Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling.

In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Eps15-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-1 mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.

mua-3, a gene required for mechanical tissue integrity in Caenorhabditis elegans, encodes a novel transmembrane protein of epithelial attachment complexes.

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force through a series of mechanical linkages from the myofibrillar lattice of the body wall muscles, across an intervening extracellular matrix and epithelium (the hypodermis) to the cuticle. Mutations in mua-3 cause a separation of the hypodermis from the cuticle, suggesting this gene is required for maintaining hypodermal-cuticle attachment as the animal grows in size postembryonically. mua-3 encodes a predicted 3,767 amino acid protein with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein modules: 5 low density lipoprotein type A, 52 epidermal growth factor, 1 von Willebrand factor A, and 2 sea urchin-enterokinase-agrin modules. MUA-3 localizes to the hypodermal hemidesmosomes and to other sites of mechanically robust transepithelial attachments, including the rectum, vulva, mechanosensory neurons, and excretory duct/pore. In addition, it is shown that MUA-3 colocalizes with cytoplasmic intermediate filaments (IFs) at these sites. Thus, MUA-3 appears to be a protein that links the IF cytoskeleton of nematode epithelia to the cuticle at sites of mechanical stress.

The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans.

Three known genes guide circumferential migrations of pioneer axons and mesodermal cells on the nematode body wall. unc-5 affects dorsal migrations, unc-40 primarily affects ventral migrations, and unc-6 affects migrations in both directions. Circumferential movements still occur, but are misdirected whereas longitudinal movements are normal in these mutants. Pioneer growth cones migrating directly on the epidermis are affected; growth cones migrating along established axon fascicles are normal. Thus these genes affect cell guidance and not cell motility per se. We propose that two opposite, adhesive gradients guide circumferential migrations on the epidermis. unc-5, unc-6, and unc-40 may encode these adhesion molecules or their cellular receptors. Neurons have access to the basal lamina and the basolateral surfaces of the epidermis, but mesodermal cells contact only the basal lamina. These genes probably identify molecular cues on the basal lamina that guide mesodermal migrations. The same basal lamina cues, or perhaps related molecules on the epidermal cell surfaces, guide pioneer neurons.

Active currents regulate sensitivity and dynamic range in C. elegans neurons.

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.

The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans.

In Caenorhabditis elegans three pairs of neurons, AFD, AIY, and AIZ, play a key role in thermosensation. The LIM homeobox gene ceh-14 is expressed in the AFD thermosensory neurons. ceh-14 mutant animals display athermotactic behaviors, although the neurons are still present and differentiated. Two other LIM homeobox genes, ttx-3 and lin-11, function in the two interneurons AIY and AIZ, respectively. Thus, the three key thermosensory neurons are specified by three different LIM homeobox genes. ceh-14 ttx-3 lin-11 triple mutant animals have a basic cryophilic thermotaxis behavior indicative of a second thermotaxis pathway. Misexpression of ceh-14 in chemosensory neurons can restore thermotactic behavior without impairing the chemosensory function. Thus, ceh-14 confers thermosensory function to neurons.

Cytokine-induced programmed death of cultured sympathetic neurons.

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic "ladder" on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.

mab-7 encodes a novel transmembrane protein that orchestrates sensory ray morphogenesis in C. elegans.

The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis.

Common genetic variation in the type A endothelin-1 receptor is associated with ambulatory blood pressure: a family study.

