Exploiting mixtures of H<Subscript>2</Subscript>, CO<Subscript>2</Subscript>, and O<Subscript>2</Subscript> for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered <Emphasis Type="Italic">Cupriavidus necator</Emphasis> strains

Current manufacturing of most bulk chemicals through petrochemical routes considerably contributes to common concerns over the depletion of fossil carbon sources and greenhouse gas emissions. Sustainable future production of commodities thus requires the shift to renewable feedstocks in combination with established or newly developed synthesis routes. In this study, the potential of Cupriavidus necator H16 for autotrophic synthesis of the building block chemical 2-hydroxyisobutyric acid (2-HIBA) is evaluated. A novel biosynthetic pathway was implemented by heterologous expression of the 2-hydroxyisobutyryl-coenzyme A (2-HIB-CoA) mutase from Aquincola tertiaricarbonis L108, relying on a main intermediate of strain H16’s C4 overflow metabolism, 3-hydroxybutyryl-CoA. The intention was to direct the latter to 2-HIBA instead or in addition to poly-3-hydroxybutyrate (PHB). Autotrophic growth and 2-HIBA (respectively, PHB) synthesis of wild-type and PHB-negative mutant strains were investigated producing maximum 2-HIBA titers of 3.2 g L^?1 and maximum specific 2-HIBA synthesis rates (q _2-HIBA) of about 16 and 175 μmol g^?1 h^?1, respectively. The obtained specific productivity was the highest reported to date for mutase-dependent 2-HIBA synthesis from heterotrophic and autotrophic substrates. Furthermore, expression of a G protein chaperone (MeaH) in addition to the 2-HIB-CoA mutase subunits yielded improved productivity. Analyzing the inhibition of growth and product synthesis due to substrate availability and product accumulation revealed a strong influence of 2-HIBA, when cells were cultivated at high titers. Nevertheless, the presented results imply that at the time the autotrophic synthesis route is superior to thus far established heterotrophic routes for production of 2-HIBA with C. necator.     

Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis of large collagen fragments. First, we show that collagen that has been pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native collagen by uPARAP/Endo180-positive cells. Second, we demonstrate that this preference is governed by the acquisition of a gelatin-like structure by the collagen, occurring upon collagenase-mediated cleavage under native conditions. Third, we demonstrate that the growth of uPARAP/Endo180-deficient fibroblasts on a native collagen matrix leads to substantial extracellular accumulation of well defined collagen fragments, whereas, wild-type fibroblasts possess the ability to direct an organized and complete degradation sequence comprising both the initial cleavage, the endocytic uptake, and the intracellular breakdown of collagen.     

Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA

Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.     

Recombinant human bile salt-stimulated lipase: an example of defectiveO-glycosylation of a protein produced in milk of transgenic mice

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprisingbssl cDNA, or alternatively genomicbssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomicbssl sequences mediated high levels (0.5–1, mg ml^–1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree ofO-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same, stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavilyO-glycosylated makes species-related restrictions less attractive as an explanation for the reducedO-glycosylation.     

Monitoring therapeutical intervention with ezetimibe using targeted near-infrared fluorescence imaging in experimental atherosclerosis

Ezetimibe (EZE), an inhibitor of cholesterol absorption, reduces atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. The matrix protein ED-B fibronectin (ED-B) is upregulated in atherosclerotic lesions. Using a novel conjugate for near-infrared fluorescence (NIRF) imaging targeting ED-B, we studied the effect of EZE on plaque lesion formation in apoE(-/-) mice. ApoE(-/-) mice received EZE (5 mug/kg/d) or chow up to the age of 4, 6, and 8 months. NIRF imaging of aortic lesions was performed 24 hours after intravenous application ex vivo and in vivo. Plaque lesion formation was analyzed by histology and immunohistochemistry. Aortic lesion formation detected by Sudan staining and NIRF imaging was significantly reduced at 6 and 8 months (p < .001). Plaque areas determined by NIRF imaging significantly correlated with Sudan staining (p < .001). EZE treatment resulted in a significant reduction in plaque macrophage and ED-B immunoreactivity (both p < .05) in brachiocephalic lesions. There was a significant reduction in plaque size in brachiocephalic arteries in 8-month-old mice treated with EZE compared with mice during short-term treatment (p < .05), indicating EZE plaque regression. Targeted NIRF imaging showed a correlation to histologic lesion extension during therapeutical intervention in experimental atherosclerosis.     

