Diversity of coexisting <Emphasis Type="Italic">Planktothrix</Emphasis> (Cyanobacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopeptides

Cyanobacteria are reported to produce secondary metabolites of which toxic and bioactive peptides are of scientific and public interest. Many peptides are synthesized by the non-ribosomal peptide synthesis pathway and their presence is a stable feature of individual clones. We isolated 18 clonal strains of Planktothrix from a single water sample from lake Maxsee near Berlin and analyzed them by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, HPLC, and PCR for their production of peptides and the presence of microcystin synthetase genes. Microcystins could be detected in seven of the strains with considerable variability of contents and numbers of structural variants. Other known peptides like anabaenopeptins B and E/F, microviridin I, and prenylagaramide B and new variants of known peptide classes like aeruginosins and cyanopeptolins were detected in some strains while lacking in others. The 18 strains represented 15 chemotypes with respect to their peptide patterns. In contrast, all strains were morphologically very similar with respect to cell dimensions and pigmentation. Given the diversity of chemotypes among the randomly selected isolates, an immense diversity of chemotypes in the entire population can be assumed.     

Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability

Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human zipcode-binding protein IMP1 on targets in the 3′-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a ‘locked’ stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities within RNP granules and with the localization apparatus.     

<Emphasis Type="Italic">Yr32</Emphasis> for resistance to stripe (yellow) rust present in the wheat cultivar Carstens V

Stripe or yellow rust of wheat, caused by Puccinia striiformis f. sp. tritici, is an important disease in many wheat-growing regions of the world. A number of major genes providing resistance to stripe rust have been used in breeding, including one gene that is present in the differential tester Carstens V. The objective of this study was to locate and map a stripe rust resistance gene transferred from Carstens V to Avocet S and to use molecular tools to locate a number of genes segregating in the cross Savannah/Senat. One of the genes present in Senat was predicted to be a gene that is present in Carstens V. For this latter purpose, stripe rust response data from both seedling and field tests on a doubled haploid population consisting of 77 lines were compared to an available molecular map for the same lines using a non-parametric quantitative trait loci (QTL) analysis. Results obtained in Denmark suggested that a strong component of resistance with the specificity of Carstens V was located in chromosome arm 2AL, and this was consistent with chromosome location work undertaken in Australia. Since this gene segregated independently of Yr1, the only other stripe rust resistance gene known to be located in this chromosome arm, it was designated Yr32. Further QTLs originating from Senat were located in chromosomes 1BL, 4D, and 7DS and from Savannah on 5B, but it was not possible to characterize them as unique resistance genes in any definitive way. Yr32 was detected in several wheats, including the North American differential tester Tres.     

Effects of climate on incidence of Campylobacter spp. in humans and prevalence in broiler flocks in Denmark

Campylobacter infections are increasing and pose a serious public health problem in Denmark. Infections in humans and broiler flocks show similar seasonality, suggesting that climate may play a role in infection. We examined the effects of temperature, precipitation, relative humidity, and hours of sunlight on Campylobacter incidence in humans and broiler flocks by using lag dependence functions, locally fitted linear models, and cross validation methods. For humans, the best model included average temperature and sunlight 4 weeks prior to infection; the maximum temperature lagged at 4 weeks was the best single predictor. For broilers, the average and maximum temperatures 3 weeks prior to slaughter gave the best estimate; the average temperature lagged at 3 weeks was the best single predictor. The combined effects of temperature and sunlight or the combined effects of temperature and relative humidity predicted the incidence in humans equally well. For broiler flock incidence these factors explained considerably less. Future research should focus on elements within the broiler environment that may be affected by climate, as well as the interaction of microclimatic factors on and around broiler farms. There is a need to quantify the contribution of broilers as a source of campylobacteriosis in humans and to further examine the effect of temperature on human incidence after this contribution is accounted for. Investigations should be conducted into food consumption and preparation practices and poultry sales that may vary by season.     

Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability

Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from apoptosis when transdifferentiating into osteocytes. By examination of osteoblasts and osteocytes embedded in calvarial bone in the MT1-MMP deficient mice, we found that MT1-MMP deficient mice had 10-fold higher levels of apoptotic osteocytes than wild-type controls. We have previously shown that MT1-MMP activates latent Transforming Growth Factorbeta (TGF-beta). These findings strongly suggest that MT1-MMP-activated TGF-beta maintains osteoblast survival during transdifferentiation into osteocytes, and maintains mature osteocyte viability. Thus, the interrelationship of MMPs and TGF-beta may play an important role in bone formation and maintenance.     

