The mode of action of primycin

Primycin, an antibiotic active against Gram-positive microorganisms increased the permeability ofBacillus subtilis cell membranes when used in bacteriostatic concentrations. On addition of the antibiotic to the washed cell suspension, a dose-dependent increase in the conductivity was observed. Furthermore, an enhanced leakage of the nucleotides (measured by the³²P-ATP release from the³²P-labelled culture) could be detected.To get more information about the mechanism of the primycin-membrane interaction, the effect of the antibiotic on the ATPase activity of membrane vesicles prepared from bothBacillus subtilis andEscherichia coli B was studied. Activation was found at about 0.5 nmol antibiotic/g protein and its extent was approximately the same as with sonicated membranes used as controls. Stimulation of ATPase activity was also achieved with vesicles prewashed with 3 mM Tris-HCl buffer.Purified membrane ATPase fromBacillus subtilis could not be activated by primycin at all; above 0.3 nmol/g protein concentration the enzyme was inhibited. When acting on membrane vesicles isolated fromEscherichia coli B, inhibition without previous activation was observed, although sonication caused a substantial activation on the ATPase of these membranes.These observations confirmed our suggestion that the primary target of primycin action is the cell membrane in Gram-positive microorganisms.Abbreviations OD Optical density     

Kinetics and dynamics of annealing during sub-gel phase formation in phospholipid bilayers: A saturation transfer electron spin resonance study

The saturation transfer electron spin resonance (STESR) spectra of spin-labeled phosphatidylcholine have been used to follow the kinetics of conversion from the gel phase to the sub-gel phase in aqueous bilayers of dipalmitoyl phosphatidylcholine. This is a simple, well-defined model system for lipid domain formation in membranes. The integrated intensity of the STESR spectrum from the chain-labeled lipid first increases and then decreases with time of incubation in the gel phase at 0°C. The first, more rapid phase of the kinetics is attributed to the conversion of germ nuclei to growth nuclei of the sub-gel phase. The increase in STESR intensity corresponds to the reduction in chain mobility of spin labels located in the gel phase at the boundaries of the growth nuclei and correlates with the increase in the diagnostic STESR line height ratios over this time range. The second, slower phase of the kinetics is attributed to growth of the domains of the sub-gel phase. The decrease in STESR intensity over this time regime corresponds to exclusion of the spin-labeled lipids from the tightly packed sub-gel phase and correlates quantitatively with calibrations of the spin label concentration dependence of the STESR intensity in the gel phase. The kinetics of formation of the sub-gel phase are consistent with the classical model for domain formation and growth. At 0°C, the half-time for conversion of germ nuclei to growth nuclei is ∼7.7 h and domain growth of the sub-gel phase is characterized by a rate constant of 0.025 h^-1. The temperature dependence of the STESR spectra from samples annealed at 0°C suggests that the subtransition takes place via dissolution of sub-gel phase domains, possibly accompanied by domain fission.     

Platelet vinculin: a substrate of activated factor XIII

In addition to plasma, Factor XIII of blood coagulation (FXIII) is also present in the cytosol of platelets, monocytes and macrophages. However, its intracellular function has not yet been revealed. Activated Factor XIII (FXIIIa) is a transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) of highly restricted substrate specificity with only a few known protein substrates. In this report, we showed that FXIIIa can link dansylcadaverine, radiolabelled histamine and putrescine to vinculin. Quantitative determinations revealed that in the vinculin molecule a single glutamine residue can serve as acyl donor for the incorporation of small-molecular-weight amines. Vinculin could not be crosslinked to another vinculin molecule. It could be covalently bound, however, to fibrinogen, which indicates that the acyl donor glutamine residue can be engaged in an epsilon-(gamma-glutamyl)lysyl crosslink formation. Since it has been shown that platelet actin and myosin, two main components of cytoskeleton, are also substrates for FXIIIa, and that vinculin is associated to the cytoskeleton during platelet activation, the involvement of FXIII in the stabilization of cytoskeleton at certain phases of cellular function is a likely possibility.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

Erythropoietin Receptor Expression Is a Potential Prognostic Factor in Human Lung Adenocarcinoma

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.     

Different Desensitization Patterns for Sensory and Vascular TRPV1 Populations in the Rat: Expression,Localization and Functional Consequences

Background and purpose

TRPV1 is expressed in sensory neurons and vascular smooth muscle cells, contributing to both pain perception and tissue blood distribution. Local desensitization of TRPV1 in sensory neurons by prolonged, high dose stimulation is re-engaged in clinical practice to achieve analgesia, but the effects of such treatments on the vascular TRPV1 are not known.

Experimental approach

Newborn rats were injected with capsaicin for five days. Sensory activation was measured by eye wiping tests and plasma extravasation. Isolated, pressurized skeletal muscle arterioles were used to characterize TRPV1 mediated vascular responses, while expression of TRPV1 was detected by immunohistochemistry.

Key results

Capsaicin evoked sensory responses, such as eye wiping (3.6±2.5 versus 15.5±1.4 wipes, p<0.01) or plasma extravasation (evans blue accumulation 10±3 versus 33±7 µg/g, p<0.05) were reduced in desensitized rats. In accordance, the number of TRPV1 positive sensory neurons in the dorsal root ganglia was also decreased. However, TRPV1 expression in smooth muscle cells was not affected by the treatment. There were no differences in the diameter (192±27 versus 194±8 µm), endothelium mediated dilations (evoked by acetylcholine), norepinephrine mediated constrictions, myogenic response and in the capsaicin evoked constrictions of arterioles isolated from skeletal muscle.

Conclusion and implications

Systemic capsaicin treatment of juvenile rats evokes anatomical and functional disappearance of the TRPV1-expressing neuronal cells but does not affect the TRPV1-expressing cells of the arterioles, implicating different effects of TRPV1 stimulation on the viability of these cell types.     

Expanding the DNA alphabet in the fruit fly

DNA integrity is under the control of multiple pathways of nucleotide metabolism and DNA damage recognition and repair. Unusual sets of protein factors involved in these control mechanisms may result in tolerance and accumulation of non-canonical bases within the DNA. We investigate the presence of uracil in genomic DNA of Drosophila melanogaster. Results indicate a developmental pattern and strong correlations between uracil-DNA levels, dUTPase expression and developmental fate of different tissues. The intriguing lack of the catalytically most efficient uracil-DNA glycosylase in Drosophila melanogaster may be a general attribute of Holometabola and is suggested to be involved in the specific characteristics of uracil-DNA metabolism in these insects.