Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts.To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17±4 g hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8±2.3 to 45.1±3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7±89.8 to 2,181±135.4 mmHg/s in the contraction rate, and 713±60.2 to 1,364±137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.     

Orofacial evaluation of individuals with temporomandibular disorder after LED therapy associated or not of occlusal splint: a randomized double-blind controlled clinical study

Lasers in Medical Science - This study compared the effects of LED therapy associated with occlusal splint (OS) on the signs and symptoms of temporomandibular disorder (TMD). In this randomized,...     

Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase

Nitrogenase catalyzes the reduction of N₂ and protons to yield two NH₃ and one H₂. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH₃), or hydrazine as substrate recently was shown to trap a common intermediate, designated I. It was further concluded that the two N-atoms of N₂ are hydrogenated alternately (“Alternating” (A) pathway). In the present work, Q-band CW EPR and ⁹⁵Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of ^14,15N isotopologs plus ^1,2H isotopologs of methyldiazene. It is concluded that: H has NH₂ bound to FeMo-co and corresponds to the penultimate intermediate of N₂ hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH₃; I corresponds to the final intermediate in N₂ reduction, the state formed after accumulation of eight electrons/protons, with NH₃ still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N₂ reduction by nitrogenase.     

Housing instability and health: Findings from the Michigan recession and recovery study

The recession of the late 2000s has increased interest in the consequences of housing instability. Previous research has shown poorer health among those experiencing housing instability, but extant studies generally have focused on selected populations (e.g., homeowners or renters) or studied only one type of housing instability (e.g. homelessness). Using new data from the Michigan Recession and Recovery Study, a population-based sample of working-aged adults from Southeastern Michigan, U.S.A., in late 2009–early 2010, we found that about one-third of respondents recently experienced some type of housing instability. Many, but not all, types of instability were associated with health. Even after adjustment for sociodemographic characteristics and earlier health, individuals who had moved for cost reasons in the past three years were more likely than those with no housing instability to report a recent anxiety attack, while those who experienced homelessness in the past year had a higher likelihood of reporting fair/poor self-rated health and of meeting criteria for major or minor depression. Renters behind on rental payments were more likely to meet criteria for depression, while mortgage-holders behind on their mortgage or in foreclosure had a higher likelihood of reporting fair/poor self-rated health or a recent anxiety attack. Among respondents who had ever owned a home, those who completed a foreclosure recently were more likely to report major or minor depression or an anxiety attack. However, frequent moves were not associated with poorer health, and doubling up and eviction were not associated with poorer health after adjustment for characteristics that sort people into different housing instability experiences. Our findings suggest the importance of considering multiple types of housing instability and using appropriate risk groups and comparison categories.     

