CAM Regulation Research Articles

Commentary on legislative regulation of complementary and alternative medicine in Ghana: The journey so far

Despite immense progress made in the area of Complementary and Alternative Medicine in the Ghanaian healthcare system, the Traditional Medicine Practice Council still seeks to regulate CAM practitioners as Traditional Medicine Practitioners under Act 575, despite the clear differences between both professions. This situation has led to numerous conflicts and agitations between practitioners of both forms of medical practice. This paper presents a commentary on the evolution of CAM legislation and regulation in Ghana.

Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

Pathogenic variants in KCNQ2 encoding for Kv7.2 potassium channel subunits have been found in patients affected by widely diverging epileptic phenotypes, ranging from Self-Limiting Familial Neonatal Epilepsy (SLFNE) to severe Developmental and Epileptic Encephalopathy (DEE). Thus, understanding the pathogenic molecular mechanisms of KCNQ2 variants and their correlation with clinical phenotypes has a relevant impact on the clinical management of these patients. In the present study, the genetic, biochemical, and functional effects prompted by two variants, each found in a non-familial SLNE or a DEE patient but both affecting nucleotides at the KCNQ2 intron 6-exon 7 boundary, have been investigated to test whether and how they affected the splicing process and to clarify whether such mechanism might play a pathogenetic role in these patients. Analysis of KCNQ2 mRNA splicing in patient-derived lymphoblasts revealed that the SLNE-causing intronic variant (c.928-1G > C) impeded the use of the natural splice site, but lead to a 10-aa Kv7.2 in frame deletion (Kv7.2 p.G310Δ10); by contrast, the DEE-causing exonic variant (c.928G > A) only had subtle effects on the splicing process at this site, thus leading to the synthesis of a full-length subunit carrying the G310S missense variant (Kv7.2 p.G310S). Patch-clamp recordings in transiently-transfected CHO cells and primary neurons revealed that both variants fully impeded Kv7.2 channel function, and exerted strong dominant-negative effects when co-expressed with Kv7.2 and/or Kv7.3 subunits. Notably, Kv7.2 p.G310S, but not Kv7.2 p.G310Δ10, currents were recovered upon overexpression of the PIP2-synthesizing enzyme PIP5K, and/or CaM; moreover, currents from heteromeric Kv7.2/Kv7.3 channels incorporating either Kv7.2 mutant subunits were differentially regulated by changes in PIP2 availability, with Kv7.2/Kv7.2 G310S/Kv7.3 currents showing a greater sensitivity to PIP2 depletion when compared to those from Kv7.2/Kv7.2 G310Δ10/Kv7.3 channels. Altogether, these results suggest that the two variants investigated differentially affected the splicing process at the intron 6-exon 7 boundary, and led to the synthesis of Kv7.2 subunits showing a differential sensitivity to PIP2 and CaM regulation; more studies are needed to clarify how such different functional properties contribute to the widely-divergent clinical phenotypes.

\u201cThen I went to a hospital abroad\u201d: acknowledging implications of stakeholders\u2019 differing risk understandings related to use of complementary and alternative medicine in European health care contexts

BackgroundComplementary and alternative medicine (CAM) is a rather novel issue within public healthcare and health policy-making. CAM use in Europe is widespread, patient-initiated, and patient-evaluated, and the regulation across countries has been evaluated as disharmonized. CAM users are left in an uncertain position, and patient safety may be threatened. How “risk” is understood by individuals in health policy-making and clinical encounters involving the use of CAM has not yet been much debated. The aim of this article is to explore and discuss the existence and possible consequences of differing risk understandings among stakeholders maneuvering in the complex landscape of CAM practice and CAM regulation contextualized by European public healthcare systems.MethodsQualitative data were derived from two studies on CAM in European healthcare contexts. Findings from the EU project CAMbrella on legislation and regulation of CAM were mixed with data from an interview study exploring risk understandings, communication, and decision-making among Scandinavian CAM users and their doctors. In a secondary content analysis, we constructed the case Sara as a typology to demonstrate important findings with regard to risk understandings and patient safety involving European citizens’ use of CAM in differing contexts.ResultsBy combining and comparing individual and structural perspectives on risk and CAM use, we revealed underexplored gaps in risk understandings among individuals involved in European CAM regulation and legislation, and between CAM users and their medical doctors. This may cause health risks and uncertainties associated with CAM use and regulation. It may also negatively influence doctor-CAM user communication and CAM users’ trust in and use of public healthcare.ConclusionAcknowledging implications of stakeholders’ differing risk understandings related to CAM use and regulation may positively influence patient safety in European healthcare. Definitions of the concept of risk should include the factors uncertainty and subjectivity to grasp the full picture of possible risks associated with the use of CAM. To transform the findings of this study into practical settings, we introduce sets of questions relevant to operationalize the important question “What is risk?” in health policy-making, clinical encounters and risk research involving European patients’ use of CAM.

