Cyanobactins—ribosomal cyclic peptides produced by cyanobacteria

Cyanobactins are small cyclic peptides that are produced by a diverse selection of cyanobacteria living in symbioses as well as terrestrial, marine, or freshwater environments. They include compounds with antimalarial, antitumor, and multidrug reversing activities and potential as pharmaceutical leads. Cyanobactins are produced through the proteolytic cleavage and cyclization of precursor peptides coupled with further posttranslational modifications such as heterocyclization, oxidation, or prenylation of amino acids. Cyanobactin gene clusters encode two proteases which cleave and cyclisize the precursor peptide as well as proteins participating in posttranslational modifications. The bioinformatic mining of cyanobacterial genomes has led to the discovery of novel cyanobactins. Heterologous expression of these gene clusters provided insights into the role of the genes participating in the biosynthesis of cyanobactins and facilitated the rational design of novel peptides. Enzymes participating in the biosynthesis of cyanobactins may prove useful as catalysts for producing novel cyclic peptides in the future. The recent discovery of the cyanobactin biosynthetic pathway in cyanobacteria extends our knowledge of their potential as producers of interesting metabolites.     

VCP Associated Inclusion Body Myopathy and Paget Disease of Bone Knock-In Mouse Model Exhibits Tissue Pathology Typical of Human Disease

Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. X-ray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCP^R155H/+ knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies.     

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.     

Procollagen VII self-assembly depends on site-specific interactions and is promoted by cleavage of the NC2 domain with procollagen C-proteinase

Procollagen VII is a homotrimer of 350-kDa proalpha1(VII) chains. Each chain has a central collagenous domain flanked by a noncollagenous amino-terminal NC1 domain and a carboxy-terminal NC2 domain. After secretion from cells, procollagen VII molecules form antiparallel dimers with a 60 nm overlap. These dimers are stabilized by disulfide bonds formed between cysteines present in the NC2 domain and cysteines present in the triple-helical domain. Electron microscopy has provided direct evidence for the existence of collagen VII dimers, but the dynamic process of dimer formation is not well understood. In the present study, we tested the hypothesis that, during dimer formation, the NC2 domain of one procollagen VII molecule specifically recognizes and binds to the triple-helical region adjacent to Cys-2625 of another procollagen VII molecule. We also investigated the role of processing of the NC2 domain by the procollagen C-proteinase/BMP-1 in dimer assembly. We engineered mini mouse procollagen VII variants consisting of intact NC1 and NC2 domains and a shortened triple helix in which the C-terminal region encompassing Cys-2625 was either preserved or substituted with the region encompassing Cys-1448 derived from the N-terminal part of the triple-helical domain. The results indicate that procollagen VII self-assembly depends on site-specific interactions between the NC2 domain and the triple-helical region adjacent to Cys-2625 and that this process is promoted by the cleavage of the NC2 by procollagen C-proteinase/BMP1.     

Probing the fetal genome: progress in non-invasive prenatal diagnosis

Progress in our understanding of the molecular basis of heritable diseases, through identification of specific mutations, has provided a foundation for the development of DNA-based prenatal diagnosis. Genetic analysis of fetal DNA is now routinely performed from chorionic villus samples obtained as early as the tenth week of gestation or by amniocentesis from week 15 onwards. However, both of these approaches involve invasive procedures with increased risk of fetal loss. To avoid such complications, attempts have been made to develop non-invasive tests through the identification, characterization and isolation of fetal cells or free fetal DNA from the maternal circulation. Recently, progress has been made towards the development of novel strategies that are expected to provide non-invasive means for early prenatal diagnosis in pregnancy.     

Isolation,characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.     

Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy

Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.     

CRITERIA OF FEMALE MATE CHOICE IN DROSOPHILA LITTORALIS,D. MONTANA,AND D. EZOANA

We examined sexual selection by Drosophila littoralis, D. montana, and D. ezoana females on male courtship sounds to determine whether the females use absolute or relative criteria when choosing their mates. Behavior of the females was observed, when they were courted by a single male producing normal sounds, or by a single wing-manipulated male producing abnormal sounds; and when they were courted by one or both of these males in a choice situation. The females usually accepted short-winged (but not wingless) males producing abnormal sounds, if they had no alternatives. However, if they heard the sound produced by a normal male, they rejected the deficient male. Drosophila littoralis and D. ezoana females selected between two wing-manipulated males with different wing areas. Our results suggest that the females choose their mates on the basis of relative criteria if the signals emitted by the courting males are within the range of acceptable cues.     

Population- and ecosystem-level effects of predation on microbial-feeding nematodes

We studied the role of nematode predation in the functioning of detrital food webs assembled in microcosms. The microcosms contained defaunated humus and litter materials, a diverse microbial community with bacteria, fungi and protozoa, and a birch (Betula pendula) seedling infected with mycorrhizal fungi. Different levels of top-down control upon microbivorous nematodes were set up by assembling food webs either without predators, or in combinations with a specialist and a non-specialist predatory mite (Mesostigmata). The nematode community was composed of either (1) three species of bacterivorous, or (2) three species of fungivorous nematodes or (3) both groups together. After two growing periods for the birch (38 weeks), the microcosms were destructively sampled for animal and microbial biomasses, concentration of mineral N in the soil, plant biomass and plant N concentration. The specialist predator reduced biomasses of both bacterial- and fungal-feeding nematodes by more than 50%, whereas the non-specialist predator weakly increased the biomass of fungivorous nematodes. Thus, under high predation pressure, the biomass of microbivores changed as predicted by trophic dynamic models assuming strong top-down control and uniformly behaving trophic levels. Despite this, microbial biomass was unaffected by the predators. However, microbial respiration increased slightly in the presence of predators. Assuming that microbial respiration correlates with microbial productivity, the increase in microbial respiration indicates a cascading productivity regulation. The composition of the microbivore community had only a minor effect on the outcome of the top-down control on microbes. The >50% reduction in nematode biomass and respiration coincided with <16% increase in microbial respiration and did not affect microbial biomass. Presence of the specialist predator slightly reduced soil NH⁺ ₄ concentration in communities with fungivore nematodes but plant growth and N uptake remained unchanged. Thus, the structure of the community only weakly controlled nutrient mineralisation. Received: 18 May 1998 / Accepted: 3 May 1999     

Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

Background  

Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis.