Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components.

Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine beta-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut.

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.

Regulation of gene expression by action potentials: dependence on complexity in cellular information processing.

Nervous system development and plasticity are regulated by neural impulse activity, but it is not well understood how the pattern of action potential firing could regulate the expression of genes responsible for long-term adaptive responses in the nervous system. Studies on mouse sensory neurons in cell cultures equipped with stimulating electrodes show that specific genes can be regulated by different patterns of action potentials, and that the temporal dynamics of intracellular signalling cascades are critical in decoding and integrating information contained in the pattern of neural impulse activity. Functional consequences include effects on neurite outgrowth, cell adhesion, synaptic plasticity and axon-glial interactions. Signalling pathways involving Ca2+, CaM KII, MAPK and CREB are particularly important in coupling action potential firing to the transcriptional regulation of both neurons and glia, and in the conversion of short-term to long-term memory. Action potentials activate multiple convergent and divergent pathways, and the complex network properties of intracellular signalling and transcriptional regulatory mechanisms contribute to spike frequency decoding.

Purification and characterization of a human bradykinin binding protein from inflammatory cells.

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

Regulation of gene expression during meiosis in Saccharomyces cerevisiae: SPR3 is controlled by both ABFI and a new sporulation control element.

The SPR3 gene encodes a sporulation-specific homolog of the yeast Cdc3/10/11/12 family of bud neck filament proteins. It is expressed specifically during meiosis and sporulation in Saccharomyces cerevisiae. Analysis of the sporulation-specific regulation of SPR3 has shown that it is strongly activated under sporulating conditions but shows low levels of expression under nonsporulating conditions. A palindromic sequence located near the TATA box is essential to the developmental regulation of this gene and is the only element directly activating SPR3 at the right time during sporulation. Within the palindrome is a 9-bp sequence, gNCRCAAA(A/T) (midsporulation element [MSE]), found in the known control regions of three other sporulation genes. A previously identified ABFI element is also needed for activation. The MSE has been shown to activate a heterologous promoter (CYC1) in a sporulation-specific manner. Related sequences, including an association of MSE and ABFI elements, have been found upstream of other genes activated during the middle stage of S. cerevisiae sporulation. One group of these may be involved in spore coat formation or maturation.

The SPR3 gene encodes a sporulation-specific homologue of the yeast CDC3/10/11/12 family of bud neck microfilaments and is regulated by ABFI.

The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdc11p and Cdc12p), Candida albicans proteins that accumulate in the hyphal phase (CaCdc3p and CaCdc10p), mouse brain-specific (H5p) and lymphocyte (Diff6p) proteins, Drosophila melanogaster (Dm) protein Pnutp (which is localized to the cleavage furrow of dividing cells), a Diff6p homologue (DmDiff6p), and the Sc septin protein (Sep1hp), a homologue of the 10-nm filament proteins of Sc. One strongly conserved region contains a potential ATP-GTP-binding domain. Primer extension analysis revealed six major transcription start points (tsp) beginning at -142 relative to the ATG start codon. The sequence immediately upstream from the tsp contains consensus binding sites for the HAP2/3/4 and ABFI transcription factors, a T-rich sequence and two putative novel elements for mid to late sporulation, termed SPR3 and PAL. Electrophoretic mobility shift assay (EMSA) and footprint analyses demonstrated that the ABFI protein binds to a region containing the putative ABFI site in vitro, and site-directed mutagenesis showed that the ABFI motif is essential for expression of SPR3 at the appropriate stage in sporulating cells.

The Drosophila melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans.

Mutations at the flightless-I locus (fliI) of Drosophila melanogaster cause flightlessness or, when severe, incomplete cellularization during early embryogenesis, with subsequent abnormalities in mesoderm invagination and in gastrulation. After chromosome walking, deficiency mapping, and transgenic analysis, we have isolated and characterized flightless-I cDNAs, enabling prediction of the complete amino acid sequence of the 1256-residue protein. Data base searches revealed a homologous gene in Caenorhabditis elegans, and we have isolated and characterized corresponding cDNAs. By using the polymerase chain reaction with nested sets of degenerate oligonucleotide primers based on conserved regions of the C. elegans and D. melanogaster proteins, we have cloned a homologous human cDNA. The predicted C. elegans and human proteins are, respectively, 49% and 58% identical to the D. melanogaster protein. The predicted proteins have significant sequence similarity to the actin-binding protein gelsolin and related proteins and, in addition, have an N-terminal domain consisting of a repetitive amphipathic leucine-rich motif. This repeat is found in D. melanogaster, Saccharomyces cerevisiae, and mammalian proteins known to be involved in cell adhesion and in binding to other proteins. The structure of the maternally expressed flightless-I protein suggests that it may play a key role in embryonic cellularization by interacting with both the cytoskeleton and other cellular components. The presence of a highly conserved homologue in nematodes, flies, and humans is indicative of a fundamental role for this protein in many metazoans.