Papildomosios ir alternatyviosios medicinos reglamentavimas Lietuvoje: problemos ir perspektyvos

Lietuvoje ir visoje Europoje pastebimas papildomosios ir alternatyviosios medicinos (toliau - PAM) paslaugų poreikio augimas, suvokiamas išnaudojimo potencialas, esant reglamentuotai teisinei bazei. Diskusijos kyla dėl PAM sąvokos, sričių apibrėžties, kuriamų paslaugų teikimo metodikų, vadinamųjų standartų, specialistų kvalifikacijos, licencijavimo tvarkos. Darbo tikslas – išanalizuoti dabartinę papildomosios ir alternatyviosios medicinos reguliavimo situaciją, esančias problemas bei galimas perspektyvas. Atlikta mokslinės literatūros analizė, apžvelgiant sveikatos, žmogaus gerovės, esmines PAM terminijas. Pateikiamos įžvalgos iš šiandienos Sveikatos apsaugos ministerijoje svarstomų teisinio reguliavimo dokumentų bei numatomų scenarijų. Tyrimo rezultatai leidžia teigti, kad Pasaulio sveikatos organizacija apibrėžia žmogaus sveikatą kaip visapusę fizinę, socialinę ir dvasinę gerovę, orientuojantis, jog sveikata yra holistinės medicinos sampratos sąvoka. Europos Sąjungos sveikatos strategijoje (trečioji programa 2014-2020) kalbama apie žmogaus sveikatingumą, prevenciją, rekreacines sveikatos funkcijas, užtikrinant jų integraciją į visų sričių politiką. PAM galėtų tapti sveikatos sampratos holistiškumo aspektu, kuomet per nustatytus sveikatinimo pogrupius būtų įgyvendinamos rekreacijos, natūralios medicinos ir liaudies medicinos bei kita papildomosios ir alternatyviosios medicinos veiklos. Svarbu kalbėti apie tradicinės ir papildomosios bei alternatyviosios medicinos integralumą. Mokslinio diskurso analizė atskleidė, jog Europos šalyse yra pasirinktos skirtingos papildomosios ir alternatyviosios medicinos reglamentavimo strategijos su tam tikrais bendrumais, taip pat skirtingos ir medicininės veiklos sritys pasaulio šalyse yra laikomos papildomąja ir alternatyviąja medicina. Kaip atsvaros tašku būtina laikytis Pasaulinės sveikatos organizacijos siūlomos krypties bei Europos regiono komiteto metinės sesijos (2012 m.) priimtos Europos sveikatos politikos strategijos „Sveikata 2020“, kurioje yra numatomas integralumas bei holistinis sveikatos supratimas.

Molecular Events Beyond apoCAM Preassociation in the CAM Regulation of Cav1.3 Channels

CaV channel regulation by calmodulin (CaM) is a central prototype for ion-channel modulation. Despite long study, relatively little is known mechanistically, beyond the initial preassociation of Ca2+-free CaM (apoCaM) with an IQ domain on the carboxy-terminus of channels. Most studies have focused on the IQ domain and immediate upstream ‘preIQ’ regions, despite hints that further upstream elements in the carboxy-terminus could be important. Accordingly, we here undertake alanine scanning mutagenesis of the entire carboxy-terminus upstream of the IQ domain (the proximal CI-region, PCI). For analysis, we chose CaV1.3 channels (highly homologous to classic CaV1.2), because they exhibit robust CaM-mediated inactivation (CDI), with particularly well-resolved profiles for both N- and C-lobe forms of inactivation. Several unexpected results were obtained. First, mutations throughout the preIQ domain left CDI essentially unchanged, at odds with functional hotspots in the homologous region of CaV1.2. Second, newly identified segments, situated upstream of the preIQ region, proved selectively critical for the C-lobe form of CDI. Specifically, we argue that the PCI region is the Ca2+/CaM effector site for C-lobe CDI, as revealed by quantitative comparison of the effect of PCI mutations on CDI, to their effect on PCI binding with Ca2+/CaM (‘Ψ-analysis’). Third, we further exploit Ψ-analysis to extend and confirm that the Ca2+/CaM effector site for the N-lobe form of CDI is structurally distinct, residing in the NSCaTE element of the channel amino terminus (Nature 451:830). Finally, while the IQ-domain is a primary site for apoCaM preassociation, our scan surprisingly reveals that PCI harbors additional preassociation sites, especially important for the N-lobe of apoCaM. Overall, this alanine scan of the CaV1.3, together with that of the IQ domain (companion abstract), outlines the long-sought molecular events beyond the initial apoCaM preassociation with the channel.

