both cyberchrons and PMNs may often be initiated by brief stimulating of a single PMN in cultured ganglia. This is in marked contrast to normal ganglia in which PMNs possess a limited ability to generate such activity. It is concluded that isolation of the buccal ganglia results in a predictable, functional alteration of its neuronal circuitry. Such a perturbation of connectivity indicates that a significant degree of plasticity can be exhibited by adult molluscan neurons.     

Structure and Function of Human Xylulokinase,an Enzyme with Important Roles in Carbohydrate Metabolism

d-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as K_m(Xu) = 24 ± 3 μm and k_cat = 35 ± 5 s⁻¹. Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease.     

Sensorin-A immunocytochemistry reveals putative mechanosensory neurons inLymnaea CNS

The pond snailLymnaea stagnalis is a useful model system for studying the neural basis of behaviour but the mechanosensory inputs that impact on behaviours such as respiration, locomotion, reproduction and feeding are not known. InAplysia, the peptide sensorin-A appears to be specific to a class of central mechanosensory neurons. We show that in theLymnaea central nervous system sensorin-A immunocytochemistry reveals a discrete pattern of staining involving well over 100 neurons. Identifiable sensorin positive clusters of neurons are located in the buccal and cerebral ganglia, and a single large neuron is immunopositive in each pedal ganglion. These putative mechanosensory neurons are not in the same locations as previously identified motoneurons, interneurons or neurosecretory cells. As would be expected for a mechanoafferent, sensorin positive fibres were found in nerve tracts innervating the body wall. This study lays the foundation for future electrophysiological and behavioural analysis of these putative mechanosensory neurons.     

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.      

Role of interflash intervals in a firefly courtship (Photinus macdermotti)

The flash code of Photinus macdermotti fireflies has been measured over a temperature range of 16 to 25°C. The code changes in characteristics fashion during different phases of firefly courtship. Males produce rhythmic patrolling flashes while flying, and when answered, shift to courtship flash pairs of significantly shorter interval. Females will answer some consecutive patrolling flashes and normally respond after the second flash of each male courtship pair. A possible behavioural role for the shifting of male patrolling and courtship flash intervals and for the female's response patterns is proposed.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.