Diastolic dysfunction in human cardiac allografts is related with reduced SERCA2a gene expression.

Previous studies demonstrated that impaired left ventricular (LV) relaxation in cardiac allografts limits exercise tolerance post-transplant despite preserved systolic ejection fraction (EF). This study tested in human cardiac allografts whether the isovolumic relaxation time (IVRT), which provides the basis for most of diastolic LV filling, relates with gene expression of regulatory proteins of calcium homeostasis or cardiac matrix proteins. Gene expression was studied in 31 heart transplant recipients (25 male, 6 female) 13-83 months post-transplant with LVEF >50%, LV end-diastolic pressure <20 mmHg, normal LV mass index and without allograft rejection or significant cardiac pathology. IVRT related with the other diastolic parameters e-wave velocity (r = -0.46; p = 0.01), e/a-wave ratio (r = -0.5; p < 0.01) but not with heart frequency (r = -0.16; p = 0.4). No relation of IVRT was observed for immunosuppression, mean rejection grade or other medication. IVRT was not related with gene expression of desmin, collagen I, phospholamban, the Na+-Ca2+ exchanger, the ryanodine receptor or interstitial fibrosis but correlated inversely with SERCA2a (r = -0.48; p = 0.02). Prolonged IVRT is associated with decreased SERCA2a expression in cardiac allografts without significant other pathology. Similar observations in non-transplanted patients with diastolic failure suggest that decreased SERCA2a expression is an important common pathomechanism.

A central neural circuit for experience-independent olfactory and courtship behavior in Drosophila melanogaster.

We have studied the function of the major central olfactory pathway in fruit flies. Key elements of this pathway, the projection neurons (PNs), connect the antennal lobes with the lateral protocerebrum both directly and indirectly, the latter via the mushroom bodies (MBs). Transgenic expression of tetanus toxin in the majority of PNs and few MB neurons leads to defects in odor detection and male courtship. Considering behavioral data from flies lacking MBs, our results argue that the direct PN-to-lateral protocerebrum pathway is necessary and sufficient to process these experience-independent behaviors. Moreover, the involvement of an olfactory pathway in male courtship suggests a role of volatile attractive female pheromones in Drosophila.

Smell and taste perception in Drosophila melanogaster larva: toxin expression studies in chemosensory neurons.

GAL4-driven targeted expression of tetanus toxin light chain (UAS-TeTxLC) in a subset of chemosensory neurons of the larval antennomaxillary complex (AMC) and pharynx causes abnormal chemosensory behavior in Drosophila melanogaster. Consistent with strongest staining in the dorsal organ (DO), the presumed olfactory organ of the AMC, tetanus toxin-expressing larvae subjected to an olfactory preference assay show anosmic behavior to most volatile substances tested. Furthermore, we observed reduced responses to sodium chloride, fructose, and sucrose in gustatory plate assays. Surprisingly, the entire subset of labeled sensory neurons from the terminal (maxillary) organ (TO) of the AMC was found to project via the antennal nerve to the larval antennal lobe region. The maxillary nerve remained completely unstained. Hence, a subset of neurons from the TO builds an anatomical entity with projections from the DO. Our results suggest that the AMC contains both olfactory and gustatory sensilla, and that the DO is the main olfactory organ in larvae.

Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons.

Hydroxyurea (HU) treatment of early first instar larvae in Drosophila was previously shown to ablate a single dividing lateral neuroblast (LNb) in the brain. Early larval HU application to P[GAL4] strains that label specific neuron types enabled us to identify the origins of the two major classes of interneurons in the olfactory system. HU treatment resulted in the loss of antennal lobe local interneurons and of a subset of relay interneurons (RI), elements usually projecting to the calyx and the lateral protocerebrum (LPR). Other RI were resistant to HU and still projected to the LPR. However, they formed no collaterals in the calyx region (which was also ablated), suggesting that their survival does not depend on targets in the calyx. Hence, the ablated interneurons were derived from the LNb, whereas the HU-resistant elements originated from neuroblasts which begin to divide later in larval life. Developmental GAL4 expression patterns suggested that differentiated RI are present at the larval stage already and may be retained through metamorphosis.

