Isolation, pharmacology and gene organization of KPSFVRFamide: a neuropeptide from Caenorhabditis elegans

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH]+) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, >/=0.1 microM) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, >/=10 nM).     

Large scale cultivation of a free-living nematode (Caenorhabditis elegans)

A method is presented for the large scale cultivation of the free-living nematode Caenorhabditis elegans, using continuous aeration and agitation in glass ware (stirrer flasks) developed for the continuous culture of suspended cells. With this technique, populations up to 10(9) nematodes may be obtained in a 10 1 culture in less than 6 weeks with an inoculum of some 50 worms. Costs can be reduced by using an inexpensive yeast extract, available from the food industry.     

Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: evidence for the presence of more than one FMRFamide-related peptide-encoding gene

Numerous FMRFamide-related peptides (FaRPs) have been isolated and sequenced from extracts of free-living and parasitic nematodes. The most abundant FaRP identified in ethanolic/methanolic extracts of the parasitic forms, Ascaris suum and Haemonchus contortus and from the free-living nematode, Panagrellus redivivus, was KHEYLRFamide (AF2). Analysis of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris identified a series of related FaRPs which did not include AF2. An acid-ethanol extract of Caenorhabditis elegans was screened radioimmunometrically for the presence of FaRPs using a C-terminally directed FaRP antiserum. Approximately 300 pmols of the most abundant immunoreactive peptide was purified to homogeneity and 30 pmols was subjected to Edman degradation analysis and gas-phase sequencing. The unequivocal primary structure of the heptapeptide, Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a time-of-flight mass spectrometer and was found to be 920 (MH+)+, which was consistent with the theoretical mass of C-terminally amidated AF2. These results indicate that C. elegans possesses more than one FaRP gene.     

Isolation of actin-associated proteins from Caenorhabditis elegans oocytes and their localization in the early embryo

The actin cytoskeleton plays an important, but poorly understood, role in the development of multicellular organisms. To help illuminate this role, we used actin filament affinity chromatography to isolate actin binding proteins from large quantities of Caenorhabditis elegans oocytes. To examine how these proteins might be involved in early development, we prepared antibodies against some of them and determined their distribution in fixed embryos. Three of these proteins co-localize with different subsets of the embryonic actin cytoskeleton. One co-localizes with actin to all cell cortices. The second oscillates between the nucleus and cortex in a cell-cycle-dependent manner. The third is asymmetrically enriched at the anterior cortex of one-cell embryos, showing a temporal and spatial localization suggestive of a function in generating developmental asymmetry. We conclude that biochemistry is a feasible and useful approach in the study of early C. elegans development, and that the embryonic actin cytoskeleton is regulated in a complex fashion in order to carry out multiple, simultaneous functions.     

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 10(9) primary bovine aortic endothelial cells and structurally characterized by immunological and chemical methods. Glucosyl- and lactosylceramide were detected as the main neutral glycosphingolipids (28% and 40% of total orcinol stain, respectively); LcOse3Cer and nLcOse4Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse6Cer occurred only in trace quantities. No neutral glycosphingolipids of the ganglio-series (GgOse3Cer and GgOse4Cer) and the globo-series (GbOse4Cer and the Forssman antigen) have been detected; only traces of GbOse3Cer were identified by TLC immunostaining. Positive CD15 bands obtained by TLC overlay with anti-Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-R antibody indicated the presence of lipid bound Lewisx antigen, whereas the isomeric Lewis(a) structure (Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-R) was not detectable. G(M3) substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole ganglioside fraction. G(M3)-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives. C18-sphingosine was the only long chain base, whereas variation occurred due to C(24:0,24:1) and C16 fatty acids. Terminally alpha2-3 sialylated neolacto-series gangliosides with nLcOse4- and nLcOse6Cer (<5% of total resorcinol stain) were found in almost equal quantities, whereas no alpha2-6 sialylated counterparts were detected. Fucosylated gangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis[x], sialyl Lewisa, and VIM-2 antigen) and sulfoglucuronylneolacto series structures with HNK-1 epitope were not detectable in the acidic glycosphingolipid fraction by TLC immunostaining. Gangliotetraose-type gangliosides G(M1) and G(D1a) (<1 % of total resorcinol stain) as well as traces of G(D1b) and G(T1b) have been distinctly identified by combined choleragenoid-TLC-immunostaining and previous neuraminidase treatment. The expression of dominant glycosphingolipids lactosylceramide and G(M3)(Neu5Gc) was proved by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The results provide the basis for investigation of the role of glycosphingolipids as cell surface antigens of cellular interaction as well as receptors for blood components and macromolecules of the extracellular matrix.     

Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata)

Bacterial feeding nematodes in the order Rhabditida including Zeldia punctata (Cephalobidae) and Caenorhabditis elegans (Rhabditidae) differ profoundly in the buccal capsule parts and associated cells. We carried out a range of tests to determine which buccal capsule parts and cells are evolutionarily homologous between the representative species of the two families. Tests included reconstruction of the buccal capsule and procorpus with transmission electron microscopy (TEM), nuclei position and morphology using 4, 6-diamidino-2-phenylindole (DAPI) staining, and cell lineage using four dimensional (4D) microscopy. The lining of the buccal capsule of Z. punctata and additional Cephalobidae includes four sets of muscular radial cells, ma, mb, mc and md, in contrast to C. elegans and additional Rhabditidae, which has two sets of epithelial cells (e1, e3) and two sets of muscle cells (m1, m2). Cell lineage of a nematode closely related to Z. punctata, Cephalobus cubaensis, supports the hypothesis that in cephalobids the e1 and e3 cells become hypodermal cells or are programmed to die. Our findings contradict all previous hypotheses of buccal capsule homology, and suggest instead that ma and mb in Z. punctata are homologous to m1 and m2 in C. elegans respectively. We also hypothesize that ma and mb could be homologous to primary and secondary sets of stylet-protractor muscle cells in the plant parasitic Tylenchida.     

Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans. Isolation and characterization

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluClalpha and GluClbeta) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegans strain with a Tc1 transposable element insertion that functionally inactivated the GluClalpha gene (GluClalpha::Tc1). GluClalpha::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity. Xenopus oocytes expressing GluClalpha::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluClalpha::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced Bmax. These experiments suggest that GluClalpha is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluClalpha2) encoding a chloride channel that shares 75% amino acid identity with GluClalpha. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate. GluClalpha2 coassembles with GluClbeta to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluClalpha may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.     

cDNA cloning and expression of a family of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase sequence homologs from Caenorhabditis elegans

The initiation of mucin-type O-glycosylation is catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTase) (EC 2.4.1.41). By screening two mixed-stage Caenorhabditis elegans cDNA libraries, a total of 11 distinct sequence homologs of the ppGaNTase gene family were cloned, sequenced, and expressed as truncated recombinant proteins (gly-3, gly-4, gly-5a, gly-5b, gly-5c, gly-6a, gly-6b, gly-6c, gly-7, gly-8, and gly-9). All clones encoded type II membrane proteins that shared 60-80% amino acid sequence similarity with the catalytic domain of mammalian ppGaNTase enzymes. Two sets of cDNA clones (gly-5 and gly-6) contained variants that appeared to be produced by alternative message processing. gly-6c contained a reading frameshift and premature termination codon in the C-terminal lectin-like domain found in most other ppGaNTase proteins, and a second clone (gly-8) lacked the typical C-terminal region completely. Homogenates of nematodes and immunopurified preparations of the recombinant GLY proteins demonstrated that worms express functional ppGaNTase enzymes (GLY-3, GLY-4, GLY-5A, GLY-5B, and GLY-5C), which can O-glycosylate mammalian apomucin peptide sequences in vitro. In addition to demonstrating the existence of ppGaNTase enzymes in a nematode organism, the substantial diversity of these isoforms in C. elegans suggests that mucin O-glycosylation is catalyzed by a complex gene family, which is conserved among evolutionary-distinct organisms.     

The generation and genetic analysis of suppressors of lethal mutations in the Caenorhabditis elegans rol-3(V) gene

