Microfluidic Analysis for the Determination of Protein Content in Different Types of Plant-Based Drinks.

The widespread consumption of plant-based drinks, driven by health and dietary reasons (including cow's milk allergy, lactose intolerance, milk protein intolerance, following a vegetarian or vegan diet) necessitates automated and accurate test methods. Our study demonstrates the simultaneous determination of protein components and total protein concentrations in plant-based milk alternatives using a rapid and reproducible microchip gel electrophoretic method. As expected, the electrophoretic profiles of each plant-based drink differed. Based on our analyses and statistical evaluation, it can be determined that the protein profiles of different plant-based beverages do not differ significantly between different manufacturers or different expiry dates. The measured total protein content was compared with the nominal values, i.e., the values stated on the beverage labels. As the number of consumers of functional and specialized plant-based milk alternatives continues to rise, it is important to prioritize methods that provide qualitative and quantitative information on protein composition and other nutrients.

Effect of Culture Conditions on Fatty Acid Profiles of Bacteria and Lipopolysaccharides of the Genus Pseudomonas-GC-MS Analysis on Ionic Liquid-Based Column.

The profiling of bacterial fatty acids is a well-established technique in identifying and classifying bacteria. Cultivation conditions may affect the biosynthesis, thereby, changing the fatty acid profile in bacteria. The effect of the culture conditions on the fatty acid components of Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa polyresistant and Pseudomonas putida all are aligned to the genus Pseudomonas. The fatty acids in the lipopolysaccharides of Pseudomonas aeruginosa PAO1 were also examined. The effects of the cultivation conditions were followed by using agar and blood agar media at the characteristic temperatures, 25 °C, 37 °C and 42 °C, respectively, and an analysis was made during the 1st, 3rd and 5th day following inoculation. In addition to quantitative differences, we also experienced qualitative differences in the fatty acid profiles which detect newly appearing fatty acids, due to changes in environmental factors. The application of ionic liquid-based column unveils new possibilities for the analyses of fatty acids in GC-MS experiments for bacterial fatty acid profiling. The validation results (response linearity, limit of detection, limit of quantification, system suitability, intraday and interday repeatability and accuracy) show the high separation efficiency of the ionic liquid-based column in the analyses.

Chemotherapy induced PRL3 expression promotes cancer growth via plasma membrane remodeling and specific alterations of caveolae-associated signaling.

BACKGROUND: The outcome of cancer therapy is greatly defined by the ability of a tumor cell to evade treatment and re-establish its bulk mass after medical interventions. Consequently, there is an urgent need for the characterization of molecules affecting tumor reoccurrence. The phosphatase of regenerating liver 3 (PRL3) protein was recently emerged among the targets that could affect such a phenomenon. METHODS: The expression induction of PRL3 in melanoma cells treated with chemotherapeutic agents was assessed by western blotting. The effect of PRL3 expression on cancer growth was investigated both in vitro and in vivo. The association of PRL3 with the caveolae structures of the plasma membrane was analyzed by detergent free raft purification. The effect of PRL3 expression on the membrane organization was assayed by electron microscopy and by membrane biophysical measurements. Purification of the plasma membrane fraction and co-immunoprecipitation were used to evaluate the altered protein composition of the plasma membrane upon PRL3 expression. RESULTS: Here, we identified PRL3 as a genotoxic stress-induced oncogene whose expression is significantly increased by the presence of classical antitumor therapeutics. Furthermore, we successfully connected the presence of this oncogene with increased tumor growth, which implies that tumor cells can utilize PRL3 effects as a survival strategy. We further demonstrated the molecular mechanism that is connected with the pro-growth action of PRL3, which is closely associated with its localization to the caveolae-type lipid raft compartment of the plasma membrane. In our study, PRL3 was associated with distinct changes in the plasma membrane structure and in the caveolar proteome, such as the dephosphorylation of integrin ß1 at Thr788/Thr789 and the increased partitioning of Rac1 to the plasma membrane. These alterations at the plasma membrane were further associated with the elevation of cyclin D1 in the nucleus. CONCLUSIONS: This study identifies PRL3 as an oncogene upregulated in cancer cells upon exposure to anticancer therapeutics. Furthermore, this work contributes to the existing knowledge on PRL3 function by characterizing its association with the caveolae-like domains of the plasma membrane and their resident proteins.

Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Membrane fluidity matters: hyperthermia from the aspects of lipids and membranes.

Hyperthermia is a promising treatment modality for cancer in combination both with radio- and chemotherapy. In spite of its great therapeutic potential, the underlying molecular mechanisms still remain to be clarified. Due to lipid imbalances and 'membrane defects' most of the tumour cells possess elevated membrane fluidity. However, further increasing membrane fluidity to sensitise to chemo- or radiotherapy could have some other effects. In fact, hyperfluidisation of cell membrane induced by membrane fluidiser initiates a stress response as the heat shock protein response, which may modulate positively or negatively apoptotic cell death. Overviewing some recent findings based on a technology allowing direct imaging of lipid rafts in live cells and lipidomics, novel aspects of the intimate relationship between the 'membrane stress' of tumour cells and the cellular heat shock response will be highlighted. Our findings lend support to both the importance of membrane remodelling and the release of lipid signals initiating stress protein response, which can operate in tandem to control the extent of the ultimate cellular thermosensitivity. Overall, we suggest that the fluidity variable of membranes should be used as an independent factor for predicting the efficacy of combinational cancer therapies.

Nutritional lipid supply can control the heat shock response of B16 melanoma cells in culture.

The in vitro culture of cells offers an extremely valuable method for probing biochemical questions and many commonly-used protocols are available. For mammalian cells a source of lipid is usually provided in the serum component. In this study we examined the question as to whether the nature of the lipid could become limiting at high cell densities and, therefore, prospectively influence the metabolism and physiology of the cells themselves. When B16 mouse melanoma cells were cultured, we noted a marked decrease in the proportions of n-3 and n-6 polyunsaturated fatty acids (PUFAs) with increasing cell density. This was despite considerable quantities of these PUFAs still remaining in the culture medium and seemed to reflect the preferential uptake of unesterified PUFA rather than other lipid classes from the media. The reduction in B16 total PUFA was reflected in changes in about 70% of the molecular species of membrane phosphoglycerides which were analysed by mass spectrometry. The importance of this finding lies in the need for n-3 and n-6 PUFA in mammalian cells (which cannot synthesize their own). Although the cholesterol content of cells was unchanged the amount of cholesterol enrichment in membrane rafts (as assessed by fluorescence) was severely decreased, simultaneous with a reduced heat shock response following exposure to 42°C. These data emphasize the pivotal role of nutrient supply (in this case for PUFAs) in modifying responses to stress and highlight the need for the careful control of culture conditions when assessing cellular responses in vitro.

The Crystal Structure of the Hsp90-LA1011 Complex and the Mechanism by Which LA1011 May Improve the Prognosis of Alzheimer's Disease.

Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer's disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-ß (Aß) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.

Can the stress protein response be controlled by 'membrane-lipid therapy'?

In addition to high temperature, other stresses and clinical conditions such as cancer and diabetes can lead to the alteration of heat-shock protein (HSP) levels in cells. Moreover, HSPs can associate with either specific lipids or with areas of special membrane topology (such as lipid rafts), and changes in the physical state of cellular membranes can alter hsp gene expression. We propose that membrane microheterogeneity is important for regulating the HSP response. In support of this hypothesis, when particular membrane intercalating compounds are used to alter membrane properties, the simultaneous normalization of dysregulated expression of HSPs causes beneficial responses to disease states. Therefore, these compounds (such as hydroxylamine derivatives) have the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy'.

Distinct Cellular Tools of Mild Hyperthermia-Induced Acquired Stress Tolerance in Chinese Hamster Ovary Cells.