The endothelins are among the most potent vasoconstrictors known. Pharmacological inhibition of endothelin receptors lowers blood pressure (BP). It is unknown whether naturally occurring genetic variation in the endothelin receptors influences BP. We have evaluated the type A endothelin receptor (EDNRA) as a candidate gene for hypertension in a large family study. A total of 1425 members of 248 families selected via a proband with hypertension were studied. Ambulatory BP monitoring was conducted using the A&D TM2421 device. Four haplotype-tagging single nucleotide polymorphisms (SNPs) spanning the EDNRA gene were typed. There was evidence of association between genotype at the rs5335 (C+70G) SNP and night systolic blood pressure (+1.24% (s.e. 0.64) per G allele; P=0.05); night diastolic blood pressure (+1.64% (s.e. 0.71) per G allele; P=0.021) and night mean BP (+1.51% (s.e. 0.64) per G allele; P=0.017). Borderline significant trends in the same direction were seen for daytime BPs. Proportions of hypertensives in each of the three genotype groups were C/C 34.7%, C/G 37.9%, G/G 42.4% yielding an odds ratio for hypertension per G allele of 1.19 (95% confidence interval 1.00-1.41; P=0.05). In conclusion, the rs5335 (C+70G) polymorphism of the EDNRA gene has small effects on the risk of hypertension. Natural variation in other genes in the endothelin-signalling pathway should be explored to identify additional influences on BP regulation.

The Caenorhabditis elegans autosomal dominant polycystic kidney disease gene homologs lov-1 and pkd-2 act in the same pathway.

Autosomal dominant polycystic kidney disease (ADPKD) strikes 1 in 1000 individuals and often results in end-stage renal failure. Mutations in either PKD1 or PKD2 account for 95% of all cases [1-3]. It has recently been demonstrated that polycystin-1 and polycystin-2 (encoded by PKD1 and PKD2, respectively) assemble to form a cation channel in vitro [4]. Here we determine that the Caenorhabditis elegans PKD1 and PKD2 homologs, lov-1 [5] and pkd-2, act in the same pathway in vivo. Mutations in either lov-1 or pkd-2 result in identical male sensory behavioral defects. Also, pkd-2;lov-1 double mutants are no more severe than either of the single mutants, indicating that lov-1 and pkd-2 act together. LOV-1::GFP and PKD-2::GFP are expressed in the same male-specific sensory neurons and are concentrated in cilia and cell bodies. Cytoplasmic, nonnuclear staining in cell bodies is punctate, suggesting that one pool of PKD-2 is localized to intracellular membranes while another is found in sensory cilia. In contrast to defects in the C. elegans autosomal recessive PKD gene osm-5 [6-8], the cilia of lov-1 and pkd-2 single mutants and of lov-1;pkd-2 double mutants are normal as judged by electron microscopy, demonstrating that lov-1 and pkd-2 are not required for ultrastructural development of male-specific sensory cilia.

Estimating pollutant removal requirements for landfills in the UK: III. Policy analysis and operational implications.

The policy analysis and management implications for achieving landfill equilibrium status within a sustainable timescale (decades rather than centuries) are presented based on modelled results reported previously. Until relatively recently, timescale estimates suggested that equilibrium or landfill completion could be achieved within 40-60 years i.e. the same order of magnitude as financial provision for aftercare. However results of modelling in this study (reported in previous paper) suggest that timescales may be considerably longer (many centuries in some instances) suggesting that financial provision may be inadequate. The role of the most promising and available waste treatment technologies and strategic waste management options in contributing towards achieving equilibrium status are discussed. Results suggest that a re-examination of techniques for accelerating landfill stabilisation, including aerobic and bioreactor landfill, is warranted.

Estimating pollutant removal requirements for landfills in the UK: II. Model development.

