Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.     

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated exchange and less efficiently mediated Cl^–/H⁺ exchange. Mutating the “gating glutamate” Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl^– than . Replacing the “proton glutamate” Glu-270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl^–/H⁺ exchange, their transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated accumulation in plant vacuoles requires tight coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl^–/H⁺ exchange of AtClC-a proceeded as efficiently as exchange, suggesting a role of this residue in exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl^–/H⁺ exchanger gained efficient coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for exchange and conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either mediate electrogenic anion/proton exchange or function as chloride channels (1). In mammals, the roles of plasma membrane CLC Cl^– channels include transepithelial transport (2–5) and control of muscle excitability (6), whereas vesicular CLC exchangers may facilitate endocytosis (7) and lysosomal function (8–10) by electrically shunting vesicular proton pump currents (11). In the plant Arabidopsis thaliana, there are seven CLC isoforms (AtClC-a–AtClC-g)² (12–15), which may mostly reside in intracellular membranes. AtClC-a uses the pH gradient across the vacuolar membrane to transport the nutrient nitrate into that organelle (16). This secondary active transport requires a tightly coupled exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1 (one of the two CLC isoforms in Escherichia coli) display tightly coupled Cl^–/H⁺ exchange, but anion flux is largely uncoupled from H⁺ when is transported (17–21). The lack of appropriate expression systems for plant CLC transporters (12) has so far impeded structure-function analysis that may shed light on the ability of AtClC-a to perform efficient exchange. This dearth of data contrasts with the extensive mutagenesis work performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues (22, 23) and the investigation of mutants (17, 19–21, 24–29) have yielded important insights into their structure and function. CLC proteins form dimers with two largely independent permeation pathways (22, 25, 30, 31). Each of the monomers displays two anion binding sites (22). A third binding site is observed when a certain key glutamate residue, which is located halfway in the permeation pathway of almost all CLC proteins, is mutated to alanine (23). Mutating this gating glutamate in CLC Cl^– channels strongly affects or even completely suppresses single pore gating (23), whereas CLC exchangers are transformed by such mutations into pure anion conductances that are not coupled to proton transport (17, 19, 20). Another key glutamate, located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark of CLC anion/proton exchangers. Mutating this proton glutamate to nontitratable amino acids uncouples anion transport from protons in the bacterial EcClC-1 protein (27) but seems to abolish transport altogether in mammalian ClC-4 and -5 (21). In those latter proteins, anion transport could be restored by additionally introducing an uncoupling mutation at the gating glutamate (21).The functional complementation by AtClC-c and -d (12, 32) of growth phenotypes of a yeast strain deleted for the single yeast CLC Gef1 (33) suggested that these plant CLC proteins function in anion transport but could not reveal details of their biophysical properties. We report here the first functional expression of a plant CLC in animal cells. Expression of wild-type (WT) and mutant AtClC-a in Xenopus oocytes indicate a general role of gating and proton glutamate residues in anion/proton coupling across different isoforms and species. We identified a proline in the CLC signature sequence of AtClC-a that plays a crucial role in exchange. Mutating it to serine, the residue present in mammalian CLC proteins at this position, rendered AtClC-a Cl^–/H⁺ exchange as efficient as exchange. Conversely, changing the corresponding serine of ClC-5 to proline converted it into an efficient exchanger. When proline replaced the critical serine in Torpedo ClC-0, the relative conductance of this model Cl^– channel was drastically increased, and “fast” protopore gating was slowed.     

A cost-effective solution to reduce dead volume of a standard dispenser system by a factor of 5

A key trend in high-throughput screening is assay miniaturization to control reagent costs and increase throughput. For this purpose, liquid-handling devices are used that transfer nano-to low-microliter volumes into all currently used microtiter well plates. One drawback of many available dispenser and pipetting systems are high dead volumes. Therefore, the authors were looking for an easy and simple solution to modify their standard liquid-handling device, PerkinElmer's FlexDrop Precision IV, allowing for a dead volume reduction to receive maximum benefit from miniaturized assay formats. Internal reservoirs were developed and constructed by Schering's Technical Development Laboratory (TDL), which are directly connected to the dispenser banks of FlexDrop without tubing. Using these newly built reservoirs, the dead volume was decreased by a factor of 5 in comparison to the manufacturer's reservoirs without compromising liquid-handling parameters such as accuracy and precision. The modified system displayed a high robustness and reliability under routine high-throughput screening conditions.     

Highly efficient transport of carboxyfluorescein diacetate succinimidyl ester into COS7 cells using human papillomavirus-like particles

Human papillomavirus virus-like particles (VLPs) have recently been used to deliver genes into mammalian cells in vitro and in vivo. Here, we investigated whether VLPs may serve as an efficient carrier of low molecular weight compounds (e.g. hormones, vitamins, peptides etc.) into cells. COS7 cells were incubated with recombinant HPV-16L1/L2 VLPs labelled with the fluorescence dye carboxyfluorescein diacetate succinimidyl ester. Using flow cytometry, we demonstrate that labelled VLPs can specifically bind to the cell surface followed by their complete internalisation. Our results indicate that VLPs are promising vehicles for highly efficient delivery of low molecular weight compounds into cells.     

Forest fragmentation and selective logging have inconsistent effects on multiple animal-mediated ecosystem processes in a tropical forest

Forest fragmentation and selective logging are two main drivers of global environmental change and modify biodiversity and environmental conditions in many tropical forests. The consequences of these changes for the functioning of tropical forest ecosystems have rarely been explored in a comprehensive approach. In a Kenyan rainforest, we studied six animal-mediated ecosystem processes and recorded species richness and community composition of all animal taxa involved in these processes. We used linear models and a formal meta-analysis to test whether forest fragmentation and selective logging affected ecosystem processes and biodiversity and used structural equation models to disentangle direct from biodiversity-related indirect effects of human disturbance on multiple ecosystem processes. Fragmentation increased decomposition and reduced antbird predation, while selective logging consistently increased pollination, seed dispersal and army-ant raiding. Fragmentation modified species richness or community composition of five taxa, whereas selective logging did not affect any component of biodiversity. Changes in the abundance of functionally important species were related to lower predation by antbirds and higher decomposition rates in small forest fragments. The positive effects of selective logging on bee pollination, bird seed dispersal and army-ant raiding were direct, i.e. not related to changes in biodiversity, and were probably due to behavioural changes of these highly mobile animal taxa. We conclude that animal-mediated ecosystem processes respond in distinct ways to different types of human disturbance in Kakamega Forest. Our findings suggest that forest fragmentation affects ecosystem processes indirectly by changes in biodiversity, whereas selective logging influences processes directly by modifying local environmental conditions and resource distributions. The positive to neutral effects of selective logging on ecosystem processes show that the functionality of tropical forests can be maintained in moderately disturbed forest fragments. Conservation concepts for tropical forests should thus include not only remaining pristine forests but also functionally viable forest remnants.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Determinants of anion-proton coupling in mammalian endosomal CLC proteins

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.     

Computational Pathology to Discriminate Benign from Malignant Intraductal Proliferations of the Breast

The categorization of intraductal proliferative lesions of the breast based on routine light microscopic examination of histopathologic sections is in many cases challenging, even for experienced pathologists. The development of computational tools to aid pathologists in the characterization of these lesions would have great diagnostic and clinical value. As a first step to address this issue, we evaluated the ability of computational image analysis to accurately classify DCIS and UDH and to stratify nuclear grade within DCIS. Using 116 breast biopsies diagnosed as DCIS or UDH from the Massachusetts General Hospital (MGH), we developed a computational method to extract 392 features corresponding to the mean and standard deviation in nuclear size and shape, intensity, and texture across 8 color channels. We used L1-regularized logistic regression to build classification models to discriminate DCIS from UDH. The top-performing model contained 22 active features and achieved an AUC of 0.95 in cross-validation on the MGH data-set. We applied this model to an external validation set of 51 breast biopsies diagnosed as DCIS or UDH from the Beth Israel Deaconess Medical Center, and the model achieved an AUC of 0.86. The top-performing model contained active features from all color-spaces and from the three classes of features (morphology, intensity, and texture), suggesting the value of each for prediction. We built models to stratify grade within DCIS and obtained strong performance for stratifying low nuclear grade vs. high nuclear grade DCIS (AUC = 0.98 in cross-validation) with only moderate performance for discriminating low nuclear grade vs. intermediate nuclear grade and intermediate nuclear grade vs. high nuclear grade DCIS (AUC = 0.83 and 0.69, respectively). These data show that computational pathology models can robustly discriminate benign from malignant intraductal proliferative lesions of the breast and may aid pathologists in the diagnosis and classification of these lesions.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.