TbMP44 is essential for RNA editing and structural integrity of the editosome in Trypanosoma brucei

RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation of TbMP44 gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.     

Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.     

Inheritance of cortical ER in yeast is required for normal septin organization

How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein-associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

The impact of a 6-year comprehensive community trial on the awareness, treatment and control rates of hypertension in Iran: experiences from the Isfahan healthy heart program

Background

Acute mental stress may contribute to the cardiovascular disease progression via autonomic nervous system controlled negative effects on the endothelium. The joint effects of stress-induced sympathetic or parasympathetic activity and endothelial function on atherosclerosis development have not been investigated. The present study aims to examine the interactive effect of acute mental stress-induced cardiac reactivity/recovery and endothelial function on the prevalence of carotid atherosclerosis.

Methods

Participants were 81 healthy young adults aged 24-39 years. Preclinical atherosclerosis was assessed by carotid intima-media thickness (IMT) and endothelial function was measured as flow-mediated dilatation (FMD) using ultrasound techniques. We also measured heart rate, respiratory sinus arrhythmia (RSA), and pre-ejection period (PEP) in response to the mental arithmetic and speech tasks.

Results

We found a significant interaction of FMD and cardiac RSA recovery for IMT (p = 0.037), and a significant interaction of FMD and PEP recovery for IMT (p = 0.006). Among participants with low FMD, slower PEP recovery was related to higher IMT. Among individuals with high FMD, slow RSA recovery predicted higher IMT. No significant interactions of FMD and cardiac reactivity for IMT were found.

Conclusions

Cardiac recovery plays a role in atherosclerosis development in persons with high and low FMD. The role of sympathetically mediated cardiac activity seems to be more important in those with impaired FMD, and parasympathetically mediated in those with relatively high FMD. The development of endothelial dysfunction may be one possible mechanism linking slow cardiac recovery and atherosclerosis via autonomic nervous system mediated effect.     

Double labeling of transferrin: tritium labeling of sialic acid and 125I or 59Fe labeling of the protein moiety

A method is described in which the glycoprotein transferrin was double labeled. Its sialic acid residues were labeled with 3H through a consecutive oxidation-reduction technique utilizing tritiated NaBH4. Its protein moiety was labeled with either 125I or 59Fe. Incubation of this double-labeled molecule at 4 degrees C with K562 cells gave overlapping curves, indicating identical patterns of binding for all labels. At 37 degrees C, 3H and 125I demonstrated identical patterns while 59Fe was cummulatively retained. This method can be used to follow the fate of other glycoproteins and their possible desialation in vivo.     

Characterisation of Schizosaccharomyces pombe rad31, a UBA-related gene required for DNA damage tolerance.

The fission yeast rad31-1 mutant is sensitive to both UV and ionising radiation and exhibits a growth defect at 35 degrees C. In addition, the mutant displays defects in cell morphology and nuclear division at 26 degrees C which are exaggerated at 35 degrees C. We have cloned the rad31 gene and have shown that it is not essential for viability, although cells containing a disrupted rad31 gene grow slowly. The null allele has similar cell and nuclear morphologies to the original allele and displays an extremely high frequency of loss of minichromosomes. rad31 is not required for either the S/M or G2/M checkpoint, however double mutant analysis indicates that rad31 acts in a process which is defective in the checkpoint rad mutants and which involves hus5 . Sequence analysis indicates that rad31 encodes a protein which is related to ubiquitin activating proteins and more particularly to an ORF in Saccharomyces cerevisiae and to the Arabidopsis thaliana AXR1 and human APP-BP1 genes. We have isolated the S.cerevisiae sequence, which we have named RHC31 ( ad31homologue in S. erevisiae), since we show that it can complement the slow growth phenotype and radiation sensitivity of S.pombe rad31.     

Transendothelial transport (transcytosis) of iron-transferrin complex in the bone marrow

To determine the transport pathway of iron-transferrin complex (Fe-TF) across the marrow-blood barrier, we labeled Fe-TF with colloidal gold and perfused rat femoral marrow with this probe. At 4 degrees C, the probe bound to the luminal surface of marrow sinus endothelium. The binding was inhibitable in the presence of excess native Fe-TF indicating the specificity of the binding. At 37 degrees C, the probe was internalized largely via a system of coated pits and vesicles and transported across the endothelium via a system of tubules and endosomal vesicles. It could not be ascertained if all Fe-TF was still associated with the colloidal gold probe within the endothelium, but the probe appeared to be externalized on the abluminal side into the interstitium where it subsequently bound to the surface of marrow erythroblasts and was internalized. Endothelium appeared to store part of the probe within a large vesicular system. No transport of Fe-TF was noted through diaphragmed fenestrations, diaphragmed vesicles, or interendothelial junctions. No endothelial uptake of this magnitude was noted when native gold particles or gold-labeled bovine serum albumin was used. Our findings indicate that in the bone marrow, gold-labeled Fe-TF is first taken up by sinus endothelium through a receptor-mediated mechanism and is possibly transported transendothelially via a vesicular system (transcytosis).     

Immunohistochemical demonstration of cytochrome b5 and hemopexin in rat liver parenchymal cells using horseradish peroxidase

The membrane-bound hemoprotein, cytochrome b₅, and the serum protein, hemopexin, were demonstrated in rat liver parenchymal cells using an immunohistochemical approach. Other liver cells contained neither protein. The immunological “sandwich” technique employs a complex of horseradish peroxidase with anti-horseradish peroxidase sequentially coupled to other antibodies including those to rat cytochrome b₅ and hemopexin.