Mild stress could help cells to survive more severe environmental or pathophysiological conditions. In the current study, we investigated the cellular mechanisms which contribute to the development of stress tolerance upon a prolonged (0-12 h) fever-like (40 °C) or a moderate (42.5 °C) hyperthermia in mammalian Chinese Hamster Ovary (CHO) cells. Our results indicate that mild heat triggers a distinct, dose-dependent remodeling of the cellular lipidome followed by the expression of heat shock proteins only at higher heat dosages. A significant elevation in the relative concentration of saturated membrane lipid species and specific lysophosphatidylinositol and sphingolipid species suggests prompt membrane microdomain reorganization and an overall membrane rigidification in response to the fluidizing heat in a time-dependent manner. RNAseq experiments reveal that mild heat initiates endoplasmic reticulum stress-related signaling cascades resulting in lipid rearrangement and ultimately in an elevated resistance against membrane fluidization by benzyl alcohol. To protect cells against lethal, protein-denaturing high temperatures, the classical heat shock protein response was required. The different layers of stress response elicited by different heat dosages highlight the capability of cells to utilize multiple tools to gain resistance against or to survive lethal stress conditions.

Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane.

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-G(M1), which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

Lipidomics reveals membrane lipid remodelling and release of potential lipid mediators during early stress responses in a murine melanoma cell line.

Membranes are known to respond rapidly to various environmental perturbations by changing their composition and microdomain organization. In previous work we showed that a membrane fluidizer benzyl alcohol (BA) could mimic the effects of heat stress and enhance heat shock protein synthesis in different mammalian cells. Here we explore heat- and BA-induced stress further by characterizing stress-induced membrane lipid changes in mouse melanoma B16 cells. Lipidomic fingerprints revealed that membrane stress achieved either by heat or BA resulted in pronounced and highly specific alterations in lipid metabolism. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of the activation of phopholipases (mainly phopholipase A(2) and C). A phospholipase C-diacylglycerol lipase-monoacylglycerol lipase pathway was identified in B16 cells and contributed significantly to the production of several lipid mediators upon stress including the potent heat shock modulator, arachidonic acid. The accumulation of cholesterol, ceramide and saturated phosphoglyceride species with raft-forming properties observed upon both heat and BA treatments of B16 cells may explain the condensation of ordered plasma membrane domains previously detected by fluorescence microscopy and may serve as a signalling platform in stress responses or as a primary defence mechanism against the noxious effects of stresses.

HSP72 protects against obesity-induced insulin resistance.

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation.

PARP2 is a DNA repair protein. The deletion of PARP2 induces mitochondrial biogenesis and mitochondrial activity by increasing NAD+ levels and inducing SIRT1 activity. We show that the silencing of PARP2 causes mitochondrial fragmentation in myoblasts. We assessed multiple pathways that can lead to mitochondrial fragmentation and ruled out the involvement of mitophagy, the fusion-fission machinery, SIRT1, and mitochondrial unfolded protein response. Nevertheless, mitochondrial fragmentation was reversed by treatment with strong reductants, such as reduced glutathione (GSH), N-acetyl-cysteine (NAC), and a mitochondria-specific antioxidant MitoTEMPO. The effect of MitoTEMPO on mitochondrial morphology indicates the production of reactive oxygen species of mitochondrial origin. Elimination of reactive oxygen species reversed mitochondrial fragmentation in PARP2-silenced cells.

Genetic modification of the Salmonella membrane physical state alters the pattern of heat shock response.

