Ion transport across the mitochondrial inner and outer membranes is central to mitochondrial function, including regulation of oxidative phosphorylation and cell death. Although essential for ATP ...production by mitochondria, recent findings have confirmed that the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane and cell death. This review will discuss recent advances in understanding the molecular components of mPTP, its regulatory mechanisms and how these contribute directly to its physiological as well as pathological roles.
Department of Pediatrics, Divisions of 1 Cardiology and
2 Endocrinology, Yale University School of Medicine, New
Haven, Connecticut 06520
Catecholamines, acetylcholine, and adenosine are known
to ...influence cardiac function, yet the effects of these agents on
mammalian embryonic myocardium are largely unknown. To address this
issue, we compared the chronotrophic effects of adenosinergic,
adrenergic, and muscarinic agents on cultured murine embryos from
postcoital day (PC) 8.0, when the fusing heart tubes first begin to
beat, to PC 14, when cardiogenesis is essentially complete. At PC 8.0 and older, A 1 -adenosine receptor (A 1 AR)
activation significantly decreased heart rates. Adrenergic stimulation
caused modest increases in heart rates (145-155% of baseline)
beginning at PC 9.0. Muscarinic activation decreased heart rates only
after PC 13. When receptor gene expression was examined,
A 1 ARs and 1 ARs were expressed in isolated
hearts as early as PC 9.0, and 2 ARs and
m 2 -muscarinic receptor genes were expressed at PC 11.0. These results identify the adenosinergic system as the earliest
and most potent regulator of embryonic cardiac function and show
that prenatal responsiveness to catecholamines and acetylcholine
develops at later embryonic stages.
adenosine; catecholamines; acetylcholine; embryo
Study of the parasite mosquito stages of
Plasmodium and its use in the production of sporozoite vaccines against malaria has been hampered by the technical difficulties of in vitro development. Here, ...we show the complete axenic development of the parasite mosquito stages of
Plasmodium yoelii. While we demonstrate that matrigel is not required for parasite development, soluble factors produced and secreted by
Drosophila melanogaster S2 cells appear to be crucial for the ookinete to oocyst transition. Parasites cultured axenically are both morphologically and biologically similar to mosquito-derived ookinetes, oocysts, and sporozoites. Axenically derived sporozoites were capable of producing an infection in mice as determined by RT-PCR; however, the parasitemia was significantly much less than that produced by mosquito-derived sporozoites. Our cell free system for development of the mosquito stages of
P. yoelii provides a simplified approach to generate sporozoites that may be for biological assays and genetic manipulations.
The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive ...protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.
Phosphorus is a common additive used in food processing that is typically consumed in excess of the recommended daily allowance; however, our knowledge of its effects on health, in the context of ...normal renal function, is limited. Unlike phosphorus, calcium intake is generally less than recommended, and it has been hypothesized that the calcium to phosphorus ratio may be partly responsible for the proposed negative health consequences. Therefore, this study sought to determine the effects of increased phosphorus additive intake, in the context of high calcium consumption, on endocrine markers of mineral metabolism and cardiometabolic health. An outpatient feeding study was performed in which healthy adults were fed a run-in control diet for 2 weeks followed by a phosphorus additive enhanced diet with supplemental calcium to an approximate ratio of 1 (experimental diet) for 2 weeks. Blood and urine samples were collected, and participants had brachial flow-mediated dilatation measured, with analyses comparing follow-up measures to baseline. Two weeks of experimental diet increased serum fibroblast growth factor 23 concentrations but lowered body weight and serum leptin; however, other phosphorus responsive factors such as osteopontin and osteocalcin did not increase. A complementary study in male mice also demonstrated that the regulation of known dietary phosphorus responsive factors was mostly abrogated when dietary calcium was raised in parallel with phosphorus. In conclusion, the study identifies weight, leptin and insulin as responsive to dietary phosphorus and that certain aspects of the systemic phosphorus response are attenuated by a corresponding high calcium intake.
This study of targeted temperature interventions in 295 children who were comatose after cardiac arrest showed no significant difference between the hypothermia group (33.0°C) and the normothermia ...group (36.8°C) with respect to 1-year survival with a good functional outcome.
Out-of-hospital cardiac arrest in children often results in death or in poor long-term functional outcome in survivors.