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in β-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of β1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAcβ1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.     

SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome

Linkage analysis and DNA sequencing in a family exhibiting an X-linked mental retardation (XLMR) syndrome, characterized by microcephaly, epilepsy, ataxia, and absent speech and resembling Angelman syndrome, identified a deletion in the SLC9A6 gene encoding the Na(+)/H(+) exchanger NHE6. Subsequently, other mutations were found in a male with mental retardation (MR) who had been investigated for Angelman syndrome and in two XLMR families with epilepsy and ataxia, including the family designated as having Christianson syndrome. Therefore, mutations in SLC9A6 cause X-linked mental retardation. Additionally, males with findings suggestive of unexplained Angelman syndrome should be considered as potential candidates for SLC9A6 mutations.     

A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.     

Acoustic and Temporal Variation in Gelada (<Emphasis Type="Italic">Theropithecus gelada</Emphasis>) Loud Calls Advertise Male Quality

Many animals rely on information from vocal signals to assess potential competitors and mates. For example, in primates, males use loud calls to assess rivals when the acoustic properties of the calls reliably indicate the condition or quality of the sender. Here, we investigate whether the loud calls of male geladas (Theropithecus gelada) function as a quality signal. Gelada males produce loud calls during ritualistic chases with rival males. Given the physically taxing nature of these displays, we hypothesize that variation in the acoustic properties of loud calls reliably signal male stamina or competitive ability. To test this hypothesis, we examined whether the acoustic properties of the gelada loud call varied in relation to individual, age, status, and exhaustion. Specifically, we examined 12 call parameters (e.g., fundamental frequency) and 3 bout parameters (e.g., number of calls per bout), that have been previously shown to vary across condition in male primates. We found that several acoustic features varied consistently across age and status such that males deemed higher quality in gelada society (e.g., high status) produced more calls per bout, produced calls that were lower in overall frequency measures, and exhibited a greater vocal range. In addition, we found that similar acoustic features varied with exhaustion; after a long chase event, males produced both fewer calls per bout and calls with higher spectral measures. Results from this study are consistent with the hypothesis that gelada loud calls are quality signals, contributing to the growing evidence that primates may use acoustic information to assess the quality of a rival or a potential mate.     

Vascular damage after fractionated whole-brain irradiation in rats

Whole-brain irradiation of animals and humans has been reported to lead to late delayed structural (vascular damage, demyelination, white matter necrosis) and functional (cognitive impairment) alterations. However, most of the experimental data on late delayed radiation-induced brain injury have been generated with large single doses or short fractionation schemes that may provide a less accurate indication of the events that occur after clinical whole-brain radiotherapy. The pilot study reported here investigates cerebral vascular pathology in male Fischer 344 rats after whole-brain irradiation with a fractionated total dose of 137Cs gamma rays that is expected to be biologically similar to that given to brain tumor patients. The brains of young adult rats (4 months old) were irradiated with a total dose of 40 Gy, given as eight 5-Gy fractions twice per week for 4 weeks. Brain capillary and arteriole pathology was studied using an alkaline phosphatase enzyme histochemistry method; vessel density and length were quantified using a stereology method with computerized image processing and analysis. Vessel density and length were unchanged 24 h after the last dose, but at 10 weeks postirradiation, both were substantially decreased. After 20 weeks, the rate of decline in the vessel density and length in irradiated rats was similar to that in unirradiated age-matched controls. No gross gliosis or demyelination was observed 12 months postirradiation using conventional histopathology techniques. We suggest that the early (10-week) and persistent vascular damage that occurs after a prolonged whole-brain irradiation fractionation scheme may play an important role in the development of late delayed radiation-induced brain injury.