It is now recognized that membranes are not simple physical barriers but represent a complex and dynamic environment that affects membrane protein structures and their functions. Recent data emphasize the role of membranes in sensing temperature changes, and it has been shown that the physical state of the plasma membrane influences the expression of a variety of genes such as heat shock genes. It has been widely shown that minor alterations in lipid membranes are critically involved in the conversion of signals from the environment to the transcriptional activation of heat shock genes. Previously, we have proposed that the composition, molecular arrangement, and physical state of lipid membranes and their organization have crucial roles in cellular responses during stress caused by physical and chemical factors as well as in pathological states. Here, we show that transformation of Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) with a heterologous Delta(12)-desaturase (or with its trans-membrane regions) causes major changes in the pathogen's membrane dynamic. In addition, this pathogen is strongly impaired in the synthesis of major stress proteins (heat shock proteins) under heat shock. These data support the hypothesis that the perception of temperature in Salmonella is strictly controlled by membrane order and by a specific membrane lipid/protein ratio that ultimately causes transcriptional activation of heat shock genes. These results represent a previously unrecognized mode of sensing temperature variation used by this pathogen at the onset of infection.

Atypical transcriptional regulation and role of a new toxin-antitoxin-like module and its effect on the lipid composition of Bradyrhizobium japonicum.

A toxin-antitoxin (TA)-like system (designated as bat/bto genes) was identified in Bradyrhizobium japonicum, based on sequence homology and similarities in organization and size to known TA systems. Deletion of the bat/bto module resulted in pleiotropic alterations in cell morphology and metabolism. The generation time of the mutant was considerably decreased in rich media. Atomic force microscopy revealed the modified shape (shorter and wider) and softness of mutant cells. The synthesis of phosphatidylcholine was completely blocked in the mutant bacteria, and vaccenic acid, the predominant fatty acid of membranes of the wild-type cell, was replaced by palmitic acid in the mutant membranes. The mutant bacteria synthesized incomplete lipopolysaccharide molecules. Remarkable changes in the membrane lipid composition may explain the observed morphological alterations and growth properties of the mutant bacteria. The overlapping promoter region of bat/bto and glpD (coding for the aerobic sn-glycerol-3-phosphate dehydrogenase) genes suggests a complex regulation and the involvement of bat/bto in the control of main metabolic pathways and an important role in the maintenance of a normal physiological state of B. japonicum. These data reveal new aspects of the role of TA systems in bacteria.

Membrane-associated stress proteins: more than simply chaperones.

The protein- and/or lipid-mediated association of chaperone proteins to membranes is a widespread phenomenon and implicated in a number of physiological and pathological events that were earlier partially or completely overlooked. A temporary association of certain HSPs with membranes can re-establish the fluidity and bilayer stability and thereby restore the membrane functionality during stress conditions. The fluidity and microdomain organization of membranes are decisive factors in the perception and transduction of stresses into signals that trigger the activation of specific HS genes. Conversely, the membrane association of HSPs may result in the inactivation of membrane-perturbing signals, thereby switch off the heat shock response. Interactions between certain HSPs and specific lipid microdomains ("rafts") might be a previously unrecognized means for the compartmentalization of HSPs to specific signaling platforms, where key signaling proteins are known to be concentrated. Any modulations of the membranes, especially the raft-lipid composition of the cells can alter the extracellular release and thus the immuno-stimulatory activity of certain HSPs. Reliable techniques, allowing mapping of the composition and dynamics of lipid microdomains and simultaneously the spatio-temporal localization of HSPs in and near the plasma membrane can provide suitable means with which to address fundamental questions, such as how HSPs are transported to and translocated through the plasma membrane. The possession of such information is critical if we are to target the membrane association principles of HSPs for successful drug development in most various diseases.

The impact of dihydropyridine derivatives on the cerebral blood flow response to somatosensory stimulation and spreading depolarization.