A modelling methodology using a leachate source term has been produced for estimating the timescales for achieving environmental equilibrium status for landfilled waste. Results are reported as the period of active management required for modelled scenarios of non-flushed and flushed sites for a range of pre-filling treatments. The base scenario against which results were evaluated was raw municipal solid waste (MSW) for which only cadmium failed to reach equilibrium. Flushed raw MSW met our criteria for stabilisation with active leachate management for 40 years, subject to each of the leachate species being present at or below their average UK concentrations. Stable non-reactive wastes, meeting EU waste acceptance criteria, fared badly in the non-flushed scenario, with only two species stabilising after a management period within 1000 years and the majority requiring > 2000 years of active leachate management. The flushing scenarios showed only a marginal improvement, with arsenic still persisting beyond 2000 years management even with an additional 500 mm y(-1) of infiltration. The stabilisation time for mechanically sorted organic residues (without flushing) was high, and even with flushing, arsenic and chromium appeared to remain a problem. Two mechanical biological treatment (MBT) scenarios were examined, with medium and high intensity composting. Both were subjected to the non-flushing and flushing scenarios. The non-flushing case of both options fell short of the basic requirements of achieving equilibrium within decades. The intense composting option with minimal flushing appeared to create a scenario where equilibrium could be achieved. For incinerator bottom ash (raw and subjected to various treatments), antimony, copper, chloride and sulphate were the main controls on achieving equilibrium, irrespective of treatment type. Flushing at higher flushing rates (500 mm y(-1)) failed to demonstrate a significant reduction in the management period required.

Estimating pollutant removal requirements for landfills in the UK: I. Benchmark study and characteristics of waste treatment technologies.

Introduction of the EU Landfill Directive is having a significant impact on waste management in the UK and in other member states that have relied on landfilling. This paper considers the length of the aftercare period required by the municipal solid waste streams that the UK will most probably generate following implementation of the Landfill Directive. Data were derived from literature to identify properties of residues from the most likely treatment processes and the probable management times these residues will require within the landfill environment were then modelled. Results suggest that for chloride the relevant water quality standard (250 mg l(-1)) will be achieved with a management period of 40 years and for lead (0.1 mg I(-1)), 240 years. This has considerable implications for the sustainability of landfill and suggests that current timescales for aftercare of landfills may be inadequate.

Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons.

Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). Here we show that the UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific Caenorhabditis elegans motor neurons. UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in the mutant unc-4(e120) confirms that SV number in the presynaptic zone is reduced ( approximately 40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, we propose that these effects are performed via an intermediate gene. Our results are consistent with a model in which this unc-4 target gene ("gene-x") functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons.

Functional and sequence analysis of splicing defective nrdB mutants of bacteriophage T4 reveal new bases and a new sub-domain required for group I intron self-splicing.

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron. In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analyzed for both sequence and functional changes. These mutants cluster towards the ends in regions of conserved structural elements of the intron. These 23 mutants have single base changes at 14 different sites. Interestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron. A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron structure. The mutation sites in pairing regions P3, P7, P8, P9 and between P6[3'] and P7[5'] are identical to changes found in the well studied td (encoding dTMP synthase) intron. However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrdB intron and disrupt self-splicing. A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in this pairing, thus defining a new sub-domain essential for RNA splicing. Like the td intron, the mutation site in P9 of the nrdB intron is a hot spot for mutations, but unlike td, the nrdB intron does not show a mutational hot spot in the P6[5'] region. Our molecular dissection of the nrdB intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3' splice site selection, cleavage and exon ligation.

Suppressors of mutations in the rII gene of bacteriophage T4 affect promoter utilization.

Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in lambda lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH2 reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L2 decreases the folate analog resistant and the inability of sip2 to grow on CTr5X. L2 itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L2 affects another regulatory gene related to the mot gene.

Genetic evidence for physical interactions between enzymes of nucleotide synthesis and proteins involved in DNA replication in bacteriophage T4.