The Caenorhabditis elegans rol-3(e754) mutation is a member of a general class of mutations affecting gross morphology, presumably through disruption of the nematode cuticle. Adult worms homozygous for rol-3(e754) exhibit rotation about their long axis associated with a left-hand twisted cuticle, musculature, gut and ventral nerve cord. Our laboratory previously isolated 12 recessive lethal alleles of rol-3. All these lethal alleles cause an arrest in development at either early or mid-larval stages, suggesting that the rol-3 gene product performs an essential developmental function. Furthermore, through the use of the heterochronic mutants lin-14 and lin-29, we have established that the expression of rol-3(e754)'s adult specific visible function is not dependent on the presence of an adult cuticle. In an attempt to understand rol-3's developmental role we sought to identify other genes whose products interact with that of rol-3. Toward this end, we generated eight EMS induced and two gamma irradiation-induced recessive suppressors of the temperature sensitive (ts) mid-larval lethal phenotype of rol-3(s1040ts). These suppressors define two complementation groups srl-1 II and srl-2 III; and, while they suppress the rol-3(s1040) lethality, they do not suppress the adult specific visible rolling phenotype. Furthermore, there is a complex genetic interaction between srl-2 and srl-1 such that srl-2(s2506) fails to complement all srl alleles tested. These results suggest that srl-1 and srl-2 may share a common function and, thus, possibly constitute members of the same gene family. Mutations in both srl-1 and srl-2 produce no obvious hermaphrodite phenotypes in the absence of rol-3(s1040ts); however, males homozygous for either srl-1 or srl-2 display aberrant tail morphology. We present evidence suggesting that the members of srl-2 are not allele specific with respect to their suppression of rol-3 lethality, and that rol-3 may act in some way to influence proper posterior morphogenesis. Finally, based on our genetic analysis of rol-3 and the srl mutations, we present a model whereby the wild-type products of the srl loci act in a concerted manner to negatively regulate the rol-3 gene.     

Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom

Several methods of external and internal fixation are used in urgent situations to lessen intrapelvic bleeding associated with unstable pelvic fractures. Pelvic stabilization limits pelvic expansion and thereby restricts the space for potential blood loss. This study compared several fixation methods using cadaveric pelves to determine which method best prevents pelvic expansion. Three methods of internal fixation and three methods of external fixation were compared. Anteroposterior fixation provided the greatest control against pelvic expansion; however, it is clinically impractical for emergency use. Therefore, external fixation provided the most reliable control of pelvic expansion in the emergency setting.     

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis. let-395. let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch. thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position.     

A Caenorhabditis elegans act-4::lacZ fusion: use as a transformation marker and analysis of tissue-specific expression

A plasmid vector that serves as a dominant marker for isolating transformed animals in Caenorhabditis elegans has been constructed as a translational fusion of the C. elegans act-4 gene (encoding actin) and the Escherichia coli lacZ gene. This gene fusion can be used as a marker in transformation rescue experiments in any fertile strain of C. elegans. Progeny of animals injected with the act-4::lacZ fusion vector are stained histochemically with XGal, and transformants turn blue. The internal eggs of stained animals remain viable, allowing recovery of the transformed strain. When the act-4::lacZ vector is co-injected with an unselected plasmid with which it shares some sequence homology, most transformants that are recovered by screening for expression of the act-4::lacZ fusion contain both plasmids. Production of active beta Gal in animals transformed with the act-4::lacZ gene fusions appears to be limited to certain tissues. A chimeric gene that contains the 5' and 3' regions of act-4 is expressed strongly in the body-wall muscles, vulval muscles, and spermathecae. Addition of the internal portion of act-4, including the protein-coding region and introns, to this chimeric gene leads to additional lacZ expression in the pharynx.     

Formation of the vulva in Caenorhabditis elegans: a paradigm for organogenesis

The genes involved in the inductive interactions that specify cell fates in the vulva of Caenorhabditis elegans are known in some detail. However, little is known about the morphogenesis of this organ. Using a combination of cell biological and anatomical approaches, we have determined a complete morphogenetic pathway of cellular events that lead to the formation of the vulva. These events include reproducible cell divisions, migrations, remodeling of adherens junctions, cell fusions and muscle attachments. In the course of these events, an epithelial channel comprising a stack of 7 toroidal cells is formed that connects the internal epithelium of the uterus with the external body epithelium, forming the vulva. Vulval muscles attach to the epithelial channel and the whole structure everts during the final molt. The mature vulva has rotational, two-fold symmetry. Using laser microsurgery, we found that the two halves of the vulva develop autonomously.     

cDNA cloning of a human homologue of the Caenorhabditis elegans cell fate-determining gene mab-21: expression, chromosomal localization and analysis of a highly polymorphic (CAG)n trinucleotide repeat