BACKGROUND AND PURPOSE: A new class of dihydropyridine derivatives, which act as co-inducers of heat shock protein but are devoid of calcium channel antagonist and vasodilator effects, has recently been developed with the purpose of selectively targeting neurodegeneration. Here, we evaluated the action of one of these novel compounds LA1011 on neurovascular coupling in the ischaemic rat cerebral cortex. As a reference, we applied nimodipine, a vasodilator dihydropyridine and well-known calcium channel antagonist. EXPERIMENTAL APPROACH: Rats were treated with LA1011 or nimodipine, either by chronic, systemic (LA1011), or acute, local administration (LA1011 and nimodipine). In the latter treatment group, global forebrain ischaemia was induced in half of the animals by bilateral common carotid artery occlusion under isoflurane anaesthesia. Functional hyperaemia in the somatosensory cortex was created by mechanical stimulation of the contralateral whisker pad under α-chloralose anaesthesia. Spreading depolarization (SD) events were elicited subsequently by 1 M KCl. Local field potential and cerebral blood flow (CBF) in the parietal somatosensory cortex were monitored by electrophysiology and laser Doppler flowmetry. KEY RESULTS: LA1011 did not alter CBF, but intensified SD, presumably indicating the co-induction of heat shock proteins, and, perhaps an anti-inflammatory effect. Nimodipine attenuated evoked potentials and SD. In addition to the elevation of baseline CBF, nimodipine augmented hyperaemia in response to both somatosensory stimulation and SD, particularly under ischaemia. CONCLUSIONS AND IMPLICATIONS: In contrast to the CBF improvement achieved with nimodipine, LA1011 seems not to have discernible cerebrovascular effects but may up-regulate the stress response.

Dihydropyridines Allosterically Modulate Hsp90 Providing a Novel Mechanism for Heat Shock Protein Co-induction and Neuroprotection.

Chaperones play a pivotal role in protein homeostasis, but with age their ability to clear aggregated and damaged protein from cells declines. Tau pathology is a driver of a variety of neurodegenerative disease and in Alzheimer's disease (AD) it appears to be precipitated by the formation of amyloid-ß (Aß) aggregates. Aß-peptide appears to trigger Tau hyperphosphorylation, formation of neurofibrillary tangles and neurotoxicity. Recently, dihydropyridine derivatives were shown to upregulate the heat shock response (HSR) and provide a neuroprotective effect in an APPxPS1 AD mouse model. The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only when a pathological state is present. We show for compounds tested herein, that they target predominantly the C-terminal domain of Hsp90, but show some requirement for its middle-domain, and that binding stimulates the chaperones ATPase activity. We identify the site for LA1011 binding and confirm its identification by mutagenesis. We conclude, that binding compromises Hsp90's ability to chaperone, by modulating its ATPase activity, which consequently induces the HSR in diseased cells. Collectively, this represents the mechanism by which the normalization of neurofibrillary tangles, preservation of neurons, reduced tau pathology, reduced amyloid plaque, and increased dendritic spine density in the APPxPS1 Alzheimer's mouse model is initiated. Such dihydropyridine derivatives therefore represent potential pharmaceutical candidates for the therapy of neurodegenerative disease, such as AD.

Poly(ADP-ribose) polymerase-2 is a lipid-modulated modulator of muscular lipid homeostasis.

There is a growing body of evidence that poly(ADP-ribose) polymerase-2 (PARP2), although originally described as a DNA repair protein, has a widespread role as a metabolic regulator. We show that the ablation of PARP2 induced characteristic changes in the lipidome. The silencing of PARP2 induced the expression of sterol regulatory element-binding protein-1 and -2 and initiated de novo cholesterol biosynthesis in skeletal muscle. Increased muscular cholesterol was shunted to muscular biosynthesis of dihydrotestosterone, an anabolic steroid. Thus, skeletal muscle fibers in PARP2-/- mice were stronger compared to those of their wild-type littermates. In addition, we detected changes in the dynamics of the cell membrane, suggesting that lipidome changes also affect the biophysical characteristics of the cell membrane. In in silico and wet chemistry studies, we identified lipid species that can decrease the expression of PARP2 and potentially phenocopy the genetic abruption of PARP2, including artificial steroids. In view of these observations, we propose a new role for PARP2 as a lipid-modulated regulator of lipid metabolism.