Identification, physiological actions, and distribution of TPSGFLGMRamide: a novel tachykinin-related peptide from the midgut and stomatogastric nervous system of Cancer crabs

In most invertebrates, multiple species-specific isoforms of tachykinin-related peptide (TRP) are common. In contrast, only a single conserved TRP isoform, APSGFLGMRamide, has been documented in decapod crustaceans, leading to the hypothesis that it is the sole TRP present in this arthropod order. Previous studies of crustacean TRPs have focused on neuronal tissue, but the recent demonstration of TRPs in midgut epithelial cells in Cancer species led us to question whether other TRPs are present in the gut, as is the case in insects. Using direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation, we found that at least one additional TRP is present in Cancer irroratus, Cancer borealis, Cancer magister, and Cancer productus. The novel TRP isoform, TPSGFLGMRamide, was present not only in the midgut, but also in the stomatogastric nervous system (STNS). In addition, we identified an unprocessed TRP precursor APSGFLGMRG, which was detected in midgut tissues only. TRP immunohistochemistry, in combination with preadsorption studies, suggests that APSGFLGMRamide and TPSGFLGMRamide are co-localized in the stomatogastric ganglion (STG), which is contained within the STNS. Exogenous application of TPSGFLGMRamide to the STG elicited a pyloric motor pattern that was identical to that elicited by APSGFLGMRamide, whereas APSGFLGMRG did not alter the pyloric motor pattern.     

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor

Two new series of EP₄ antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP₄ receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.     

REMARKABLE CONSERVATION OF INTERNALLY TRANSCRIBED SPACER SEQUENCES OF ARTHROSPIRA (“SPIRULINA” ) (CYANOPHYCEAE,CYANOBACTERIA) STRAINS FROM FOUR CONTINENTS AND OF RECENT AND 30‐YEAR‐OLD DRIED SAMPLES FROM AFRICA1

The internally transcribed spacer (ITS) sequences of 21 Arthrospira clonal strains from four continents and assigned to four different species (A. platensis, A. maxima, A. fusiformis, A. indica) in the culture collections were determined. Two main clusters, I and II, were differentiated by 49 positions out of 475 nt or 477 nt, respectively. Each cluster was further subdivided into two subclusters. Subclusters I.A and I.B were separated by two substitutions, whereas subclusters II.A and II.B were distinguished by four substitutions. After direct sequencing of the PCR products, three dried samples from Chad aged between 3 months and 35 years yielded a sequence belonging to subcluster I.A, as did a recent commercial product. The strains grown in production plants belonged to the same (sub)clusters as strains from culture collections, mainly I.A and II. PCR primers specific for each cluster and subcluster were designed and tested with crude cell lysates of Arthrospira strains. One dried sample (“dihé” 1) and a herbarium sample from Lake Sonachi (Kenya) only contained I.A sequences, whereas the commercial product was a mixture of the four genotypes and the other two dried samples contained minor polymorphisms characteristic of different clusters. Five clonal Arthrospira strains, thought to be duplicates, showed the simultaneous presence of the two forms of the four diagnostic positions that distinguish subclusters genotype II.A and genotype II.B. This is likely to be caused by multiple copies of the rDNA operon, in a intermediate stage of homogenization between subcluster II.A and subcluster II.B. The high conservation of ITS sequences is in contrast with the assignment to four different species, the great morphological variability of the strains, and their wide geographic distribution.     

Incidence and diversity of potentially highly heat-resistant spores isolated at dairy farms

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100 degrees C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.     

Learning and colonization of new niches: a first step toward speciation

Learning processes potentially play a role in speciation but are often ignored in speciation models. Learning may, for instance, play a role when a new niche is being colonized, because the learning of niche features may cause niche-specific assortative mating and a tendency to produce young in this niche. Several animal species learn about their environmental features that may be important in finding or attracting mates. We use a gene-culture coevolutionary model to look into the effect of such learning on the colonization of new niches and on the genetic divergence between groups using different niches, which are steps necessary in achieving speciation. We assume that density is regulated separately in each of the two niches and that the viability of an individual depends on its genotype as well as on which niche it exploits. Our results show that genetic adaptation to the new niche is enhanced by a high female fecundity and a low viability selection against heterozygotes. Furthermore, when initial colonization (without genetic adaptation) fails, genetic divergence is more difficult when the mating preference is stronger. In contrast, when colonization without genetic adaptation is successful, a stronger mating preference makes genetic divergence easier. An increase in the number of egg-laying mistakes by females can have a positive or negative effect on the success of genetic adaptation depending on other parameters. We show that genetic divergence can be prevented by a niche shift, which can occur only if viabilities in the two niches are asymmetrical.     

Modes of reproduction and the accumulation of deleterious mutations with multiplicative fitness effects

Mutational load depends not only on the number and nature of mutations but also on the reproductive mode. Traditionally, only a few specific reproductive modes are considered in the search of explanations for the maintenance of sex. There are, however, many alternatives. Including these may give radically different conclusions. The theory on deterministic deleterious mutations states that in large populations segregation and recombination may lead to a lower load of deleterious mutations, provided that there are synergistic interactions. Empirical research suggests that effects of deleterious mutations are often multiplicative. Such situations have largely been ignored in the literature, since recombination and segregation have no effect on mutation load in the absence of epistasis. However, this is true only when clonal reproduction and sexual reproduction with equal male and female ploidy are considered. We consider several alternative reproductive modes that are all known to occur in insects: arrhenotoky, paternal genome elimination, apomictic thelytoky, and automictic thelytoky with different cytological mechanisms to restore diploidy. We give a method that is based on probability-generating functions, which provides analytical and numerical results on the distributions of deleterious mutations. Using this, we show that segregation and recombination do make a difference. Furthermore, we prove that a modified form of Haldane's principle holds more generally for thelytokous reproduction. We discuss the implications of our results for evolutionary transitions between different reproductive modes in insects. Since the strength of Muller's ratchet is reduced considerably for several forms of automictic thelytoky, many of our results are expected to be also valid for initially small populations.     

Mutations in Lama1 Disrupt Retinal Vascular Development and Inner Limiting Membrane Formation

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin α1, a basement membrane protein. Despite normal localization of laminin α1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin α1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1^tm1.1Olf, was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.     

GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision.     

Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

Background

Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na⁺ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined.

Materials and Methods

The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers.

Results

G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005).

Discussion

Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.