The two most consistent features of the diseases caused by trinucleotide repeat expansion-neuropsychiatric symptoms and the phenomenon of genetic anticipation-may be present in forms of dementia, hereditary ataxia, Parkinsonism, bipolar affective disorder, schizophrenia and autism. To identify candidate genes for these disorders, we have screened human brain cDNA libraries for the presence of gene fragments containing polymorphic trinucleotide repeats. Here we report the cDNA cloning of CAGR1, originally detected in a retinal cDNA library. The 2743 bp cDNA contains a 1077 bp open reading frame encoding 359 amino acids. This amino acid sequence is homologous (56% amino acid identify and 81% amino acid conservation) to the Caenorhabditis elegans cell fate-determining protein mab-21. CAGR1 is expressed in several human tissues, most prominently in the cerebellum, as a message of approximately 3.0 kb. The gene was mapped to 13q13, just telomeric to D13S220. A 5'-untranslated CAG trinucleotide repeat is highly polymorphic, with repeat length ranging from six to 31 triplets and a heterozygosity of 87-88% in 684 chromosomes from several human populations. One allele from an individual with an atypical movement disorder and bipolar affective disorder type II contains 46 triplets, 15 triplets longer than any other allele detected. Though insufficient data are available to link the long repeat to this clinical phenotype, an expansion mutation of the CAGR1 repeat can be considered a candidate for the etiology of disorders with anticipation or developmental abnormalities, and particularly any such disorders linked to chromosome 13.     

Genetic and molecular analysis of fox-1, a numerator element involved in Caenorhabditis elegans primary sex determination

fox-1 was previously identified as a candidate numerator element based on its overexpression phenotype. FOX-1 is an RRM-type RNA-binding protein, which can bind RNAs in vitro. Western analysis detects FOX-1 throughout development. fox-1::lacZ comes on ubiquitously early during embryogenesis. Postembryonically, fox-1::lacZ is expressed sex specifically in a subset of cells in the head and tail. We describe a Tc1-derived deletion allele [fox-1(Delta)] that removes the RRM domain. fox-1(Delta) confers no phenotype in XXs, but can rescue XO-specific lethality and feminization caused by duplications of the left end of the X. fox-1(Delta) synergizes with putative numerators, resulting in abnormal XX development. Genetic analysis indicated that fox-1(Delta) leads to a slight increase in xol-1 activity, while fox-1(gf) leads to partial loss of xol-1 activity, and xol-1 is epistatic to fox-1. RNase protection experiments revealed increased levels of the 2.2-kb xol-1 message in fox-1(Delta) animals, and reduced levels in fox-1(gf) animals. Additionally, fox-1(Delta) impairs male mating efficiency, which, we propose, represents another function of fox-1, independent of xol-1 and its role in sex determination.     

Molecular characterization of an anchor protein (AKAPCE) that binds the RI subunit (RCE) of type I protein kinase A from Caenorhabditis elegans

Classical A kinase anchor proteins (AKAPs) preferentially tether type II protein kinase A (PKAII) isoforms to sites in the cytoskeleton and organelles. It is not known if distinct proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In Caenorhabditis elegans, a single type I PKA mediates all aspects of cAMP signaling. We have discovered a cDNA that encodes a binding protein (AKAPCE) for the regulatory subunit (RCE) of C. elegans PKAICE. AKAPCE is a novel, highly acidic RING finger protein composed of 1,280 amino acids. It binds RI-like RCE with high affinity and neither RIIalpha nor RIIbeta competitively inhibits formation of AKAPCE.RCE complexes. The RCE-binding site was mapped to a segment of 20 amino acids in an N-terminal region of AKAPCE. Several hydrophobic residues in the binding site align with essential Leu and Ile residues in the RII-selective tethering domain of prototypic mammalian AKAPs. However, the RCE-binding region in AKAPCE diverges sharply from consensus RII-binding sites by inclusion of three aromatic amino acids, exclusion of a highly conserved Leu or Ile at position 8 and replacement of C-terminal hydrophobic amino acids with basic residues. AKAPCE.RCE complexes accumulate in intact cells.     

Lessons to learn from the cell death and heat shock genes of Caenorhabditis elegans

The nematode Caenorrhabditis elegans is an applicable experimental system for simulation of complex biochemical processes of mammalian cells and tissues. The genetic pathway of programmed cell death (PCD) has been partially clarified in the nematode. Analysis of cell death genes of C. elegans led to the conclusion that PCD is conserved in the animal kingdom. Our intention is to study the role of tissue transglutaminase and heat shock proteins in the process of PCD. Tissue transglutaminase is often observed to be induced and activated during the molecular mechanism of PCD. The connection between the heat shock proteins and PCD is not well understood, but it is clear that many apoptosis inducers lead to increased synthesis of heat shock proteins and production of heat shock proteins is coupled with the appearance of apoptosis in numerous experimental systems. Our preliminary observations made by studying cell death mutants of C. elegans we suggest that transglutaminase and heat shock proteins are involved in the death program of the nematode.