Search Results (18)

β-parvalbumin (β-PV) is the primary fish allergen responsible for most allergic reactions in individuals sensitive to fish. To ensure food safety, a sandwich-based magnetic immunoassay was developed using maleimide-functionalized magnetic beads (NH-MBs). Specific anti-β-PV antibodies were immobilized on these MBs, and a screen-printed carbon electrode was employed as the electrochemical transducer. A linear concentration range from 10 to 1000 ng/mL, a limit of detection of 1.8 ng/mL, and a limit of quantification of 7.1 ng/mL were achieved. Nineteen commercial food samples were analyzed to assess the potential of the sensor for routine use in food quality control. Important factors such as protein source and food processing (e.g., boiling, grilling, and frying) and preservation (e.g., in oil, and vacuum) were evaluated. The validated results confer the usefulness of the assay for food quality control. Full article

Leptin is a hormone produced by the small intestines and adipose tissue that promotes feelings of satiety. Leptin receptors (LepRs) are highly expressed in the hypothalamus, enabling central neural control of hunger. Interestingly, LepRs are also expressed in several other regions of the body and brain, notably in the cerebral cortex and hippocampus. These brain regions mediate higher-order sensory, motor, cognitive, and memory functions, which can be profoundly altered during periods of hunger and satiety. However, LepR expression in these regions has not been fully characterized on a cell-type-specific basis, which is necessary to begin assessing their potential functional impact. Consequently, we examined LepR expression on neurons and glia in the forebrain using a LepR-Cre transgenic mouse model. LepR-positive cells were identified using a ‘floxed’ viral cell-filling approach and co-labeling immunohistochemically for cell-type-specific markers, i.e., NeuN, VGlut2, GAD67, parvalbumin, somatostatin, 5-HT3R, WFA, S100β, and GFAP. In the cortex, LepR-positive cells were localized to lower layers (primarily layer 6) and exhibited non-pyramidal cellular morphologies. The majority of cortical LepR-positive cells were neurons, while the remainder were identified primarily as astrocytes or other glial cells. The majority of cortical LepR-positive neurons co-expressed parvalbumin, while none expressed somatostatin or 5-HT3R. In contrast, all hippocampal LepR-positive cells were neuronal, with none co-expressing GFAP. These data suggest that leptin can potentially influence neural processing in forebrain regions associated with sensation and limbic-related functions. Full article

In search of novel potential drug candidates that could be used as treatments or prophylactics for memory impairment, an aporphine alkaloid magnoflorine (MAG) isolated from the root of Berberis vulgaris was proven to exhibit beneficial anti-amnestic properties. Its effects on immunoreactivity to parvalbumin in the mouse hippocampus were assessed together with a study on its safety and concentration in the brain and plasma. For this purpose, four experimental groups were created: the MAG10 group—treated with 10 mg MAG/kg b.w. i.p., the MAG20 group—treated with 20 mg MAG/kg b.w. i.p., the MAG50 group—treated with 50 mg MAG/kg b.w. i.p., and a control group—injected with saline i.p. at a volume corresponding to their weight. Our results indicated that the hippocampal fields CA1–CA3 were characterized by an elevated number of parvalbumin-immunoreactive neurons (PV-IR) and nerve fibers in mice at the doses of 10 and 20 mg/kg b.w. (i.p.). No significant changes to the levels of IL-1β, IL-6 or TNF-α were observed for the above two doses; however, the administration of 50 mg/kg b.w. i.p. caused a statistically significant elevation of IL-6, IL-1beta plasma levels and an insignificant raise in the TNF-alpha value. The HPLC–MS analysis showed that the alkaloid’s content in the brain structures in the group treated with 50 mg/kg b.w. did not increase proportionally with the administered dose. The obtained results show that MAG is able to influence the immunoreactivity to PV-IR in hippocampal neurons and might act as a neuroprotective compound. Full article

Parvalbumins (PVALBs) are low molecular weight calcium-binding proteins. In addition to their role in many biological processes, PVALBs play an important role in regulating Ca2+ switching in muscles with fast-twitch fibres in addition to their role in many biological processes. The PVALB gene family is divided into two gene types, alpha (α) and beta (β), with the β gene further divided into two gene types, beta1 (β1) and beta2 (β2), carrying traces of whole genome duplication. A large variety of commonly consumed fish species contain PVALB proteins which are known to cause fish allergies. More than 95% of all fish-induced food allergies are caused by PVALB proteins. The authentication of fish species has become increasingly important as the seafood industry continues to grow and the growth brings with it many cases of food fraud. Since the PVALB gene plays an important role in the initiation of allergic reactions, it has been used for decades to develop alternate assays for fish identification. A brief review of the significance of the fish PVALB genes is presented in this article, which covers evolutionary diversity, allergic properties, and potential use as a forensic marker. Full article