Identification and characterization of a novel Chlamydia trachomatis reticulate body protein

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.     

Pore membrane and/or filament interacting like protein 1 (POMFIL1) is predominantly expressed in the nervous system and encodes different protein isoforms

We have isolated and characterized a novel differentially spliced gene predominantly expressed in the nervous system, which encodes protein isoforms with significant homology to the alpha-actinin protein superfamily, the Caenorhabditis elegans UNC-53 protein and weak homology to the nuclear membrane protein POM121. Similar to POM121 the primary structures show a hydrophobic region that is likely to form one or more adjacent transmembrane segment(s). Indirect immunofluorescence with antibodies against a synthetic peptide gave staining of the nucleus. Target experiments with EGFP (enhanced green fluorescent protein)-fusion proteins confirmed the nuclear localization. Two further members of this gene family could be isolated. All three pore membrane and/or filament interacting like (POMFIL) genes are differentially expressed in neuronal tumor cell lines. In 40% of tested primary neuroblastomas expression of POMFIL1 is strongly reduced and after brain injury POMFIL1 protein expression is upregulated, indicating that POMFIL1 is involved in the process of neuron growth and regeneration, as well as in neural tumorigenesis.     

Cardiotrophin-1 stimulates endothelin-1 via gp130 in vascular endothelial cells

Endothelin-1 (ET-1) is a vasoconstricting and mitogenic peptide released from vascular endothelial cells under normal and pathophysiological conditions, and synthesis and secretion of ET-1 are stimulated by cytokines. Cardiotrophin-1 (CT-1) is a new member of the interleukin-6-type cytokines that induce biological actions through the glycoprotein (gp) 130. The present study was designed to determine the presence of CT-1 and the gp130 cytokine system in vascular endothelial cells and to investigate whether CT-1 stimulates synthesis and secretion of ET-1 in the vascular endothelial cells. We first sought to determine gene expression and immunoreactivity of CT-1, gp130 and ET-1 in cultured canine aortic endothelial cells (CAECs) using Northern blot analysis and immunocytochemistry, which revealed the presence of CT-1 and gp130 together with ET-1 in CAECs. CT-1 increased ET-1 gene expression in CAECs, and stimulated ET-1 secretion from CAECs in a dose-dependent manner. Furthermore, inhibition of gp130 by monoclonal antibody attenuated ET-1 secretion from CAECs, suggesting that actions of CT-1 on the secretion of ET-1 are mediated through gp130 receptor system. The present study, therefore, reports the presence of CT-1 and gp130 in vascular endothelial cells and mechanisms of secretion of ET-1 related to this cytokine system.     

The development of potential screens based on shoot calcium and iron concentrations for the evaluation of tolerance in Egyptian genotypes of white lupin (Lupinus albus L.) to limed soils

European cultivars of white lupin (Lupinus albus L.) grow poorly in limed or calcareous soils. However, Egyptian genotypes are grown successfully in highly calcareous soil and show no stress symptoms. To examine their physiological responses to alkaline soil and develop potential screens for tolerance, three experiments were conducted in limed and non-limed (neutral pH) soil. Measurements included net CO2 uptake, and the partitioning of Fe2+ and Fe3+ and soluble and insoluble Ca in stem and leaf tissue. Intolerant plants showed clear symptoms of stress, whereas stress in the Egyptian genotypes and in L pilosus Murr. (a tolerant species) was less marked. Only the intolerant plants became chlorotic and this contributed to their reduced net CO2 uptake in the limed soil. In contrast, Egyptian genotypes and L pilosus showed no change in net CO2 uptake between the soils. The partitioning of Ca and Fe either resulted from the stress responses, or was itself a stress response. L pilosus and some Egyptian genotypes differed in soluble Ca concentrations compared with the intolerant cultivars, although no significant difference was apparent in the Ca partitioning of the Egyptian genotype Giza 1. In a limed soil, Giza 1 maintained its stem Fe3+ concentration at a level comparable with that of plants grown in non-limed soil, whereas stem [Fe3+] of an intolerant genotype increased. Gizal increased the percentage of plant Fe that was Fe2+ in its leaf tissue under these conditions; that of the intolerant genotype was reduced. The potential tolerance of the Egyptian genotypes through these mechanisms and the possibility of nutritional-based screens are discussed.