Structure of thylakoids in cells of Rhodopseudomonas viridis as influenced by growth conditions

A vessel was constructed for growth of photosynthetic bacteria at defined light intensity, temperature and partial pressure of oxygen.Under growth conditions at light intensities larger than 1,000 lx, the particles exposed by freeze-fracturing of thylakoids are unordered.Under growth conditions at light intensities lower than 30 lx, the particles seen are hexagonally arranged. If the oxygen partial pressure is increased from 0 to 30 mm Hg while keeping the light intensity at 30 lx, the particles seen in the thylakoids are found to be unregularly arranged.The protein pattern of thylakoids isolated from bacteria grown either at 2,000 lx or at 30 lx revealed a constant ratio of reaction centre polypeptide to either of the membrane polypeptides of 8 kdalton apparent Mr and 12 kdalton apparent Mr.Dedicated to Prof. Dr. G. Drews on occasion of his 60th birthday     

The determination of the electron density profile of the human erythrocyte ghost membrane by small-angle x-ray diffraction.

Diffraction patterns of stacked hemolyzed erythrocyte ghosts in the wet state were recorded. Three orders of a surprisingly high first-order periodicity of 600 A were detected. The scattering curves were evaluated by the Q-function method, including lattice distortions of the one-dimensional multilamellar system. The resulting electron density profile of the membrane in the wet state is strongly asymmetric. It consists of an asymmetrical bilayer-type part and an excess of positive relative electron density at the inner, cytoplasmic, side of the membrane. The extension of the whole membrane profile in the wet state is 100--120 A. We suggest that the innermost positive density peak mainly represents the loosely bound protein components spectrin and actin, located at the cytoplasmic side of the membrane and sometimes seen as "fuzzy" material on electron micrographs.     

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP–IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5–20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.     

Alcohol Impairs Predation Risk Response and Communication in Zebrafish

The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.     

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background

An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods

Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results

Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion

We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.     

Automated Entire Thrombus Density Measurements for Robust and Comprehensive Thrombus Characterization in Patients with Acute Ischemic Stroke

Background and Purpose

In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements.

Materials and Method

In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described using medians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between the median of entire thrombus measurements and commonly applied manual measurements using 3 regions of interest were determined using linear regression.

Results

Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density.

Conclusions

Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, discrimination of low and high density thrombi is improved with the automated method.     

A plasmid-based expression system to study protein–protein interactions at the Golgi in vivo

There is still an unmet need for simple methods to verify, visualize, and confirm protein–protein interactions in vivo. Here we describe a plasmid-based system to study such interactions. The system is based on the transmembrane domain (TMD) of the EF-hand Ca²⁺ sensor protein calneuron-2. We show that fusion of 28 amino acids that include the TMD of calneuron-2 to proteins of interest results in prominent localization on the cytoplasmic side of the Golgi. The recruitment of binding partners to the protein of interest fused to this sequence can then be easily visualized by fluorescent tags.     

PEtab—Interoperable specification of parameter estimation problems in systems biology

Reproducibility and reusability of the results of data-based modeling studies are essential. Yet, there has been—so far—no broadly supported format for the specification of parameter estimation problems in systems biology. Here, we introduce PEtab, a format which facilitates the specification of parameter estimation problems using Systems Biology Markup Language (SBML) models and a set of tab-separated value files describing the observation model and experimental data as well as parameters to be estimated. We already implemented PEtab support into eight well-established model simulation and parameter estimation toolboxes with hundreds of users in total. We provide a Python library for validation and modification of a PEtab problem and currently 20 example parameter estimation problems based on recent studies.     

Brevican isoforms associate with neural membranes

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.