Absence seizures are hyperexcitations within the cortico-thalamocortical (CTC) network, however the underlying causative mechanisms at the cellular and molecular level are still being elucidated and appear to be multifactorial. Dysfunctional feed-forward inhibition (FFI) is implicated as one cause of absence seizures. Previously, we reported altered excitation onto parvalbumin-positive (PV⁺) interneurons in the CTC network of the stargazer mouse model of absence epilepsy. In addition, downstream changes in GABAergic neurotransmission have also been identified in this model. Our current study assessed whether dysfunctional FFI affects GABA_A receptor (GABA_AR) subunit expression in the stargazer primary somatosensory cortex (SoCx). Global tissue expression of GABA_AR subunits α1, α3, α4, α5, β2, β3, γ2 and δ were assessed using Western blotting (WB), while biochemically isolated subcellular fractions were assessed for the α and δ subunits. We found significant reductions in tissue and synaptic expression of GABA_AR α1, 18% and 12.2%, respectively. However, immunogold-cytochemistry electron microscopy (ICC-EM), conducted to assess GABA_AR α1 specifically at synapses between PV⁺ interneurons and their targets, showed no significant difference. These data demonstrate a loss of phasic GABA_AR α1, indicating altered GABAergic inhibition which, coupled with dysfunctional FFI, could be one mechanism contributing to the generation or maintenance of absence seizures. Full article

Parvalbumins are small molecules with important functions in Ca²⁺ signaling, but their sequence comparisons to date, especially in fish, have been relatively poor. We here, characterize sequence motifs that distinguish parvalbumin subfamilies across vertebrate species, as well as those that distinguish individual parvalbumins (orthologues) in fish, and map them to known parvalbumin structures. As already observed by others, all classes of jawed vertebrates possess parvalbumins of both the α-parvalbumin and oncomodulin subfamilies. However, we could not find convincing phylogenetic support for the common habit of classifying all non-α-parvalbumins together as “β-parvalbumins.” In teleost (modern bony) fish, we here distinguish parvalbumins 1-to-10, of which the gene copy number can differ between species. The genes for α-parvalbumins (pvalb6 and pvalb7) and oncomodulins (pvalb8 and pvalb9) are well conserved between teleost species, but considerable variation is observed in their copy numbers of the non-α/non-oncomodulin genes pvalb1-to-5 and pvalb10. Teleost parvalbumins 1-to-4 are hardly distinguishable from each other and are highly expressed in muscle, and described allergens belong to this subfamily. However, in some fish species α-parvalbumin expression is also high in muscle. Pvalb5 and pvalb10 molecules form distinct lineages, the latter even predating the origin of teleosts, but have been lost in some teleost species. The present study aspires to be a frame of reference for future studies trying to compare different parvalbumins. Full article

Consumption of aquatic food, including fish, accounts for 17% of animal protein intake. However, fish consumption might also result in several side-effects such as sneezing, swelling and anaphylaxis in sensitized consumers. Fish allergy is an immune reaction to allergenic proteins in the fish muscle, for instance parvalbumin (PV), considered the major fish allergen. In this study, we characterize PV in two economically important fish species for southern European aquaculture, namely gilthead seabream and European seabass, to understand its stability during in vitro digestion and fish processing. This information is crucial for future studies on the allergenicity of processed fish products. PVs were extracted from fish muscles, identified by mass spectrometry (MS), and detected by sandwich enzyme-linked immunosorbent assay (ELISA) after simulated digestion and various food processing treatments. Secondary structures were determined by circular dichroism (CD) after purification by anion exchange and gel filtration chromatography. In both species, PVs presented as α-helical and β-sheet structures, at room temperature, were shown to unfold at boiling temperatures. In European seabass, PV detectability decreased during the simulated digestion and after 240 min (intestinal phase) no detection was observed, while steaming showed a decrease (p < 0.05) in PVs detectability in comparison to raw muscle samples, for both species. Additionally, freezing (−20 °C) for up to 12 months continued to reduce the detectability of PV in tested processing techniques. We concluded that PVs from both species are susceptible to digestion and processing techniques such as steaming and freezing. Our study obtained preliminary results for further research on the allergenic potential of PV after digestion and processing. Full article

