Oligomerization and Spatial Distribution of Kvβ1.1 and Kvβ2.1 Regulatory Subunits

Members of the regulatory Kvβ family modulate the kinetics and traffic of voltage-dependent K+ (Kv) channels. The crystal structure of Kv channels associated with Kvβ peptides suggests a α4/β4 composition. Although Kvβ2 and Kvβ1 form heteromers, evidence supports that only Kvβ2.1 forms tetramers in the absence of α subunits. Therefore, the stoichiometry of the Kvβ oligomers fine-tunes the activity of hetero-oligomeric Kv channel complexes. We demonstrate that Kvβ subtypes form homo- and heterotetramers with similar affinities. The Kvβ1.1/Kvβ2.1 heteromer showed an altered spatial distribution in lipid rafts, recapitulating the Kvβ1.1 pattern. Because Kvβ2 is an active partner of the Kv1.3-TCR complex at the immunological synapse (IS), an association with Kvβ1 would alter this location, shaping the immune response. Differential regulation of Kvβs influences the traffic and architecture of the Kvβ heterotetramer, modulating Kvβ-dependent physiological responses

Breeding performance in the Italian chicken breed Mericanel della Brianza

In Italy, 90 local avian breeds were described; the majority (61%) were classified as extinct and only 8.9% as still widely spread. Therefore, efforts for conservation of Italian avian breeds are urgently required. The aim of this study was to record the breeding performance of the Italian breed Mericanel della Brianza to multiply a small population, in order to develop a conservation programme. Fourteen females and eight males were available at the beginning of the reproductive season in 2009 and organised into eight families (1 male/1-2 females) kept in floor pens. Birds received a photoperiod of 14L:10D and were fed ad libitum. Breeding performance was recorded from March to June. Egg production and egg weight were recorded daily; eggs were set every two weeks and fertility, embryo mortality and hatchability were recorded. Mean egg production was 37% and mean egg weight was 34±3.49 g. High fertility values from 94% to 87% were recorded in the first three settings and the overall mean fertility value was 81.6%. Overall hatchability was only 49.6% owing to a high proportion of dead embryos. Embryo mortality occurred mainly between days 2 and 7 of incubation and during hatching. The highest hatchability values were recorded in settings 1 and 2, 69% and 60% respectively, and a large decrease was found in the subsequent settings. Marked variations in egg production, fertility, hatchability and embryo mortality were found among families. The present results represent the basic know ledge of reproductive parameters necessary to improve the reproductive efficiency of the breed within a conservation plan

Surface Functionalisation of biomaterials with alkaline phosphatase

Two different glasses, one biocompatible but with a low bioactivity index (G1) and the other with an higher bioactivity index (G2), the ceramic version of the second glass and a titanium alloy (Ti6Al4V) have been functionalizated by anchoring alkaline phosphatase (ALP) on their surfaces. The enzyme has been chosen because it is involved in mineralization processes of hard tissues and is a model for more complex ones. ALP has been grafted on glasses and glass-ceramics surfaces both with and without samples silanization and on metallic surfaces with and without tresyl chloride activation. Samples have been analyzed at each step of the functionalization process in order to verify i

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit+ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit+ CSCs

Amorphous WO3 as transparent conductive oxide in the near-IR

The demand for transparent conductive films (TCFs) is dramatically increasing. In this work tungsten oxide (WO3-x) is studied as a possible option additional to the existed TCFs. We introduce WO3-x thin films fabricated by a non-reactive magnetron RF-sputtering process at room temperature, followed by thermal annealing in dry air. Films are characterized morphologically, structurally, electrically, optically, and dielectrically. Amorphous WO3-x thin films are shown to be ntype conductive while the transparency extends to the near-IR. By evaluating a figure of merit for transparent-conductive performance and comparing to some most-widely used TCFs, WO3-x turns out to outperform in the near-IR optical range

