Transmission vs. Diffuse Transmission in Circular Dichroism: What to Choose for Probing Solid‐State Samples?

Recent advances in equipment enabled the collection of solid‐state electronic circular dichroism (ECD) spectra using the commercially available integrating sphere attachment for a regular ECD spectrometer. This accessory was designed to reduce negative factors occurring in solid‐state ECD measurements, and is, thereby, very useful for recording diffuse transmittance CD (DTCD) spectra using the pellet technique. In the present article, the operating principle of the integrating sphere and utility of the DTCD method in recording solid‐state ECD spectra is demonstrated. Based on illustrative examples, i.e., 10‐camphorsulfonic acid ammonium, cholest‐4‐en‐3‐one, (3R,4R,5S)‐oseltamivir, and (S)‐linezolid, ECD solid‐state measurements were performed by means of both transmission and diffusion methods and later compared. Selection of these compounds as models for comparative studies was made in view of their different chromophoric systems and the profound importance in the pharmaceutical industry. During the course of this work the benefits and limitations of the use of integrating sphere are presented. The final conclusion is that more relevant solid‐state spectra can be obtained by means of the DTCD method. Chirality 27:441–448, 2015. © 2015 Wiley Periodicals, Inc.     

An unstructured model for studies on phenanthrene bioconversion by filamentous fungus Cunninghamella elegans

An unstructured model for phenanthrene bioconversion by filamentous fungus Cunnighamella elegans in stirred tank batch bioreactors was proposed. It was observed that the process of phenanthrene bioconversion is strictly associated with exponential growth of biomass. Therefore, a Monod-type, with Contois term, unstructured model could be applied to describe the process mathematically. The inhibition of biomass growth due to high concentrations of phenanthrene present in the medium was taken into account in the form of Yerusalimski's uncompetitive inhibition term.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

NETosis occurs independently of neutrophil serine proteases

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.     

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10^?9 nor 10^?6 mol/L concentration. Ghrelin (10^?6 mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10^?6 mol/L ghrelin increased, while 10^?4 mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.     

Morphology of the epithelial cells and expression of androgen receptor in rat prostate dorsal lobe in experimental hyperprolactinemia

The effect of hyperprolactinemia on the prostate has not been well investigated. Since androgens play an important role in prostate development, growth and function, the goal of the present study was to estimate the influence of hyperprolactinemia on expression of the androgen receptor (AR) in rat epithelial cells of prostate dorsal lobe and on morphology of these cells. Studies were performed on sexually mature male Wistar rats. The experimental group rats received metoclopramide (MCP) intraperitoneally to provoke hyperprolactinemia. The control group animals were given saline in the same way. For light and electron microscopy the prostate dorsal lobes were obtained routinely. To evaluate the intensity of immunohistochemical reaction for AR in epithelial cells, the optical density was measured and computer-assisted image analysis system was used. Morphological observations of the dorsal lobe epithelial cells were carried out in transmission electron microscope. MCP caused over twofold increase in prolactin (PRL) serum levels. In rats with hyperprolactinemia, the testosterone levels (T) were twofold decreased. The intensity of immunohistochemical reaction for AR in epithelial cells of dorsal lobe in the experimental group was significantly lower than in the control group. In the dorsal lobe epithelial cells of experimental group animals, the transmission electron microscopy (TEM) revealed highly dilated RER cisternae and reduced number of microvilli on the cellular surface when compared to the control group. The results show that hyperprolactinemia in male rats causes morphological abnormalities in the dorsal lobe of prostate. The abnormalities are caused by elevated prolactin either directly or indirectly through decreased level of testosterone. Decreased expression of AR in epithelial cells of prostate dorsal lobe is likely to be caused by decreased testosterone level.     

Species diversity and phylogeography of Cornus kousa (Asian dogwood) captured by genomic and genic microsatellites

Cornus kousa (Asian dogwood), an East Asia native tree, is the most economically important species of the dogwood genus, owing to its desirable horticultural traits and ability to hybridize with North America‐native dogwoods. To assess the species genetic diversity and to better inform the ongoing and future breeding efforts, we assembled an herbarium and arboretum collection of 131 noncultivated C. kousa specimens. Genotyping and capillary electrophoresis analyses of our C. kousa collection with the newly developed genic and published nuclear genomic microsatellites permitted assessment of genetic diversity and evolutionary history of the species. Regardless of the microsatellite type used, the study yielded generally similar insights into the C. kousa diversity with subtle differences deriving from and underlining the marker used. The accrued evidence pointed to the species distinct genetic pools related to the plant country of origin. This can be helpful in the development of the commercial cultivars for this important ornamental crop with increased pyramided utility traits. Analyses of the C. kousa evolutionary history using the accrued genotyping datasets pointed to an unsampled ancestor population, possibly now extinct, as per the phylogeography of the region. To our knowledge, there are few studies utilizing the same gDNA collection to compare performance of genomic and genic microsatellites. This is the first detailed report on C. kousa species diversity and evolutionary history inference.     

Lipopeptide Biosurfactant Pseudofactin II Induced Apoptosis of Melanoma A 375 Cells by Specific Interaction with the Plasma Membrane

In the case of melanoma, advances in therapies are slow, which raises the need to evaluate new therapeutic strategies and natural products with potential cancer cell inhibiting effect. Pseudofactin II (PFII), a novel cyclic lipopeptide biosurfactant has been isolated from the Arctic strain of Pseudomonas fluorescens BD5. The aim of this study was to investigate the effect of PFII on A375 melanoma cells compared with the effect of PFII on Normal Human Dermis Fibroblast (NHDF) cells and elucidate the underlying mechanism of PFII cytotoxic activity. Melanoma A375 cells and NHDF cells were exposed to PFII or staurosporine and apoptotic death was assessed by monitoring caspase 3-like activity and DNA fragmentation. From time-dependent monitoring of lactate dehydrogenase (LDH) release, Ca²⁺ influx, and a correlation between Critical Micelle Concentration (CMC) we concluded that cell death is the consequence of plasma membrane permeabilisation by micelles. This finding suggests that pro-apoptotic mechanism of PFII is different from previously described cyclic lipopeptides. The mechanism of PFII specificity towards malignant cells remains to be discovered. The results of this study show that PFII could be a new promising anti-melanoma agent.     

Study of the Serum Copper Levels in Patients with Major Depressive Disorder

Copper may be involved in the pathophysiology of depression. Clinical data on this issue are very limited and not conclusive. The purpose of the study was to determine the copper concentration in the serum of patients with major depressive disorder and to discuss its potential clinical usefulness as a biomarker of the disease. A case–control clinical study included 69 patients with current depressive episode, 45 patients in remission and 50 healthy volunteers. Cu concentration was measured by electrothermal atomic absorption spectrometry (ETAAS). The mean serum copper level in depressed patients was slightly lower (by 11 %; not statistically significant) than in the control group. Furthermore, there was no significant difference in Cu²⁺ concentration between depressive episode and remission, nor between remission and control group. In the remission group were observed significant correlations between copper levels and the average number of relapses over the past years or time of remission. There was no correlation between serum copper and severity of depression, as measured by HDRS and MADRS. The obtained results showed no significant differences between the copper concentration in the blood serum of patients (both with current depressive episode and in remission) and healthy volunteers, as well as the lack of correlations between the copper level in the active stage of the disease and clinical features of the population. Our study is the first conducted on such a large population of patients, so the results may be particularly important and reliable source of knowledge about the potential role of copper in depression.