The anterior cingulate cortex (ACC) is particularly critical for pain information processing. Peripheral nerve injury triggers neuronal hyper-excitability in the ACC and mediates descending facilitation to the spinal dorsal horn. The mechanically gated ion channel Piezo1 is involved in the transmission of pain information in the peripheral nervous system. However, the pain-processing role of Piezo1 in the brain is unknown. In this work, we found that spared (sciatic) nerve injury (SNI) increased Piezo1 protein levels in inhibitory parvalbumin (PV)-expressing interneurons (PV-INs) but not in glutaminergic CaMKⅡ⁺ neurons, in the bilateral ACC. A reduction in the number of PV-INs but not in the number of CaMKⅡ⁺ neurons and a significant reduction in inhibitory synaptic terminals was observed in the SNI chronic pain model. Further, observation of morphological changes in the microglia in the ACC showed their activated amoeba-like transformation, with a reduction in process length and an increase in cell body area. Combined with the encapsulation of Piezo1-positive neurons by Iba1⁺ microglia, the loss of PV-INs after SNI might result from phagocytosis by the microglia. In cellular experiments, administration of recombinant rat TNF-α (rrTNF) to the BV2 cell culture or ACC neuron primary culture elevated the protein levels of Piezo1 and NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3). The administration of the NLRP3 inhibitor MCC950 in these cells blocked the rrTNF-induced expression of caspase-1 and interleukin-1β (key downstream factors of the activated NLRP3 inflammasome) in vitro and reversed the SNI-induced Piezo1 overexpression in the ACC and alleviated SNI-induced allodynia in vivo. These results suggest that NLRP3 may be the key factor in causing Piezo1 upregulation in SNI, promoting an imbalance between ACC excitation and inhibition by inducing the microglial phagocytosis of PV-INs and, thereby, facilitating spinal pain transmission. Full article

Parvalbumin (PA) is a small, acidic, mostly cytosolic Ca²⁺-binding protein of the EF-hand superfamily. Structural and physical properties of PA are well studied but recently two highly conserved structural motifs consisting of three amino acids each (clusters I and II), which contribute to the hydrophobic core of the EF-hand domains, have been revealed. Despite several decades of studies, physiological functions of PA are still poorly known. Since no target proteins have been revealed for PA so far, it is believed that PA acts as a slow calcium buffer. Numerous experiments on various muscle systems have shown that PA accelerates the relaxation of fast skeletal muscles. It has been found that oxidation of PA by reactive oxygen species (ROS) is conformation-dependent and one more physiological function of PA in fast muscles could be a protection of these cells from ROS. PA is thought to regulate calcium-dependent metabolic and electric processes within the population of gamma-aminobutyric acid (GABA) neurons. Genetic elimination of PA results in changes in GABAergic synaptic transmission. Mammalian oncomodulin (OM), the β isoform of PA, is expressed mostly in cochlear outer hair cells and in vestibular hair cells. OM knockout mice lose their hearing after 3–4 months. It was suggested that, in sensory cells, OM maintains auditory function, most likely affecting outer hair cells’ motility mechanisms. Full article

Oestrogen receptor β (ERβ) knock-out female mice display increased anxiety and decreased threshold for synaptic plasticity induction in the basolateral amygdala. This may suggest that the γ-aminobutyric acid (GABA) inhibitory system is altered. Therefore, the immunoreactivity of main GABAergic markers—i.e., calbindin, parvalbumin, calretinin, somatostatin, α1 subunit-containing GABA_A receptor and vesicular GABA transporter—were compared in the six subregions (LA, BL, BM, ME, CE and CO) of the amygdala of adult female wild-type and ERβ knock-out mice using immunohistochemistry and quantitative methods. The influence of ERβ knock-out on neuronal loss and glia was also elucidated using pan-neuronal and astrocyte markers. The results show severe neuronal deficits in all main amygdala regions in ERβ knock-out mice accompanied by astroglia overexpression only in the medial, basomedial and cortical nuclei and a decrease in calbindin-expressing neurons (CB+) in the amygdala in ERβ knock-out mice compared with controls, while other markers of the GABAergic system remain unchanged. Concluding, the lack of ERβ led to failure in the structural integrity of the CB+ subpopulation, reducing interneuron firing and resulting in a disinhibitory effect over pyramidal function. This fear-promoting excitatory/inhibitory alteration may lead to the increased anxiety observed in these mice. The impact of neuronal deficits and astroglia overexpression on the amygdala functions remains unknown. Full article