Regulatory subunits controlling the Kv1.3 channelosome

[eng] The Kv1.3 voltage-dependent potassium channel plays a crucial role in the immune response participating in various cellular functions like proliferation, activation, and apoptosis. The aberrant expression of this channel is associated with autoimmune diseases, highlighting the need for precise regulation in leukocyte physiology. Kvβ proteins, the first identified modulators of Kv channels, have been extensively studied in their regulation of α-subunit kinetics and traffic. However, limited information is available regarding their own biology. Despite their cytosolic distribution, Kvβ subunits show spatial localization near plasma membrane-Kv channels for an effective immune response. Our study focused on the structural elements influencing Kvβ distribution. We discovered that Kvβ peptides could target the cell surface independently of Kv channels. Additionally, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains via S-acylation of two C-terminal cysteines (C301/C311), concomitantly with the peptide localization at the immunological synapse. Moreover, growth factor-dependent proliferation increased the Kvβ2.1 surface targeting, whereas PKC activation disrupted lipid raft localization, but PSD95 counteracted this action. These findings elucidate the mechanisms by which Kvβ2 clusters within immunological synapses during leukocyte activation. Kvβ peptides, interacting with Kv channels, exhibited a suggested α4/β4 conformation. While Kvβ2 and Kvβ1 can form homo- and heterotetramers with similar affinities, only Kvβ2.1 forms tetramers independently of α subunits. Thus, Kvβ oligomers stoichiometry fine-tunes hetero-oligomeric Kv channel complexes. Similar to Kvβ1.1, Kvβ1.1/Kvβ2.1 heteromers did not target lipid rafts. Therefore, because Kvβ2 is an active partner of the Kv1.3-TCR complex at the immunological synapse, an association with Kvβ1 would alter its location, impacting on immune responses. Differential regulation of Kvβs influences the traffic and architecture of Kvβ heterotetramers, modulating Kvβ-dependent physiological responses. Regulatory KCNE subunits are expressed in the immune system and KCNE4 tightly regulates Kv1.3. KCNE4 modifies Kv1.3 currents altering kinetics and retaining the channel at the endoplasmic reticulum (ER). This function affects in turn membrane localization of the channel. Our research showed that KCNE4 can dimerize via the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4. This cluster serves as a competitive structural platform for Kv1.3, Ca2+/calmodulin (CaM) and KCNE4 dyads. While KCNE4 is typically retained in the ER, the association with CaM leads to COP-II-dependent forward trafficking. Consequently, CaM plays a vital role in controlling the dimerization and membrane targeting of KCNE4, affecting the regulation of Kv1.3 and, subsequently, leukocyte physiology. Kv1.3, localized in membrane lipid rafts, accumulates at immunological synapses during cell activation, influencing membrane potential and downstream calcium-signalling pathways. KCNE4 acts as a dominant negative regulatory subunit on Kv1.3, causing intracellular retention. Palmitoylation, a reversible post- translational modification, enhances protein hydrophobicity, facilitating membrane association, protein interactions, and subcellular trafficking. Our data demonstrated the S-acylation of KCNE4, resulting in spatial rearrangements that reduce ER distribution, which in turn affects Kv1.3 regulation. KCNE4 partially traffics to the cell surface with Kv1.3 in activated dendritic cells but alters immunological synapse targeting. This highlights the significance of KCNE4 palmitoylation in regulating protein subcellular localization and oligomeric state, subsequently affecting channel membrane expression. Given the role of Kv1.3 as an immunomodulatory target, these findings offer insights for future clinical and pharmacological studies

Regulatory subunits controlling the Kv1.3 channelosome

Programa de Doctorat en Biomedicina[eng] The Kv1.3 voltage-dependent potassium channel plays a crucial role in the immune response participating in various cellular functions like proliferation, activation, and apoptosis. The aberrant expression of this channel is associated with autoimmune diseases, highlighting the need for precise regulation in leukocyte physiology. Kvβ proteins, the first identified modulators of Kv channels, have been extensively studied in their regulation of α-subunit kinetics and traffic. However, limited information is available regarding their own biology. Despite their cytosolic distribution, Kvβ subunits show spatial localization near plasma membrane-Kv channels for an effective immune response. Our study focused on the structural elements influencing Kvβ distribution. We discovered that Kvβ peptides could target the cell surface independently of Kv channels. Additionally, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains via S-acylation of two C-terminal cysteines (C301/C311), concomitantly with the peptide localization at the immunological synapse. Moreover, growth factor-dependent proliferation increased the Kvβ2.1 surface targeting, whereas PKC activation disrupted lipid raft localization, but PSD95 counteracted this action. These findings elucidate the mechanisms by which Kvβ2 clusters within immunological synapses during leukocyte activation. Kvβ peptides, interacting with Kv channels, exhibited a suggested α4/β4 conformation. While Kvβ2 and Kvβ1 can form homo- and heterotetramers with similar affinities, only Kvβ2.1 forms tetramers independently of α subunits. Thus, Kvβ oligomers stoichiometry fine-tunes hetero-oligomeric Kv channel complexes. Similar to Kvβ1.1, Kvβ1.1/Kvβ2.1 heteromers did not target lipid rafts. Therefore, because Kvβ2 is an active partner of the Kv1.3-TCR complex at the immunological synapse, an association with Kvβ1 would alter its location, impacting on immune responses. Differential regulation of Kvβs influences the traffic and architecture of Kvβ heterotetramers, modulating Kvβ-dependent physiological responses. Regulatory KCNE subunits are expressed in the immune system and KCNE4 tightly regulates Kv1.3. KCNE4 modifies Kv1.3 currents altering kinetics and retaining the channel at the endoplasmic reticulum (ER). This function affects in turn membrane localization of the channel. Our research showed that KCNE4 can dimerize via the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4. This cluster serves as a competitive structural platform for Kv1.3, Ca2+/calmodulin (CaM) and KCNE4 dyads. While KCNE4 is typically retained in the ER, the association with CaM leads to COP-II-dependent forward trafficking. Consequently, CaM plays a vital role in controlling the dimerization and membrane targeting of KCNE4, affecting the regulation of Kv1.3 and, subsequently, leukocyte physiology. Kv1.3, localized in membrane lipid rafts, accumulates at immunological synapses during cell activation, influencing membrane potential and downstream calcium-signalling pathways. KCNE4 acts as a dominant negative regulatory subunit on Kv1.3, causing intracellular retention. Palmitoylation, a reversible post- translational modification, enhances protein hydrophobicity, facilitating membrane association, protein interactions, and subcellular trafficking. Our data demonstrated the S-acylation of KCNE4, resulting in spatial rearrangements that reduce ER distribution, which in turn affects Kv1.3 regulation. KCNE4 partially traffics to the cell surface with Kv1.3 in activated dendritic cells but alters immunological synapse targeting. This highlights the significance of KCNE4 palmitoylation in regulating protein subcellular localization and oligomeric state, subsequently affecting channel membrane expression. Given the role of Kv1.3 as an immunomodulatory target, these findings offer insights for future clinical and pharmacological studies

