During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2؉ homeostasis and the physiological roles of CCE.The two primary second messengers mediating rapid responses of cells to hormones, autacoids, and neurotransmitters are cyclic nucleotides and Ca 2ϩ . Cyclic nucleotides act, for the most part, by activating protein kinases. The actions of Ca 2ϩ are more complex, in that this cation acts in two ways: directly, by binding to effector proteins, and indirectly, by first binding to regulatory proteins such as calmodulin, troponin C, and recoverin, which in turn associate and modulate effector proteins. Effector proteins regulated in these manners by Ca 2ϩ include not only protein kinases and protein phosphatases but also phospholipases and adenylyl cyclases, which are signaling enzymes in their own right, and an array of proteins involved in cellular responses that range from muscle contraction to glycogenolysis, endo-, exo-, and neurosecretion, cell differentiation, and programmed cell death. A common mechanism used by hormones and growth factors to signal through cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) is activation of a rather complex reaction cascade that begins with stimulation of phosphoinositide-specific phospholipase C (PLC) enzymes, PLC␤ and PLC␥, and is followed sequentially by formation of diacylglycerol plus inositol 1,4,5-trisphosphate (IP3), liberation of Ca 2ϩ from intracellular stores, and finally, entry of Ca 2ϩ from the external milieu. The basic mechanisms used to signal through [Ca 2ϩ ] i are determined by the fact that the resting level of cytosolic Ca 2ϩ is very low, in the neighborhood of 100 nM, while that in intracellular stores and in the surrounding extracellular milieu is in the neighborhood of 2 mM, that is, Ϸ10,000-fold higher. As a result, [Ca 2ϩ ] i is set by the balance of two opposing forces. One is passive influx into the cytoplasm. It is driven by the electrochemical gradient and causes cytosolic [Ca 2ϩ ] i to rise without expenditure of energy. This influx is carefully controlled both at the level of the plasma membrane and at the level of the membranes, which delimit the internal storage compartment. Entry of Ca 2ϩ from the extracellular space occurs through three classes of Ca 2ϩ permeable gates: voltagedependent Ca 2ϩ...

Several classes of voltage-gated Ca 2؉ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca 2؉ channels the executing arm of the activated G protein is its ␤␥ dimer (G␤␥). We report below the existence of two G␤␥-binding sites on the A-, B-, and E-type ␣ 1 subunits that form non-L-type Ca 2؉ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both G␤␥-binding regions also bind the Ca 2؉ channel ␤ subunit (CC␤), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in ␣ 1E of loop 1 with that of the G proteininsensitive and G␤␥-binding-negative loop 1 of ␣ 1C did not abolish inhibition by G proteins, but the exchange of the ␣ 1E C terminus with that of ␣ 1C did. This and properties of ␣ 1E C-terminal truncations indicated that the G␤␥-binding site mediating the inhibition of Ca 2؉ channel activity is the one in the C terminus. Binding of G␤␥ to this site was inhibited by an ␣ 1 -binding domain of CC␤, thus providing an explanation for the functional antagonism existing between CC␤ and G protein inhibition. The data do not support proposals that G␤␥ inhibits ␣ 1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.Studies on stimulation-evoked release of norepinephrine from sympathetic terminals of the cat's nictitating membrane before and after ␣-adrenergic blockade led to the discovery in 1971 of an inhibitory presynaptic ␣ adrenoreceptor, now known as one of the ␣ 2 -adrenoreceptors (1). Presynaptic inhibition of neurosecretion by the released neurotransmitter (2) or by neuropeptides (3), all acting through G protein-coupled receptors, is now recognized as an important regulatory feedback mechanism utilized throughout the central and the peripheral nervous system. Evidence has accumulated to indicate that this type of inhibition of neurotransmitter release is due to inhibition of presynaptic N-and P͞Q-type Ca 2ϩ channels (4-9) by a mechanism that is likely to use the ␤␥ signaling arm of activated G proteins (10, 11).Voltage-gated Ca 2ϩ channels are multisubunit complexes formed of a pore-forming and voltage-sensing ␣ 1 subunit, a regulatory ␣ 2 ␦, and one or possibly two (12) regulatory ␤ subunits. Voltage dependence; fundamental aspects of activation, deactivation, and inactivation; feedback inhibition by Ca 2ϩ ; and sensitivity to various Ca 2ϩ channel blockers are all encoded in ␣ 1 subunits, of which there are six major types (S, A, B, C, D, and E). Each is subject to modulation to variable degrees by the named regulatory subunits, and each is expressed in alternatively spliced forms (reviewed in refs. 13-15). The N-and P͞Q-type Ca 2ϩ channels regulated negatively by a G pro...

ABSTRACT␤ subunits of voltage-gated Ca 2؉ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for ␤2a, which differs in that it does not support prepulse facilitation of ␣ 1C Ca 2؉ channels, inhibits voltage-induced inactivation of neuronal ␣ 1E Ca 2؉ channels, and is more effective in blocking inhibition of ␣ 1E channels by G protein-coupled receptors. We show that the distinguishing properties of ␤2a, rather than interaction with a distinct site of ␣ 1 , are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of ␤2a were lost in a mutant that could not be palmitoylated [␤2a(Cys 3,4 Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three ␤2 splice variants differing only in their N-termini.Voltage-gated Ca 2ϩ channels are multiprotein complexes made up of at least three distinct types of subunits: an ␣ 1 , which senses voltage changes and spans the membrane multiple times to form the pore and the ␤ and ␣ 2 ␦ subunits, which modulate almost all aspects of ␣ 1 function (1). In addition, ␤ and ␣ 2 ␦ play structural roles that are important, but not well understood, in channel maturation and accumulation at the cell surface. Voltage-gated Ca 2ϩ channels are molecularly diverse. Six ␣ 1 and four ␤ genes are known, and many genes exhibit additional heterogeneity in their translated proteins because of alternative splicing. In contrast, only one gene encoding the ␣ 2 ␦ complex has been found so far, but it also yields transcripts that are spliced alternatively to give slightly differing proteins (2, 3)Although in most cases it has been difficult to ascribe a functional correlate to specific Ca 2ϩ channel splice variants, there is one striking exception: the a-type splice variant of the ␤2 subunit from rat brain (␤2a) acts differently on inactivation of ␣ 1E , on prepulse-induced long lasting facilitation of ␣ 1C , and also, to some extent, on G protein-mediated inhibition of neuronal Ca 2ϩ channels. In ␣ 1E , brain ␤2a reduces the rate at which ␣ 1E inactivates in response to depolarization and causes a right shift in the steady-state inactivation curve. All other ␤ subunits, including the b-type splice variant of ␤2, accelerate channel inactivation and cause steady-state inactivation curves to be left-shifted along the voltage axis (4, 5). In contrast, ␤2a is indistinguishable from its homologs in terms of ␣ 1E activation (5).Prepulse facilitation is a phenomenon in which a train of depolarizations, or a long and strong depolarizing pulse, induces a form of the Ca 2ϩ channel that exhibits an increased opening probability in response to a given test potential that persist...

IntroductionRecent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).MethodsWe used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.Resultsp16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.ConclusionsWe disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

BackgroundHealthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcare-associated pathogens, thus indicating the need for new and sustainable strategies.AimThis study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting.MethodsThe effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected.ResultsMicrobial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3–4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m2 when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities.ConclusionsThis study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAI-related microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.