Regulation of FMN Subdomain Interactions and Function in Neuronal Nitric Oxide Synthase

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.

Unified Mechanisms of Ca 2+ Regulation across the Ca 2+ Channel Family

L-type (Ca V1.2) and P/Q-type (Ca V2.1) calcium channels possess lobe-specific CaM regulation, where Ca 2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the Ca V1-2 channel family, R-type (Ca V2.3) and N-type (Ca V2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca 2+-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca 2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in Ca V1-2 channels—independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca 2+ activity, respectively.

Functionally Important Calmodulin-binding Sites in Both NH2- and COOH-terminal Regions of the Cone Photoreceptor Cyclic Nucleotide-gated Channel CNGB3 Subunit

Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.

CDNA cloning and characterization of a novel squid rhodopsin kinase encoding multiple modular domains

Rhodopsin phosphorylation is one of the key mechanisms of inactivation in vertebrate and invertebrate visual signal transduction. Here we report the cDNA cloning and protein characterization of a 70-kDa squid rhodopsin kinase, SQRK. The cDNA encoding the 70-kDa protein demonstrates high sequence identity with octopus rhodopsin kinase (92%) and mammalian beta-adrenergic receptor kinases (63-65%), but only 33% similarity with bovine rhodopsin kinase, suggesting that invertebrate rhodopsin kinases may be structurally similar to beta-adrenergic receptor kinases. This cDNA encodes three distinct modular domains: RGS, S/TKc, and PH domains. The native SQRK is an eye-specific protein that is only expressed in photoreceptor cells and the optic ganglion as determined by immunoblotting. Purified SQRK is able to phosphorylate both squid and bovine rhodopsin. Squid rhodopsin phosphorylation by purified SQRK was sensitive to both Mg2+ and GTPgammaS but was insensitive to Ca2+/CaM regulation. The ability of SQRK to phosphorylate rhodopsin was totally lost in the presence of SQRK-specific antibodies. Our results suggest that SQRK plays an important role in squid visual signal termination.

Altered control of calcium in coronary smooth muscle cells by exercise training

The increase in myoplasmic free Ca (Cam) is a primary trigger of contraction in vascular smooth muscle. We review data showing that the sarcoplasmic reticulum (SR) can buffer (attenuate) increases in Cam by: 1) sequestering a fraction of Ca entering the cell via sarcolemmal influx pathways, and 2) slowly releasing Ca from the SR toward the sarcolemma for extrusion from the cell, thereby decreasing subsequent agonist-induced Cam transients and contraction--so called "SR Ca unloading." Endurance exercise trained (EX), not sedentary (SED), Yucatan miniature pigs show SR Ca unloading via a ryanodine-sensitive SR Ca release pathway. The slow release of Ca from SR of EX cells may allow for efflux from the cell by close functional association with sarcolemmal Ca efflux mechanisms. In contrast, rapid, bolus release and resequestration of Ca by the SR of SED cells prevents Ca efflux from the cell. The endothelin-sensitive SR Ca store, a subset of the caffeine- and ryanodine-sensitive SR, is decreased in EX cells. Mildly increased resting Cam in EX cells may reflect a constant leak of Ca from the SR. The endothelin-sensitive SR Ca store was loaded above basal levels by depolarization-induced Ca influx. Collectively, these data indicate altered Cam regulation by the SR in coronary artery of EX animals. Future studies should focus on the molecular mechanisms of altered Cam regulation.

Water relation parameters of the CAM plant Kalanchoë daigremontiana in relation to diurnal malate oscillations.

In the CAM plant Kalanchoë daigremontiana, kept in an environmental rhythm of 12 h L: 12 h D in a growth chamber at 60% relative humidity and well watered in the root medium, decreasing water potentials and osmotic potentials of the leaves are correlated with malate accumulation in the dark. In the light increasing water and osmotic potentials (ψ W and ψ S ) are associated with decreasing malate levels. Transpiratory H2O loss is high in dark and low in light.In continuous light, the CAM rhythm rapidly disappears in the form of a highly damped endogenous oscillation. Malate levels, and water and osmotic potentials of the leaves remain correlated as described above. However, transpiration is very high as malate levels decrease and water and osmotic potentials increase.It can concluded, that water relation parameters like total water potential (ψ W ) and osmotic potential (ψ S ) change in close correlation with changes of malic acid levels. As an important osmotically active solute in CAM plants, malic acid appears to affect water relations independently of and in addition to transpiration. The question remains open, whether turgor (ψ P ) is involved in CAM regulation in intact plants in a similar way as it determines malate fluxes in leaf slices.