The Potassium Channel Odyssey: Mechanisms of Traffic and Membrane Arrangement

Ion channels are transmembrane proteins that conduct specific ions across biological membranes. Ion channels are present at the onset of many cellular processes, and their malfunction triggers severe pathologies. Potassium channels (KChs) share a highly conserved signature that is necessary to conduct K+ through the pore region. To be functional, KChs require an exquisite regulation of their subcellular location and abundance. A wide repertoire of signatures facilitates the proper targeting of the channel, fine-tuning the balance that determines traffic and location. These signature motifs can be part of the secondary or tertiary structure of the protein and are spread throughout the entire sequence. Furthermore, the association of the pore-forming subunits with different ancillary proteins forms functional complexes. These partners can modulate traffic and activity by adding their own signatures as well as by exposing or masking the existing ones. Post-translational modifications (PTMs) add a further dimension to traffic regulation. Therefore, the fate of a KCh is not fully dependent on a gene sequence but on the balance of many other factors regulating traffic. In this review, we assemble recent evidence contributing to our understanding of the spatial expression of KChs in mammalian cells. We compile specific signatures, PTMs, and associations that govern the destination of a functional channel

Pellet cryopreservation for chicken semen: Effects of sperm working concentration, cryoprotectant concentration, and equilibration time during in vitro processing

a b s t r a c t The aim of the study was to standardize the pellet cryopreservation procedure for chicken semen. Mericanel della Brianza male chicken breeders (Italian breed) were used. Pooled semen samples were processed according to the following conditions: (1) dilution in prefreezing extender to 1 versus 1.5 bill cells/mL sperm working concentration (SWC); (2) 6% versus 9% dimethyl acetamide (DMA) concentration (DMAco); (3) 1 versus 30 minutes DMA equilibration (DMAeq) at 4 C. Sperm viability and motility were assessed in semen (four replicates/treatment) soon after collection (time 0), after DMAeq (time D), and after freezing/thawing (time FT). The recovery rates (%) of viable and motile sperm after freezing/thawing were also calculated. The low SWC (1 bill/mL) and the low DMAco (6%) indicated a positive significant effect on the proportion of motile sperm (1 bill/mL ¼ 53% vs. 1.5 bill/mL ¼ 48%; 6% DMA ¼ 55% vs. 9% DMA ¼ 47%). Very short DMAeq (1 minute) did not significantly change sperm viability during processing (from time 0 to time D) before freezing whatever the DMAco, and, in contrast, the longer DMAeq showed a significant negative effect on sperm viability. The highest proportion of motile sperm was recorded in semen samples diluted to 1 bill/mL and added with 6% DMA; in this condition, DMAeq had no effect (57% 1 minute and 61% 30 minutes). Increasing SWC to 1.5 bill/mL and adding again 6% DMA, a significant effect of DMAeq was observed, and the higher proportion of motile sperm (58% vs. 43%) was recorded after 1 minute DMAeq. A general decrease in sperm motility was shown in semen samples with 9% DMA (47% vs. 55%), and different conditions in SWC and DMAeq were not effective in the prevention of such decrease