The Drosophila Eip78C gene is not vital but has a role in regulating chromosome puffs.

We have generated a number of chromosomal aberrations that disrupt the early-late ecdysone-induced 78C puff gene (Eip78C, ecdysone-induced protein, FlyBase name for the E78 gene of Stone and Thummel 1993), which encodes the two members of the nuclear hormone receptor superfamily Eip78C-A and Eip78C-B. The aberrations include deletions of the ligand-binding/dimerization domain of both, inversions that split Eip78C-A but retain residual Eip78C-B expression, and a small deletion specific for Eip78C-B. We find that wild-type Eip78C functions are completely dispensable for normal development under laboratory conditions. However, we show that Eip78C-B is required for the maximal puffing activity of a subset of late puffs (63E and 82F) since these puffs are reduced in size in Eip78C-B mutant backgrounds. Paradoxically the same late puffs are reduced, as well as at least one other, when the Eip78C-B cDNA is overexpressed from a heat shock promoter. These data indicate either that Eip78C function is redundant or that it plays a subtle modulating role in the regulation of chromosome puffing.

Invertebrate synapsins: a single gene codes for several isoforms in Drosophila.

Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.

The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals.

Proteins expressed specifically in neurons and transported to synaptic terminals are likely to constitute important molecular elements of nervous system function. In an effort to characterize synapse-associated proteins (SAPs) of Drosophila, we have isolated from a hybridoma library several monoclonal antibodies (MABs) that selectively stain synaptic terminals in immunohistochemical preparations. MAB nc46 binds to most but not all synaptic terminals of the Drosophila nervous system, it also recognizes a protein with homologous distribution in other dipteran flies and binds to large parts of fish CNS. In Western blots the antibody labels a Drosophila brain protein of 47 kDa and cross-reacts with brain proteins from several species including insects, fish, mouse and man. From these data we conclude that the corresponding gene has been conserved in evolution at least among diptera. Using MAB nc46 and expression cloning we have identified the 'sap47' gene coding for the 'synapse-associated protein of 47 kDa' of Drosophila melanogaster. Sequence analysis of genomic and cDNA clones reveals the intron-exon structure of the gene and characterizes the complete open reading frames of two alternatively spliced transcripts. The sap47 gene is located in 89A8-B3 on chromosome 3R and codes for two almost identical inferred polypeptides of 347 and 351 amino acids with no significant sequence homology to known proteins.

An invertebrate calcium-binding protein of the calbindin subfamily: protein structure, genomic organization, and expression pattern of the calbindin-32 gene of Drosophila.

Antisera against vertebrate calcium-binding proteins cross-react with Drosophila nervous and muscle tissue. We have used an antiserum against carp parvalbumin to isolate from a Drosophila head cDNA library immunopositive expression clones. Tissue in situ hybridization identified a clone that labeled specific neurons and muscles similar to the parvalbumin-like immunohistochemical staining pattern. Five independent cDNAs derive from an mRNA whose open reading frame codes for a 310 amino acid polypeptide. Sequence analysis identifies six EF-hand calcium-binding domains and reveals 42% and 37% homology to chicken calretinin and calbindin D-28k, respectively. Since the positions of 9 out of 10 introns within the ORF are conserved from the Drosophila gene to both vertebrate genes, we conclude that we have identified the first invertebrate member of the calbindin sub-family of calcium-binding protein genes of the EF-hand homolog family. The calbindin-32 gene (cbn) maps to 53E on the second chromosome. It is expressed through most of ontogenesis with a selective distribution in the nervous system and in a few small adult thoracic muscles. The cloning of a Drosophila homolog to vertebrate neuronal Ca(2+)-binding proteins opens new routes to study the so far largely elusive function of these brain molecules.

A cysteine-string protein is expressed in retina and brain of Drosophila.

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.