Human macrophage colony-stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing

The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV-1 cells expressing a short M-CSF clone secrete only 24 kd recombinant M-CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M-CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M-CSF protein heterogeneity. Most secreted MIA PaCa-2 M-CSF reacts with long-clone-specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa-2 M-CSF size heterogeneity. In addition, cell lysates also contain larger M-CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M-CSF protein heterogeneity results from both pre- and post-translational processing.     

Broad-host-range shuttle vectors for screening of regulated promoter activity in viridans group streptococci: isolation of a pH-regulated promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.     

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.     

Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase

Singlet oxygen ((1)O(2)) is a reactive oxygen species generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g., myeloperoxidase and eosinophil peroxidase). (1)O(2) oxidizes the free amino acids Trp, Tyr, His, Cys, and Met, and those species present on peptides/proteins, with this resulting in modulation of protein structure and function. Impairment of the activity of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathologies involving either light exposure or inflammation. In this study, the effects of (1)O(2) on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (an established source of (1)O(2)) resulted in significant, photolysis time-dependent reductions in enzyme activity (10-40%, P<0.05). More extensive inhibition (ca. 2-fold) was detected when the reactions were carried out in D(2)O, consistent with the intermediacy of (1)O(2). No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts rapidly with (1)O(2) (k～10(7)M(-1) s(-1))(,) significantly reduced photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH(4), DTT, GSH, or NADPH) added after the cessation of (1)O(2) formation were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of nonreducible protein aggregates and/or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes GPx and TrxR can be irreversibly inactivated by (1)O(2).     

Improvement of production,assay and purification of streptavidin

Summary The production of streptavidin byStreptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.     

The use of light-directed combinatorial peptide synthesis in epitope mapping

The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptidtes each allowed for the independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations. A binary synthesis strategy was used to assemble the arrays, resulting in all deletions and truncations possible within the FLRRQFKVVT sequence being present and available for screening. The relative binding interactions of each peptide was determined by incubating the arrays with either mAb D32.39 and goat antimouse immunoglobulin G–FITC or mAb D32.39 Fab-FITC conjugate, followed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVT was found to bind lightly to both the mAb and Fab fragment while tethered to the surface, and was measured to have 0.49 n M affinity in solution. The frame-shifted RRQFKVV sequence was found to have lower affinity both in solution (1.3 m M) and on the surface. The fragment RQFKVV was determined to be responsible for antibody recognition and was found to bind tightly when tethered to the surface, yet exhibited no binding in solution as the free acid, suggesting the requirement of an amidated C-terminus or an additional flunking residue. A deletion analysis revealed that the novel RQFKVT sequence exhibited higher affinity than the RQFKVV sequence while tethered to the surface. © 1994 John Wiley & Sons, Inc.     

Indirect effects shape macroalgal epifaunal communities

We tested the response of algal epifauna to the direct effects of predation and the indirect consequences of habitat change due to grazing and nutrient supply through upwelling using an abundant intertidal rhodophyte, Gelidium pristoides. We ran a mid‐shore field experiment at four sites (two upwelling sites interspersed with two non‐upwelling sites) along 450 km of the south coast of South Africa. The experiment was started in June 2014 and ran until June 2015. Four treatments (predator exclusion, grazer exclusion, control, and procedural control) set out in a block design (n = 5) were monitored monthly for algal cover for the first 6 months and every 2 months for the last 6 months. Epifaunal abundance, species composition, algal cover, and algal architectural complexity (measured using fractal geometry) were assessed after 12 months. Predation had no significant effect on epifaunal abundances, while upwelling interacted with treatment. Grazing reduced the architectural complexity of algae, with increased fractal dimensions in the absence of grazers, and also reduced algal cover at all sites, though the latter effect was only significant for upwelling sites. Epifaunal community composition was not significantly affected by the presence of herbivores or predators but differed among sites independently of upwelling; sites were more similar to nearby sites than those farther away. In contrast, total epifaunal abundance was significantly affected by grazing, when normalized to algal cover. Grazing reduced the cover of algae; thus, epifaunal abundances were not affected by the direct top‐down effects of predation but did respond to the indirect effects of grazing on habitat availability and quality. Our results indicate that epifaunal communities can be strongly influenced by the indirect consequences of biotic interactions.