We have found that mutations in phage T4 genes 41 (five of five) and 61 (three of three) cause resistance to the folate analogue pyrimethamine that inhibits T4 dihydrofolate (FH2) reductase. These genes code for subunits of a T4 primase and are part of a putative T4 replication complex. In contrast to many previously isolated folate analogue-resistant (Far) T4 mutants, these T4 primase mutants do not overproduce FH2 reductase nor do they alter its primary structure. A new mutant with a single lesion in gene 41 was isolated which proved resistant to the folate analogue at 30 degrees and was lethal at 42 degrees. This mutant induced normal levels of FH2 reductase (encoded by the frd gene) and appeared to have normal expression of other T4 genes at 30 degrees. Like other mutations in gene 41, tsP129 reduced phage-induced DNA synthesis to about 15% that of wild-type T4 as measured by thymidine incorporation under restrictive conditions. Double mutants carrying mutations in genes 41 and 61, 41 and frd or 61 and frd showed allele-specific suppression suggesting that the products of these genes interact. We suggest that abnormal interactions between components of the replication complex and a DNA precursor synthesizing complex cause folate analog resistance by allosterically altering the T4 FH2 reductase.

Visualization of fibrillar amyloid deposits in living, transgenic Caenorhabditis elegans animals using the sensitive amyloid dye, X-34.

Transgenic Caenorhabditis elegans animals can be engineered to express high levels of the human beta amyloid peptide (Abeta). Histochemistry of fixed tissue from these animals reveals deposits reactive with the amyloid-specific dyes Congo Red and thioflavin S (Fay et al., J. Neurochem 71:1616, 1998). Here we show by immuno-electron microscopy that these animals contain intracellular immunoreactive deposits with classic amyloid fibrillar ultrastructure. These deposits can be visualized in living animals using the newly developed, intensively fluorescent, amyloid-specific dye X-34. This in vivo staining allows monitoring of amyloid deposition in individual animals over time. The specificity of this staining is demonstrated by examining transgenic animals expressing high levels of a non-fibrillar beta peptide variant, the beta single-chain dimer. These animals have deposits immunoreactive with anti-beta antibodies, but do not have X-34 deposits or deposits with a fibrillar ultrastructure. X-34 can also be used in vivo to visualize putative amyloid deposits resulting from accumulation of human transthyretin, another amyloidic protein. In vivo amyloid staining with X-34 may be a useful tool for monitoring anti-amyloidic treatments in real time or screening for genetic alterations that affect amyloid formation.

Immuno-EM localization of GFP-tagged yolk proteins in C. elegans using microwave fixation.

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures.

The role of the p53 protein in the selective vulnerability of the inner retina to transient ischemia.

PURPOSE: To determine whether the p53 protein plays a role in the selective vulnerability of the inner retina to transient ischemia. METHODS: Transient retinal ischemia was induced using a high intraocular pressure (HIOP) model in the Sprague-Dawley rat for 60 minutes. Histopathologic outcome was determined 7 days after ischemia. In addition, analysis for evidence for apoptosis (TdT-dUTP terminal nick-end label [TUNEL] staining) and p53 protein expression (immunohistochemistry) was performed at several points during the reperfusion period. In a separate set of experiments, wild-type mice and two groups of transgenic mice, one homozygous and the other heterozygous for the p53 null gene, were also subjected to HIOP for 60 minutes, and histopathology was performed 7 days later. RESULTS: At 7 days subsequent to 60 minutes of ischemia in the rat, there was marked thinning of the inner retinal layers. There were scattered TUNEL-positive cells within the inner retina, peaking at 24 to 48 hours and persisting for at least 7 days. p53 immunochemistry demonstrated elevated protein levels within the inner retina; this finding peaked at 24 to 48 hours but was no longer present at 4 days after ischemia. TUNEL staining of the inner retina of the mouse was most prominent 24 hours subsequent to ischemia but persisted at 48 hours. Seven days subsequent to 60 minutes of ischemia in the wild-type and transgenic mice, histopathologic evaluation demonstrated preservation of the retinal histoarchitecture in the heterozygous group compared with the wild-type or homozygous animals. CONCLUSIONS: These data further support the hypothesis that the delayed cell death that occurs after transient retinal ischemia is, in part, apoptotic. In addition, they suggest a role for the p53 protein in the selective vulnerability of the inner retina to transient ischemia. p53 protein may be a target for future therapeutic agents in the treatment of disorders of the retina where ischemia plays a pathogenetic role.