Single channel characterization of the mitochondrial ryanodine receptor in heart mitoplasts

Heart mitochondria utilize multiple Ca(2+) transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca(2+) uptake pathway across the inner membrane to control "excitation and metabolism coupling." In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ～100, ～225, ～700, and ～1,000 picosiemens (pS) in symmetrical 150 mm CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels.     

Maternal Determinants of Birth Weight in Northern Ghana

Objectives

Weight at birth is usually considered as an indicator of the health status of a given society. As a result this study was designed to investigate the association between birth weight and maternal factors such as gestational weight gain, pre—pregnancy BMI and socio—economic status in Northern Ghana.

Methods

The study was a facility-based cross-sectional survey conducted in two districts in the Northern region of Ghana. These districts were purposively sampled to represent a mix of urban, peri—urban and rural population. The current study included 419 mother-infant pairs who delivered at term (37–42 weeks). Mother’s height, pre-pregnancy weight and weight changes were generated from the antenatal records. Questionnaires were administered to establish socio-economic and demographic information of respondents. Maternal factors associated with birth weight were examined using multiple and univariate regressions.

Results

The mothers were generally well nourished before conception (Underweight 3.82%, Normal 57.76%, Overweight 25.06% and Obesity 13.37%) but approximately half of them could not gain adequate weight according to Institute of Medicine recommendations (Low weight gain 49.64%, Adequate weight gain 42.96% and Excessive weight gain 7.40%). Infants whose mothers had excess weight gain were 431g (95% CI 18–444) heavier compared to those whose mothers gained normal weight, while those whose mothers gained less were 479g (95% CI -682– (-276) lighter. Infants of mothers who were overweight and obese before conception were 246g (95% CI 87–405) and 595g (95% CI 375–815) respectively heavier than those of normal mothers, whereas those whose mothers were underweight were 305g (95% CI -565 –(-44) lighter. The mean birth weight observed was 2.98 ± 0.68 kg.

Conclusion

Our findings show that pre-pregnancy body mass index and weight gain during pregnancy influence birth weight. Therefore, emphasis should be placed on counseling and assisting pregnant women to stay within the recommended weight gain ranges.     

Diagnosis and therapeutic monitoring of inborn errors of creatine metabolism and transport using liquid chromatography–tandem mass spectrometry in urine,plasma and CSF

Biochemical detection of inborn errors of creatine metabolism or transport relies on the analysis of three main metabolites in biological fluids: guanidinoacetate (GAA), creatine (CT) and creatinine (CTN). Unspecific clinical presentation of the diseases might be the cause that only few patients have been diagnosed so far. We describe a LC–MS/MS method allowing fast and reliable diagnosis by simultaneous quantification of GAA, CT and CTN in urine, plasma and cerebrospinal fluid (CSF) and established reference values for each material.     

DNA end resection is needed for the repair of complex lesions in G1-phase human cells

Repair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy.     

The small protein floodgates are opening; now the functional analysis begins

Aside from a few serendipitous discoveries, small proteins of less than 50 amino acids in bacteria and 100 amino acids in eukaryotes were largely ignored due to challenges in their genetic and biochemical detection. However, with the ever-increasing availability of completed genome sequences and deep sequencing, which allows analysis of genome-wide ribosome occupancy, hundreds of small proteins are now being identified. This brings to the forefront the challenges and opportunities associated with the characterization of these proteins.See research article: http://www.biomedcentral.com/1471-2164/15/946.     

Loss of Hippocampal Neurons after Kainate Treatment Correlates with Behavioral Deficits

Treating rats with kainic acid induces status epilepticus (SE) and leads to the development of behavioral deficits and spontaneous recurrent seizures later in life. However, in a subset of rats, kainic acid treatment does not induce overt behaviorally obvious acute SE. The goal of this study was to compare the neuroanatomical and behavioral changes induced by kainate in rats that developed convulsive SE to those who did not. Adult male Wistar rats were treated with kainic acid and tested behaviorally 5 months later. Rats that had experienced convulsive SE showed impaired performance on the spatial water maze and passive avoidance tasks, and on the context and tone retention tests following fear conditioning. In addition, they exhibited less anxiety-like behaviors than controls on the open-field and elevated plus-maze tests. Histologically, convulsive SE was associated with marked neuron loss in the hippocampal CA3 and CA1 fields, and in the dentate hilus. Rats that had not experienced convulsive SE after kainate treatment showed less severe, but significant impairments on the spatial water maze and passive avoidance tasks. These rats had fewer neurons than control rats in the dentate hilus, but not in the hippocampal CA3 and CA1 fields. Correlational analyses revealed significant relationships between spatial memory indices of rats and neuronal numbers in the dentate hilus and CA3 pyramidal field. These results show that a part of the animals that do not display intense behavioral seizures (convulsive SE) immediately after an epileptogenic treatment, later in life, they may still have noticeable structural and functional changes in the brain.     

