The force-frequency relationship is dependent on Ca2+-influx via L-type- and SR-Ca2+-channels in human heart

The present study investigated the influence of Bay K 8644 and nifedipine (Nif) on the force-frequency relationship and on tetanic tension and force of contraction of failing human myocardium (PAP, n = 12). In addition, ryanodine (Rya) was studied on the force-frequency relationship. Bay K 8644 (0.1 μM) increased, but Nif (0.01 μM) reduced isometric force of contraction significantly. However, both, Bay K 8644 (2 Hz vs. 0.5 Hz: Control: −31.6 ± 7.8 %; +Bay K 8644: +103 ± 30 % (% basal); p < 0.005) as well as Nif (2 Hz vs. 0.5 Hz: Control: −8.8 ± 9.7 %; +Nif: +90.9 ± 31.5 %) (% basal); p < 0.05), were able to restore a positive FFR in PAP. By measurement of tetanic tension and posttetanic potentiation in the presence of the 1,4-dihydropyridines, we support the hypothesis of the existence and functional relevance of a dihydropyridin-ryanodine receptor junctional complex. In skinned fiber preparations, Bay K 8644 showed no effect on Ca²⁺-sensitivity or caffeine induced Ca²⁺-release. Rya (10 μM) decreased force of contraction in PAP and was effective in restoring a postive FFR (2 Hz vs. 0.5 Hz: Control: −7.3 ± 5.1 %; +Rya: +98.0 ± 31.9 % (% basal); p < 0.05). Thus, the altered FFR and Ca²⁺-homeostasis in failing human myocardium may result from changes in sarcolemmal Ca²⁺-influx and/or from altered SR-Ca²⁺-load. Received: 27 August 1998, Returned for 1. revision: 24 September 1998, 1. Revision received: 23 October 1998, Returned for 2. revision: 20 November 1998, 2. Revision received: 18 December 1998, Accepted: 22 December 1998     

School Wellness Committees Are Associated With Lower Body Mass Index Z‐Scores and Improved Dietary Intakes in US Children: The Healthy Communities Study

BACKGROUND

Our objective was to examine the association between school wellness committees and implementation of nutrition wellness policies and children's weight status and obesity‐related dietary outcomes.

METHODS

A cross‐sectional study was conducted of 4790 children aged 4‐15 years recruited from 130 communities in the Healthy Communities Study. Multilevel statistical models assessed associations between school wellness policies and anthropometric (body mass index z‐score [BMIz]) and nutrition measures, adjusting for child and community‐level covariates.

RESULTS

Children had lower BMI z‐scores (?0.11, 95% confidence interval [CI]: ?0.19, ?0.03) and ate breakfast more frequently (0.14 days/week, 95% CI: 0.02‐0.25) if attending a school with a wellness committee that met once or more in the past year compared to attending a school with a wellness committee that did not meet/did not exist. Children had lower added sugar (p < .0001), lower energy‐dense foods (p = .0004), lower sugar intake from sugar‐sweetened beverages (p = .0002), and lower dairy consumption (p = .001) if attending a school with similar or stronger implementation of the nutrition components of the school wellness policies compared to other schools in the district.

CONCLUSIONS

A more active wellness committee was associated with lower BMI z‐scores in US schoolchildren. Active school engagement in wellness policy implementation appears to play a positive role in efforts to reduce childhood obesity.

     

Effects of plasma nitric oxide levels on platelet activation in single donor apheresis and random donor concentrates.

P-selectin is an useful marker to determine platelet activation and nitric oxide inhibits platelet activation, secretion, adhesion and aggregation. The aim of this study was to investigate the relationship between nitric oxide and P-selectin values in both single donor apheresis and random donor platelet concentrates. According to the results of this study, we found that the best platelet concentrate is freshly prepared single donor apheresis concentrate and it is important to prevent activation at the beginning of the donation. Nitric oxide, which is synthesized from platelets during the storage period, is not sufficient to prevent platelet activation.     

Overexpression of sorcin enhances cardiac contractility in vivo and in vitro

Sorcin (SOR), an EF-hand Ca(2+)-binding protein, interacts with the sarcolemmal proteins Annexin VII and L-type Ca(2+)-channel and with the sarcoplasmic reticulum (SR) Ca(2+)-release channel (ryanodine-receptor, RYR), and has been implicated to influence the intracellular Ca(2+)-homeostasis. The present study aimed at investigating the effects of increased SOR expression on force development and relaxation in virus transfected rat hearts and isolated cardiomyocytes. We generated an adenovirus encoding the SOR coding DNA with a separate cassette for green fluorescent protein (GFP) both driven by the CMV-promoter to induce SOR-overexpression (Ad.SOR.GFP). As control served an adenovirus carrying an empty cassette with a separate cassette for GFP also driven by CMV-promoters (Ad.GFP). Cardiomyocytes of healthy male rats were isolated, transfected and cultured for 48 h with Ad.SOR.GFP as well as Ad.GFP as control. In addition, Ad.SOR.GFP was injected into coronary arteries via a catheter-based technique and rat hearts were transfected in vivo for 12 days. Echocardiography was performed to assess cardiac function at 7 and 12 days before the animals were sacrificed. A 1.7-fold increase of the SOR protein amount in cultured myocytes treated with Ad.SOR.GFP compared to Ad.GFP-transfected cells indicated a successful overexpression of SOR. Cell-contracting experiments using infected cardiomyocytes (transfection: 48 h; frequency: 0.5 Hz) exhibited a significantly higher peak force of contraction (FOC) in the SOR-overexpression group (n = 64) vs. control (n = 21) (6.8% +/- 0.2% vs. 4.3% +/- 0.1%). Beta-adrenergic stimulation with forskolin resulted in similar increases in FOC. Echocardiography of in vivo transfected rat hearts exhibited enhanced fractional shortening (65.9 +/- 5.5% vs. 79.3 +/- 2.5%) and decreased end-systolic diameters indicating enhanced cardiac contractility. Gross morphology was similar in both groups after 14 days of transfection. These results strengthen the notion that overexpression of SOR improves cardiac contractility independent of beta-adrenergic stimulation and may prove beneficial in the treatment of decreased cardiac output such as heart failure.     

