The ultrastructural organization of the visual system of the wax moth,Galleria mellonella: The retina

Summary The ultrastructural organization of ommatidial components of the retina of the moth, Galleria mellonella are described from electron microscopic observations. Each ommatidium is composed of 12 common retinula cells and one basal eccentric cell. The retinula cells are connected together by a desmosomal strip along their length. The rhabdom occupies the basal thirty percent of the ommatidium and can be divided into nine segments of parallel microvilli. Several cells may contribute to an individual rhabdomere. The rhabdomeres are arranged in a cross with single cell rhabdomeres lying between the arms of the cross. Thin sections of ommatidium absorb polarized light differentially. The total amount of plane polarized light absorbed varies with angle of rotation for an entire ommatidium but there are also differences between the amount of absorption of adjacent rhabdomeric segments. Galleria appears to be the only lepidopteran in which the possibility of the polarized light reception has been reported.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney

The 100,000 xg supernatant of rabbit kidney contains a prostaglandin-E₂-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE₂ to PGF_2α. A broad pH profile was evident with an optimum at pH 7·5. Kinetic studies indicated a K_m of 3·2 × 10⁻⁴M PGE₂. The isoelectric point was at pH 5·65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE₂-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico,USA

BackgroundLeptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology.Methodology/Principal findingsWe collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro.We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup.Conclusions/SignificanceDiverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.     

Is Play Behavior Sexually Dimorphic in Monogamous Species?

Monogamy is a relatively rare social system in mammals, occurring only in about 3% of mammalian species. Monogamous species are characterized by the formation of pair‐bonds, biparental care, and a very low level of sexual dimorphism. Whereas in most polygynous species males engage in more rough‐and‐tumble play than females, we predicted that males and females of monogamous species would have similar, or monomorphic, play behavior. In this study, we focused on two monogamous species: coppery titi monkeys (Callicebus cupreus) and prairie voles (Microtus ochrogaster). We documented the development of play behavior in both species, and quantified different types of play behavior. We did not find any sex differences in either species in the frequencies and types of play. However, we did find sex differences in the choice of play partner in titi monkeys: female offspring spent a higher proportion of time playing with their father, while male offspring played equally with their mother and father. It is possible that rough‐and‐tumble play behavior is monomorphic in many monogamous mammals, perhaps reflecting differences from polygynous species in the effects of exposure to early androgens or in the estrogen receptor distribution. However, more subtle differences in monomorphic play behavior, such as choice of partner, may still exist.     

A Double-Isotope Procedure for Examining Protein Microheterogeneity: Multiple Forms of Fast-Transported Glycoproteins and Sulfoproteins Possess a Common Polypeptide Chain

Several fast-transported proteins that appear as single bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolve into multiple spots during isoelectric focusing. A method was devised for determining if such microheterogeneity in net charge indicates that individual polypeptides have been posttranslationally modified to differing extents. Dorsal root ganglia were pulse-labeled with [35S]methionine and either [3H]leucine or [3H]proline, proteins fast-transported into peripheral sensory axons were separated by two-dimensional gel electrophoresis, and isotope incorporation ratios of proteins associated with individual gel spots were determined. When four microheterogeneous glycoproteins were analyzed, each protein "family" showed markedly similar isotope ratios for its three to seven characteristic spots. Such ratios differed between families by almost twofold. In addition, a group of nonglycosylated, sulfate-containing proteins was identified as a family on the basis of the similar isotope incorporation ratios of its component spots. These results suggest that protein microheterogeneity can result from variable sulfation of tyrosine residues as well as from variation in sialic acid-containing oligosaccharide side-chains. More generally, the method can be utilized to test for protein microheterogeneity in cases where the amounts of protein are too low to permit peptide mapping analysis and where the nature of the charge-altering modification is unknown.     

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer)

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.