Comparison of the effect of calcium(II) and manganese(II) ions on trypsin autolysis

The effect of Mn²⁺ and Ca²⁺ ions on the rate of trypsin autolysis was studied at pH 7.0 and at 34.4-60.2°C. For comparison, the kinetic constants of esterolytic activity of trypsin in the presence of the metal ion were determined at pH 7.4 and at 36° and 40°C. There was no significant difference in the rate of autolysis between Mn²⁺ and Ca²⁺ in the temperature range 34-47°C, but at 56.8° and 60.2° autolysis was slightly more rapid in the presence of Mn²⁺. The Mn²⁺ or Ca²⁺ ion bound to trypsin is supposed to control the conformation and thereby the stability and the activity of the enzyme. This indirect effect of Mn²⁺ and Ca²⁺ is discussed on a structural basis of the enzyme molecule.     

The nature of absorbance changes following the addition of ADP to mitochondria: ATP synthesis from intramitochondrial inorganic phosphate

The transient absorbance increase induced by ADP in phosphate-loaded respiring mitochondria becomes stable and greatly amplified by inhibitors of phosphate transport. The absorbance changes are sensitive to oligomycin and to aurovertin and their extent is proportional to the amount of ADP added. Simultaneously with the ADP-dependent increase in absorbance the inorganic phosphate and K⁺-ion content of the matrix decreases. It is concluded that the optical change reflects contraction of the matrix compartment secondary to intramitochondrial solute changes.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Extracts and Steroids from the Edible Mushroom Hypholoma lateritium Exhibit Anti‐Inflammatory Properties by Inhibition of COX‐2 and Activation of Nrf2

Hypholoma lateritium is an edible macrofungus with a common distribution in Europe, North America, and the Far East. The aim of this study was to investigate the potential anti‐inflammatory effects of H. lateritium extracts and its isolated steroids: fasciculic acid B, fasciculol E, fasciculol C, lanosta‐7,9(11)‐diene‐12β,21α‐epoxy‐2α,3β,24β,25‐tetraol, fasciculol F, and demethylincisterol A2. Organic (hexane, chloroform and 50 % methanol) and water extracts of H. lateritium were subjected to in vitro assays to determine pro‐inflammatory protein levels, such as cyclooxygenase‐2 (COX‐2), cytosolic prostaglandin E2 synthase (cPGES), and antioxidant nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). Fungal extracts demonstrated significant activities on pro‐inflammatory protein levels with minor differences among the activities of the fractions of different polarities. All the compounds proved to exert notable inhibitory properties on COX‐2 and were capable to stimulate the Nrf2 pathway. Fungal extracts and the compounds exerted no cytotoxic activities on RAW 264.7 cells.     

Edwardsiella ictaluri evpP is required for colonisation of channel catfish ovary cells and necrosis in anterior kidney macrophages

Edwardsiella ictaluri is a Gram‐negative facultative anaerobe that can survive inside channel catfish phagocytes. E. ictaluri can orchestrate Type VI Secretion System (T6SS) for survival in catfish macrophages. evpP encodes one of the T6SS translocated effector proteins. However, the role of evpP in E. ictaluri is still unexplored. In this work, we constructed an E. ictaluri evpP mutant (EiΔevpP) and assessed its survival under complement and oxidative stress. Persistence of EiΔevpP in catfish as well as attachment and invasion in catfish macrophage and ovary cells were determined. Further, virulence of EiΔevpP in catfish and apoptosis it caused in macrophages were explored. EiΔevpP behaved same as wild type (EiWT) under complement and oxidative stress in complex media, whereas oxidative stress affected mutant's survival significantly in minimal media (p < .05). Persistence of EiΔevpP in live catfish and uptake and survival inside peritoneal macrophages were similar. The attachment and invasion capabilities of EiΔevpP in catfish ovary cells were significantly less than that of EiWT (p < .05). Although EiΔevpP showed reduced attenuation in catfish, causing decreased catfish mortality compared with EiWT (44.73% vs. 67.53%), this difference was not significant. The apoptosis assay using anterior kidney macrophages indicated that the number of live macrophages exposed to EiΔevpP was significantly higher compared with EiWT exposed macrophages at 24‐hr post‐treatment (p < .05). However, there were no significant differences in the early and late apoptosis. Remarkably, necrosis in EiΔevpP exposed macrophages was significantly less than that of EiWT exposed macrophages at 24 hr (p < .05). Our results demonstrated that evpP is required for colonisation of catfish ovary cells and increased apoptosis and necrosis in anterior kidney macrophages.     

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.     

Isolation and identification of sapotexanthin 5,6-epoxide and 5,8-epoxide from red mamey (Pouteria sapota)

Two new carotenoids, sapotexanthin 5,6-epoxide and sapotexanthin 5,8-epoxide, have been isolated from the ripe fruits of red mamey (Pouteria sapota). Sapotexanthin 5,6-epoxide was also prepared by partial synthesis via epoxidation of sapotexanthin, and the (5R,6S) and (5S,6R) stereoisomers were identified by high-performance liquid chromatography–electronic circular dichroism (HPLC-ECD) analysis. Spectroscopic data of the natural and semisynthetic derivatives obtained by acid-catalyzed rearrangement of cryptocapsin 5,8-epoxide stereoisomers were compared for structural elucidation.     

Trigonostemons G and H,dinorditerpenoid dimers with axially chiral biaryl linkage from Trigonostemon chinensis

Trigonostemons G and H, two novel dimeric dinorditerpenoids, were isolated from the stem barks of Trigonostemon chinensis. Their planar structures and relative configurations were established by extensive analysis of spectroscopic data. Trigonostemons G and H possess a homodimeric biaryl skeleton obtained from two rearranged chiral nonracemic abietane-type dinorditerpenes through an axially chiral biaryl 11,11′-linkage. Torsional scan and computation of the transition states were carried out to estimate the rotational energy barrier, and the axial chirality (aS) was determined by time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The positive n-π* ECD transitions of the isolated carbonyl chromophore above 300 nm could be used to determine the central chirality of trigonostemon G independently by ECD calculations of the diastereomers.     

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.