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s⁻¹) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.Anthropogenic compounds used as pesticides, solvents, and explosives often persist in the environment and can cause toxicity to humans and wildlife. In some cases, microbes have evolved the ability to partially or completely degrade such compounds. Complete degradation of anthropogenic compounds requires the evolution of new metabolic pathways that convert these compounds into metabolites that can be used by existing metabolic networks. The sequence of steps in new degradative pathways is dictated by the repertoire of enzymes in exposed microbes and/or microbial communities that can catalyze newly needed reactions. The serendipitous assembly of new degradation pathways can result in the production of intermediates that are toxic due to their intrinsic chemical reactivity or adventitious interactions between these intermediates and cellular macromolecules. The toxicity of such intermediates can pose a significant challenge to the evolution of efficient pathways for the degradation of anthropogenic compounds (1).The pathway for degradation of pentachlorophenol (PCP) by Sphingobium chlorophenolicum (Fig. 1) (2, 3) includes the remarkably toxic intermediate tetrachlorobenzoquinone (TCBQ). The LD₅₀ for Escherichia coli protoplasts is <1 µM (4). TCBQ is the most toxic of a set of 14 quinones (5), with an LD₅₀ of 18 µM for primary rat hepatocytes. (For comparison, the LD₅₀ for benzoquinone is 57 µM and for 2,3,4,6-tetramethylbenzoquinone is 800 µM.) TCBQ is a potent alkylating agent, capable of forming covalent adducts with proteins (6) and with the nucleobases of DNA (7). Thiols such as glutathione react with TCBQ particularly rapidly (3, 6). Depletion of glutathione eliminates a critical cellular mechanism for protection against alkylating agents and oxidative damage. Further, TCBQ can generate hydroxyl radicals by its interaction with hydrogen peroxide (8). Thus, TCBQ is a devastating toxin due to a plethora of molecular mechanisms.Open in a separate windowFig. 1.The initial steps in the pathway for degradation of PCP in S. chlorophenolicum via the intermediates TCBQ and TCHQ are highlighted in green. Hydroxylation of TCP by PcpB forms TCHQ directly.The pathway for degradation of PCP in S. chlorophenolicum is believed to be still in the process of evolving because PCP was introduced as a pesticide less than 100 y ago (9, 10) and some of the enzymes in the pathway function quite poorly (3, 11). The intermediacy of a dangerous compound such as TCBQ in a newly evolving pathway brings up a number of interesting questions. First, why has the pathway evolved via a route that involves TCBQ? Second, how does the bacterium protect itself from the toxic effects of TCBQ? And third, does the intermediacy of TCBQ explain the inefficiency of PCP degradation by S. chlorophenolicum?The answer to the first question emerges from a consideration of the enzymatic repertoire that exists to initiate degradation of naturally occurring phenols. Phenols are common natural products, produced by a range of organisms from microbes to insects and by degradation of lignin. Cleavage of phenols under aerobic conditions requires the introduction of a second hydroxyl group. This reaction can be carried out by cytochrome P450 enzymes or flavin monooxygenases. PCP hydroxylase (PcpB), the first enzyme in the pathway for degradation of PCP, is a flavin monooxygenase (3) and likely originated from an enzyme that hydroxylated a naturally occurring phenol. Hydroxylation of phenols at a position carrying a hydrogen results in formation of a catechol or hydroquinone rather than a benzoquinone (Fig. 1). However, when a good leaving group such as chloride is present at the position of hydroxylation, the unstable intermediate eliminates HCl to form a benzoquinone.The answer to the second question is suggested by the observation that microbes that generate benzoquinones from phenols typically have a reductase that converts a benzoquinone to a hydroquinone. The gene encoding the reductase is located immediately downstream of the gene encoding the hydroxylase (12). Examples are found in the pathways for degradation of 2,4,6-trichlorophenol (TriCP), p-nitrophenol and o-nitrophenol in Cupriavidus necator JMP134 (13), Pseudomonas sp. Strain WBC-3 (14), and Alcaligenes sp. strain NyZ215 (15), respectively. Like these microbes, S. chlorophenolicum contains a gene encoding a reductase (PcpD) immediately downstream of the gene encoding the hydroxylase (PcpB). We have previously shown that pcpD is essential for survival of S. chlorophenolicum at high concentrations of PCP but is not essential for survival at high concentrations of 2,3,5,6-tetrachlorophenol (TCP) (2). In this work, we have addressed the mechanism of that protection. As shown in Fig. 1, hydroxylation of TCP produces tetrachlorohydroquinone (TCHQ) whereas hydroxylation of PCP produces the more toxic TCBQ. PcpD is an efficient TCBQ reductase. However, its ability to prevent the escape of TCBQ to the solvent before it is reduced to TCHQ is an even more critical aspect of its function.Our results provide insights into the answer to the third question. Because PcpB and PcpD do not form a stable complex, the rate of their encounter is limited by diffusion. The slow rate of turnover by PcpB [at 0.02 s⁻¹ (3), it is the rate-limiting step of the pathway (4)] may be a result of selective pressure to prevent production of TCBQ at a rate faster than it can be intercepted by PcpD. Thus, the efficiency of the interaction between PcpB and PcpD may limit the efficiency of the degradation of PCP and prevent selection of a more catalytically robust PcpB.