1
–
3
In 2002, two trials involving adults showed that therapeutic hypothermia improved neurologic outcomes in comatose survivors after out-of-hospital cardiac arrest with ventricular fibrillation or ventricular tachycardia.
4
,
5
International guidelines recommend therapeutic hypothermia for adults with out-of-hospital cardiac arrest who have similar characteristics.
6
,
7
Recently, another trial involving adults after out-of-hospital cardiac arrest showed that therapeutic hypothermia with the use of a target temperature of 33°C, as compared with actively maintained therapeutic normothermia (36°C), did not improve outcomes.
8
The fundamental difference between this . . .
Mutations in SCN1A, encoding the voltage-gated sodium channel NaV1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with ...premature truncations of NaV1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of NaV1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) NaV1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative NaV1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-NaV1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-NaV1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional NaV1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.
Background: Nontruncating SCN1A mutations can cause severe myoclonic epilepsy of infancy (SMEI).
Results: Some SMEI-associated mutations exhibit reduced cell surface expression, which is reversible for certain alleles by pharmacological treatment.
Conclusion: Nontruncating SCN1A mutations with reduced cell surface expression may be amenable to pharmacological rescue.
Significance: Although rescue of trafficking-impaired SCN1A alleles may be beneficial, increased surface expression of dysfunctional channels could exacerbate SMEI.
Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea among travelers and pediatric populations worldwide. The tip-localized adhesin of colonization factor antigen (CFA)/I fimbriae ...was engineered as a donor strand complemented variant (dscCfaE) and delivered via transcutaneous immunization. Preclinical vaccine testing demonstrated safety, immunogenicity and efficacy. A series of open-label dose-escalating phase 1 studies evaluated a 3-dose (days 0, 21, 42) regimen via a transcutaneous skin patch. A total of forty-six subjects were enrolled into one of four vaccine dose levels (10, 50, 250, or 1250 µg) co-administered with single-mutant heat-labile enterotoxin (LTR(192G)). At the 50 µg dose level, ten subjects received the dscCfaE vaccine without LT(R192G). The vaccine was well tolerated with mild local vaccine site reactions characterized by an erythematous papular rash and pruritus, which were less frequent and reactive in the group not receiving LT(R192G). The frequency of responses to dscCfaE were moderate, whereas anti-toxin responses (serum IgA/IgG) ranged from 75 to 100% across groups that received LT(R192G). Antigen-specific antibody-secreting cell responses were elicited at all dose levels, but were generally low. Follow-on studies will optimize construct and route of delivery and assess efficacy in an ETEC challenge study.
Background
Osteogenesis imperfecta (OI) is a rare group of disorders characterized by increased susceptibility to fractures due to genetically determined bone fragility. About 90% of cases are due to ...mutations in COL1A1 (17q21.33) or COL1A2 (7q21.3) resulting in quantitative or qualitative defects in type I collagen, a key structural constituent of bone. OI due to complete COL1A1 deletion is rare.
Methods
We present a case of OI type I in a Caucasian female referred at 10 months of age for investigation of multiple fractures associated with minimal or no known trauma, small stature, and blue sclera. Her father has four to five lifetime fractures, blue sclera, normal stature, and a 14.5 kilobase (kb) deletion of COL1A1 detected by targeted array performed at an outside institution. Microarray comparative genomic hybridization was performed on the proband and all members of the family.
Results
A previously unreported 235 kb deletion at 17q21.33 encompassing COL1A1, ITGA3, PDK2, SGCA, and HILS1 was detected in the proband. Also identified in both the proband and sibling is a maternally inherited 283 kb gain at 8p21.3 encompassing CSGALNACT1 and a 163 kb loss at 10q21.3 encompassing CTNNA3. Analysis in the father revealed the same size deletion at 17q21.33 as in the proband.
Conclusion
Together with previously reported cases of COL1A1 deletions, this case report emphasizes the importance of a whole‐genome DNA copy number assessment in patients suspected for OI, which will elucidate the presence of precise COL1A1 deletions and any pathogenic secondary copy number variations.
Whole‐genome DNA copy number assessment in patients suspected for OI will elucidate the presence of precise COLIA1 deletions and any secondary pathogenic copy number variations. This case report highlights the importance of large COLIA1 deletions and secondary copy number variations in identifying clinical features which may establish as yet unknown variants of OI and lead to improved management and genetic counseling of patients.
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