Somatostatin (SST) is widely expressed in the brain and plays various, vital roles involved in neuromodulation. The purpose of this study is to characterize the organization of SST neurons in the Mongolian gerbil visual cortex (VC) using immunocytochemistry, quantitative analysis, and confocal microscopy. As a diurnal animal, the Mongolian gerbil provides us with a different perspective to other commonly used nocturnal rodent models. In this study, SST neurons were located in all layers of the VC except in layer I; they were most common in layer V. Most SST neurons were multipolar round/oval or stellate cells. No pyramidal neurons were found. Moreover, 2-color immunofluorescence revealed that only 33.50%, 24.05%, 16.73%, 0%, and 64.57% of SST neurons contained gamma-aminobutyric acid, calbindin-D28K, calretinin, parvalbumin, and calcium/calmodulin-dependent protein kinase II, respectively. In contrast, neuropeptide Y and nitric oxide synthase were abundantly expressed, with 80.07% and 75.41% in SST neurons, respectively. Our immunocytochemical analyses of SST with D₁ and D₂ dopamine receptors and choline acetyltransferase, α₇ and β₂ nicotinic acetylcholine receptors suggest that dopaminergic and cholinergic fibers contact some SST neurons. The results showed some distinguishable features of SST neurons and provided some insight into their afferent circuitry in the gerbil VC. These findings may support future studies investigating the role of SST neurons in visual processing. Full article

In the Mediterranean region, fish is a common cause of food protein-induced enterocolitis syndrome (FPIES) in children. No laboratory tests specific to FPIES are available, and oral food challenge (OFC) is the gold standard for its diagnosis and testing for achievement of tolerance. Children with FPIES to fish are usually advised to avoid all fish, regardless of the species. Fish are typically classified into bony and cartilaginous, which are phylogenetically distant species and therefore contain less cross-reacting allergens. The protein β-parvalbumin, considered a pan-allergenic, is found in bony fish, while the non-allergenic α-parvalbumin is commonly found in cartilaginous fish. Based on this difference, as a first step in the therapeutic process of children with FPIES caused by a certain fish in the bony fish category (i.e., hake, cod, perch, sardine, gilthead sea bream, red mullet, sole, megrim, sea bass, anchovy, tuna, swordfish, trout, etc.), an OFC to an alternative from the category of cartilaginous fish is suggested (i.e., blue shark, tope shark, dogfish, monkfish, skate, and ray) and vice versa. Regarding the increased mercury content in some sharks and other large species, the maximum limit imposed by the European Food Safety Authority (EFSA) for weekly mercury intake must be considered. An algorithm for the management of fish-FPIES, including alternative fish species, is proposed. Full article

Parvalbumin is considered a major fish allergen. Here, we report the molecular evolution of the parvalbumin genes in bony fishes based on 19 whole genomes and 70 transcriptomes. We found unexpectedly high parvalbumin diversity in teleosts; three main gene types (pvalb-α, pvalb-β1, and pvalb-β2, including oncomodulins) originated at the onset of vertebrates. Teleosts have further multiplied the parvalbumin gene repertoire up to nine ancestral copies—two copies of pvalb-α, two copies of pvalb-β1, and five copies of pvalb-β2. This gene diversity is a result of teleost-specific whole-genome duplication. Two conserved parvalbumin genomic clusters carry pvalb-β1 and β2 copies, whereas pvalb-α genes are located separately in different linkage groups. Further, we investigated parvalbumin gene expression in 17 tissues of the common carp (Cyprinus carpio), a species with 21 parvalbumin genes in its genome. Two pvalb-α and eight pvalb-β2 copies are highly expressed in the muscle, while two alternative pvalb-α copies show expression in the brain and the testes, and pvalb-β1 is dominant in the retina and the kidney. The recent pairs of muscular pvalb-β2 genes show differential expression in this species. We provide robust genomic evidence of the complex evolution of the parvalbumin genes in fishes. Full article

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a rapid accumulation of amyloid β (Aβ) protein in the hippocampus, which impairs synaptic structures and neuronal signal transmission, induces neuronal loss, and diminishes memory and cognitive functions. The present study investigated the impact of neuregulin 1 (NRG1)-ErbB4 signaling on the impairment of neural networks underlying hippocampal long-term potentiation (LTP) in 5xFAD mice, a model of AD with greater symptom severity than that of TG2576 mice. Specifically, we observed parvalbumin (PV)-containing hippocampal interneurons, the effect of NRG1 on hippocampal LTP, and the functioning of learning and memory. We found a significant decrease in the number of PV interneurons in 11-month-old 5xFAD mice. Moreover, synaptic transmission in the 5xFAD mice decreased at 6 months of age. The 11-month-old transgenic AD mice showed fewer inhibitory PV neurons and impaired NRG1-ErbB4 signaling than did wild-type mice, indicating that the former exhibit the impairment of neuronal networks underlying LTP in the hippocampal Schaffer-collateral pathway. In conclusion, this study confirmed the impaired LTP in 5xFAD mice and its association with aberrant NRG1-ErbB signaling in the neuronal network. Full article

Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca²⁺-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca²⁺-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pK_a of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca²⁺-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg²⁺-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca²⁺/Mg²⁺ affinity of the EF site, and accelerates Ca²⁺ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca²⁺ affinity. Full article