A horizontally transferred tRNA(Cys) gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is aminoacylated

Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.     

Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ～72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ～40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.     

Uric Acid Level Has a U-Shaped Association with Loss of Kidney Function in Healthy People: A Prospective Cohort Study

BackgroundThe relationship between hyperuricemia and chronic kidney disease (CKD) has been found in various observational studies. Although hypouricemia is associated with cardiovascular events, it has not been established as a risk factor for CKD. We investigated the relationship between serum uric acid level and the loss of kidney function and incident CKD in healthy people.ResultsThe following data was obtained: mean±SD age, male, 39.6±10.4 years, female 38.4±10.8 years; eGFR, male, 81.9±16.4 ml/min/1.73m2, female, 82.1±17.5 ml/min/1.73m2; serum uric acid level, male, 5.8±1.2 mg/dl, female, 4.1±0.9 mg/dl. Both low and high serum uric acid levels were associated with the outcome and eGFR decline in males (multivariate logistic additional additive models, linear p = 0.0001, spline p = 0.043; generalized additive models, linear p = 0.0001, spline p = 0.012). In subjects with low serum uric acid levels (male, <5 mg/dl; female, <3.6 mg/dl), multivariate linear mixed models showed that low serum uric acid levels were associated with eGFR decline in a time-dependent manner (male, p = 0.0001; female, p = 0.045).ConclusionThis study showed that low as well as high levels of uric acid are associated with the loss of kidney function. Hypouricemia is a candidate predictor of kidney function decline in healthy people.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Intestinal malrotation with suspected cow's milk allergy: a case report

ABSTRACT: BACKGROUND: Intestinal malrotation is an incomplete rotation of the intestine. Failure to rotate leads to abnormalities in intestinal positioning and attachment that leave obstructing bands across the duodenum and a narrow pedicle for the midgut loop, thus making it susceptible to volvulus. One of the important differential diagnoses for malrotation is an allergy to cow's milk. Several studies have described infants with surgical gastrointestinal diseases and cow's milk allergy. However, to our knowledge, no study has reported infants with intestinal malrotation who have been symptomatic before surgery was performed and have been examined by allergen-specific lymphocyte stimulation test and food challenge tests with long-term follow-up. CASE PRESENTATION: The patient was a Japanese male born at 39 weeks of gestation. He was breast-fed and received commercial cow's milk supplementation starting the day of birth and was admitted to our hospital at 6 days of age due to bilious vomiting. Plain abdominal radiography showed a paucity of gas in the distal bowel. Because we demonstrated malpositioning of the intestine by barium enema, we repositioned the bowel in a normal position by laparotomy. The patient was re-started on only breast milk 2 days post surgery because we suspected the presence of a cow's milk allergy, and the results of an allergen-specific lymphocyte stimulation test showed a marked increase in lymphocyte response to kappa-casein. At 5 months of age, the patient was subjected to a cow's milk challenge test. After the patient began feeding on cow's milk, he had no symptoms and his laboratory investigations showed no abnormality. In addition, because the patient showed good weight gain and no symptoms with increased cow's milk intake after discharge, we concluded that the present case was not the result of a cow's milk allergy. At 1 year, the patient showed favorable growth and development, and serum allergy investigations revealed no reaction to cow's milk. CONCLUSION: When physicians encounter infants with surgical gastrointestinal disease, including intestinal malrotation, they should consider cow's milk allergy as a differential diagnosis or complication and should utilize food challenge tests for a definitive diagnosis.     

Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy

Oral manifestations of Down syndrome include high susceptibility to gingival inflammation with early onset and rapidly progressive periodontitis. The influence of reactive oxygen species (ROS) on periodontitis of Down syndrome is unclear. The aim of this study was to characterize ROS formation in Down syndrome-gingival fibroblasts (DS-GF) using electron spin resonance (ESR) spin trapping with 5,5-dimetyl-1-pyrolline-N-oxide (DMPO), and to determine whether ROS generation plays a role in the pathogenesis of periodontitis in Down syndrome patients. We observed formation of the DMPO-OH spin adduct, indicating HO* generation from cultured DS-GF and non-DS-GF. The increased HO* generation in cultured DS-GF was strongly decreased in the presence of the H2O2 scavenger, catalase, or the iron chelator, desferal. This may due to the enzymatic ability of over-expressed CuZn-superoxide dismutase in Down syndrome to catalyze the formation of H2O2 from O2*-, thereby increasing the availability of substrate H2O2 for the iron-dependent generation of HO* via the Fenton reaction, suggesting that HO* generated from DS-GF may be involved in progressive periodontitis of Down syndrome.     

Erratum to: Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell transplants

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase

F₁-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F₁-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 10⁶ M⁻¹ s⁻¹ at 4°C to 4.3 × 10⁷ M⁻¹ s⁻¹ at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F₁-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.