Fronto-Parietal Connectivity Is a Non-Static Phenomenon with Characteristic Changes during Unconsciousness

Background

It has been previously shown that loss of consciousness is associated with a breakdown of dominating fronto-parietal feedback connectivity as assessed by electroencephalogram (EEG) recordings. Structure and strength of network connectivity may change over time. Aim of the current study is to investigate cortico-cortical connectivity at different time intervals during consciousness and unconsciousness. For this purpose, EEG symbolic transfer entropy (STEn) was calculated to indicate cortico-cortical information transfer at different transfer times.

Methods

The study was performed in 15 male volunteers. 29-channel EEG was recorded during consciousness and propofol-induced unconsciousness. EEG data were analyzed by STEn, which quantifies intensity and directionality of the mutual information flow between two EEG channels. STEn was computed over fronto-parietal channel pair combinations (10 s length, 0.5–45 Hz total bandwidth) to analyze changes of intercortical directional connectivity. Feedback (fronto → parietal) and feedforward (parieto → frontal) connectivity was calculated for transfer times from 25 ms to 250 ms in 5 ms steps. Transfer times leading to maximum directed interaction were identified to detect changes of cortical information transfer (directional connectivity) induced by unconsciousness (p<0.05).

Results

The current analyses show that fronto-parietal connectivity is a non-static phenomenon. Maximum detected interaction occurs at decreased transfer times during propofol-induced unconsciousness (feedback interaction: 60 ms to 40 ms, p = 0.002; feedforward interaction: 65 ms to 45 ms, p = 0.001). Strength of maximum feedback interaction decreases during unconsciousness (p = 0.026), while no effect of propofol was observed on feedforward interaction. During both consciousness and unconsciousness, intensity of fronto-parietal interaction fluctuates with increasing transfer times.

Conclusion

Non-stationarity of directional connectivity may play a functional role for cortical network communication as it shows characteristic changes during propofol-induced unconsciousness.     

Ocean cleaning stations under a changing climate: biological responses of tropical and temperate fish‐cleaner shrimp to global warming

Cleaning symbioses play an important role in the health of certain coastal marine communities. These interspecific associations often occur at specific sites (cleaning stations) where a cleaner organism (commonly a fish or shrimp) removes ectoparasites/damaged tissue from a ‘client’ (a larger cooperating fish). At present, the potential impact of climate change on the fitness of cleaner organisms remains unknown. This study investigated the physiological and biochemical responses of tropical (Lysmata amboinensis) and temperate (L. seticaudata) cleaner shrimp to global warming. Specifically, thermal limits (CTMax), metabolic rates, thermal sensitivity, heat shock response (HSR), lipid peroxidation [malondialdehyde (MDA) concentration], lactate levels, antioxidant (GST, SOD and catalase) and digestive enzyme activities (trypsin and alkaline phosphatase) at current and warming (+3 °C) temperature conditions. In contrast to the temperate species, CTMax values decreased significantly from current (24–27 °C) to warming temperature conditions (30 °C) for the tropical shrimp, where metabolic thermal sensitivity was affected and the HSR was significantly reduced. MDA levels in tropical shrimp increased dramatically, indicating extreme cellular lipid peroxidation, which was not observed in the temperate shrimp. Lactate levels, GST and SOD activities were significantly enhanced within the muscle tissue of the tropical species. Digestive enzyme activities in the hepatopancreas of both species were significantly decreased by warmer temperatures. Our data suggest that the tropical cleaner shrimp will be more vulnerable to global warming than the temperate Lysmata seticaudata; the latter evolved in a relatively unstable environment with seasonal thermal variations that may have conferred greater adaptive plasticity. Thus, tropical cleaning symbioses may be challenged at a greater degree by warming‐related anthropogenic forcing, with potential cascading effects on the health and structuring of tropical coastal communities (e.g. coral reefs).     

Specific Contactin N-Glycans Are Implicated in Neurofascin Binding and Autoimmune Targeting in Peripheral Neuropathies

Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to contactin (1). The complex of contactin·Caspr·neurofascin-155 (NF155) enables the formation of paranodal junctions, suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of contactin and was dependent on N-glycans. The serum of one patient was selectively directed against contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of contactin N-glycans, we have mutated each of the nine consensus N-glycosylation sites independently. We found that the mutation of three sites (N467Q/N473Q/N494Q) in Ig domain 5 of contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (Asn-467, Asn-473, and Asn-494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgGs from the four CIDP patients prevented adhesive interaction between contactin·Caspr and NF155. Importantly, we showed that the anti-contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly suggest that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.