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

The biological reduction of N₂ to NH₃ catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: k_ATP = 70 s⁻¹, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (k_ET = 140 s⁻¹, 25 °C), (ii) ATP hydrolysis (k_ATP = 70 s⁻¹, 25 °C), (iii) Phosphate release (k_Pi = 16 s⁻¹, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (k_diss = 6 s⁻¹, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe^ox(ADP)₂ protein and the reduced MoFe protein.Biological nitrogen fixation catalyzed by the Mo-dependent nitrogenase has a limiting reaction stoichiometry shown in Eq. 1 (1, 2):The ATP-driven reduction of one N₂ with evolution of one H₂ requires a minimum of 8 e⁻ and the hydrolysis of 16 ATP molecules in a complex cascade of events in which electron transfer (ET) from the nitrogenase Fe protein to the catalytic MoFe protein is coupled to the hydrolysis of two ATP molecules (1, 3, 4). The Fe protein is a homodimer with a single [4Fe–4S] cluster and two nucleotide binding sites, one in each subunit (5). The MoFe protein is an α₂β₂-tetramer, with each αβ-pair functioning as a catalytic unit that binds an Fe protein (6). Each αβ-unit contains an [8Fe–7S] cluster (abbreviated as P cluster) and a [7Fe–9S–Mo–C–R-homocitrate] cluster (abbreviated as FeMo cofactor or M cluster) (6–10). In each ET event, the Fe protein, in the reduced (1+) state with two bound ATP, first associates with the MoFe protein (Fig. 1). In a recent model, termed “deficit spending,” it is proposed that this association triggers a two-step ET event (11, 12). The first ET step occurs inside the MoFe protein, involving ET from the P cluster resting state (P^N) to the resting FeMo cofactor (M^N), resulting in an oxidized P cluster (P¹⁺) and a reduced FeMo cofactor (M^R) (12). This ET event is conformationally gated (11) with an apparent first-order rate constant (k_ET) between 100 and 140 s⁻¹ (11, 12). In the second ET step, an electron is transferred from the Fe protein [4Fe–4S] cluster to the oxidized P¹⁺ cluster, resulting in the return of the P cluster to the resting oxidation state (P^N) and an oxidized [4Fe–4S]²⁺ cluster in the Fe protein (12). This second step is fast, having a rate constant greater than 1,700 s⁻¹ (12).Open in a separate windowFig. 1.Order of events in nitrogenase complex. (A) Fe protein subunits are shown as two blue ovals (Left) with an ATP bound in each subunit and the [4Fe–4S] cluster (green cubane). (Right) MoFe protein α-subunit (orange) and β-subunit (green), with the P^N cluster shown as a gray box and the FeMo cofactor (M^N) shown as a gray diamond. (B) From left to right, order of events in the nitrogenase ET is shown with rate constants (s⁻¹) displayed where known.Transfer of one electron from the Fe protein to an αβ-unit of MoFe protein is known to be coupled to the hydrolysis of the two ATP molecules bound to the Fe protein, yielding two ADP and two Pi (2). Following the hydrolysis reaction, the two phosphates (Pi) are released from the protein complex with a first-order rate constant (k_Pi) of 22 s⁻¹ at 23 °C (13). The last event in the cycle is the release of the oxidized Fe protein with two ADP bound [Fe^ox(ADP)₂] from the MoFe protein with a rate constant (k_diss) of ∼6 s⁻¹, the rate-limiting step in catalysis at high electron flux (14). After dissociation from the MoFe protein, the [Fe^ox(ADP)₂] protein is prepared for a second round of electron delivery by one-electron reduction to [Fe^red(ADP)₂] and replacement of the two MgADP by MgATP. This cycle is repeated until enough electrons are transferred to the MoFe protein to achieve substrate reduction (15).Although the energetic coupling between ET and ATP hydrolysis is firmly established (1, 3, 4, 16), the nature of this coupling has remained unresolved: does ATP hydrolysis itself provide the principal energy input for the conformational change(s) that drive ET from Fe protein to the MoFe protein, or, does the bound ATP induce the formation of a reactive, “activated” conformation of the complex, with ET being driven by the free energy of ATP-activated protein–protein binding? These alternatives are characterized by different orders of ET and ATP hydrolysis, but the order has never been established. Some studies have indicated that ATP hydrolysis occurs after ET (13, 17, 18), whereas other studies have suggested just the opposite, namely that ATP hydrolysis occurs before ET (15, 16, 19, 20). One of the reasons for this lack of clarity in the order of these key events is the absence of direct measurement of ATP hydrolysis rates by nitrogenase within a single catalytic cycle. The rate constant for Pi release during one cycle has been measured, thereby establishing a lower limit on the rate constant for ATP hydrolysis (13). However, the rate constant for ATP hydrolysis could be much faster than Pi release, and could be faster than the rate constant for ET.Here, we have directly measured the rate constant for ATP hydrolysis for a single nitrogenase turnover cycle, as well as measuring the rate constants for each of the other key steps under the same conditions, thereby allowing an unequivocal assignment of the order of events in a single electron-transfer cycle. Establishing the order of events allows a full thermodynamic Fe–protein cycle to be constructed.     

A double logistic comparison of growth patterns of normal children and children with Down's syndrome.

A double logistic model was used to compare six parameters of growth in standing height of 31 children with Down's syndrome with 136 children from the California Guidance Study. Multivariate analysis of variance of the growth data showed that while there were significant differences in all six parameters favouring the normal over the Down's children, there were no significant differences with respect to error of fit. Multivariate analysis with final height as a covariate revealed that differences between the normal and the Down's children in the prepubertal and adolescent components were explainable by differences in final height. In summary, the double logistic model, when applied to this sample of Down's children, identified those well defined logistic components which are characteristic of the growth of normal children, the differences being those of degree, not of form.     

Reaction crystallization of pharmaceutical molecular complexes

A mechanism for cocrystal synthesis is reported whereby nucleation and growth of cocrystals are directed by the effect of the cocrystal components on reducing the solubility of the molecular complex to be crystallized. The carbamazepine:nicotinamide cocrystal (CBZ:NCT) was chosen as a model system to study the reaction cocrystallization pathways and kinetics in aqueous and organic solvents. Fiber optic Raman spectroscopy and Raman microscopy were used for in situ monitoring of the cocrystallization in macroscopic and microscopic scales in solutions, suspensions, slurries, and wet solid phases of cocrystal components. This study demonstrates the advantages of reaction cocrystallization methods to develop rational approaches for high-throughput screening of cocrystals that can be transferable to control batch and continuous cocrystallization processes.     

A methyldiazene (HN=N-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

Methyldiazene (HN=N-CH3) isotopomers labeled with 15N at the terminal or internal nitrogens or with 13C or 2H were used as substrates for the nitrogenase alpha-195Gln-substituted MoFe protein. Freeze quenching under turnover traps an S = (1/2) state that has been characterized by EPR and 1H-, 15N-, and 13C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt-Schrock mechanism for N2 reduction, H atoms sequentially add to the distal N before N-N bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to N-N bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before N-N cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in alpha-195Gln-substituted MoFe protein.