Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection

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Familial dysautonomia is caused by mutations of the IKAP gene

The defective gene DYS, which is responsible for familial dysautonomia (FD) and has been mapped to a 0.5-cM region on chromosome 9q31, has eluded identification. We identified and characterized the RNAs encoded by this region of chromosome 9 in cell lines derived from individuals homozygous for the major FD haplotype, and we observed that the RNA encoding the IkappaB kinase complex-associated protein (IKAP) lacks exon 20 and, as a result of a frameshift, encodes a truncated protein. Sequence analysis reveals a T-->C transition in the donor splice site of intron 20. In individuals bearing a minor FD haplotype, a missense mutation in exon 19 disrupts a consensus serine/threonine kinase phosphorylation site. This mutation results in defective phosphorylation of IKAP. These mutations were observed to be present in a random sample of Ashkenazi Jewish individuals, at approximately the predicted carrier frequency of FD. These findings demonstrate that mutations in the gene encoding IKAP are responsible for FD.     

The protective effect on Cu2+- and AAPH-mediated oxidation of human low-density lipoproteins depends on glycosaminoglycan structure

The effect of various glycosaminoglycans on Cu(2+)- and AAPH-induced oxidation of human low-density lipoprotein (LDL) was studied by monitoring conjugated diene formation. Heparin (Hep) increased the lag phase (t(lag)) of LDL oxidation, and fast moving and slow moving Hep species modified the kinetics of LDL oxidation to the same extent. Beef spleen heparan sulfate (HS) sample produced a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL whilst beef kidney HS species modified LDL oxidation kinetics to a lower extent. Dermatan sulfate (DS) from different sources caused a significant increase of the t(lag) and a decrease of the conjugated diene formation of LDL. Hyaluronic acid had no effect. Chondroitin sulfate (CS) from beef trachea produced a very strong protective antioxidant effect evaluated by increasing of the t(lag) and decreasing of the conjugated diene formation. Hep was completely ineffective in protecting LDL from 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation, whilst DS was moderately effective. Beef trachea CS showed a very strong ability to protect LDL oxidation induced by 1 mM AAPH. The different protective effect on Cu(2+)- and AAPH-induced LDL oxidation by glycosaminoglycans is discussed considering their various structures and properties, and their capacity to interact to different extents with hydrophobic regions of LDL protein is confirmed by measuring the LDL-tryptophan fluorescence kinetics.     

Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection

We (41) previously reported that Na-K-Cl-cotransporter (NKCC) function and microsomal protein expression are both dramatically reduced late in human cytomegalovirus (HCMV) infection of a human fibroblast cell line (MRC-5). We now report DNA microarray data showing that no significant HCMV-dependent NKCC gene repression can be detected 30 h postexposure (PE) to the virus. Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semiquantitative immunoblotting and confocal microscopy to investigate the possibility that intracellular redistribution of the NKCC protein after HCMV infection could be a cause of the HCMV-induced loss of NKCC ion transport function. Our results show that the lifetime of plasmalemmal NKCC protein in quiescent, uninfected MRC-5 cells is 48 h, and <20% of the total expressed NKCC protein are in the plasma membrane. The remainder (80%) was detected as diffusely distributed, small punctate structures in the cytoplasm. Following HCMV infection: 1) NKCC protein expression in the plasmalemma was sharply reduced (75%) within 24 h PE and thereafter continued to slowly decrease; 2) total cellular NKCC protein content remained unchanged or slightly increased during the course of the viral infection; and 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72 h PE). Thus our results imply that, in the process of productive HCMV infection, NKCC protein continues to be synthesized, but, instead of being delivered to the plasma membrane, it is clustered in a large, detergent-soluble perinuclear structure. sodium-potassium-chloride-cotransporter; human fibroblast cell line; perinuclear accumulation     

Contribution of store-operated Ca2+ entry to pHo-dependent changes in vascular tone of porcine coronary smooth muscle

Vascular smooth muscle contracts on increases of extracellular pH (pH(o)) and relaxes on pH(o) decreases possibly resulting from changes in transsarcolemmal Ca(2+) influx. Therefore, we studied store-operated Ca(2+) entry (SOCE; i.e. capacitative Ca(2+) entry (CCE)) during acidification (pH(o)=6.5) and alkalinization (pH(o)=8.0) in isolated porcine coronary smooth muscle cells (SMCs) by monitoring cytoplasmic Ca(2+) ([Ca(2+)](i)) and divalent cation entry (Mn(2+) quench) with fura-2/AM-fluorometry. Additionally, we evaluated the contribution of SOCE to pH(o)-dependent changes in isometric tension of porcine coronary smooth muscle strips. SOCE elicited in SMCs by the SERCA inhibitor BHQ was strongly modulated by pH(o) showing a decrease upon acidification and vice versa an increase upon alkalinization. BHQ-mediated tension of smooth muscle strips also revealed strong pH(o) dependence. In contrast, L-VOC-dependent tension ([K(+)](o)=20 and 40 mmol l(-1)) was remarkably less affected by pH(o) changes. Moreover, refilling of depleted Ca(2+) stores after repeated M(3)-cholinergic receptor stimulation could be almost completely inhibited by SKF 96365 and was markedly reduced by acidification and considerably enhanced by alkalinization pointing to a major role of SOCE in refilling. We conclude that vascular tone particularly responds to alterations in pH(o) whenever SOCE substantially contributes to the amount of activator Ca(2+) for contraction.     

Detection of Cyclospora cayetanensis in Wastewater

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.     

Up-Regulation of TREK-2 Potassium Channels in Cultured Astrocytes Requires De Novo Protein Synthesis: Relevance to Localization of TREK-2 Channels in Astrocytes after Transient Cerebral Ischemia

Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K⁺ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.     

Selective removal of keratan sulfate in chondroitin sulfate samples by sequential precipitation with ethanol

Keratan sulfate (KS) is present as a contaminant in chondroitin sulfate (CS) mainly extracted from shark cartilage. We report a selective removal procedure of KS in CS samples by means of sequential precipitation with ethanol. Purified shark CS containing approximately 10% to 15% KS was subjected to a precipitation procedure in the presence of increasing percentages of saturated ethanol. In contrast to other solvents, 1.0 volume of ethanol was able to selectively purify CS, with a purity of approximately 100%, from KS. The current selective and simple procedure appears to be a reliable industrial preparation of CS devoid of large amounts of the residual KS.     

Bypassing the Requirement for an Essential MYST Acetyltransferase

Histone acetylation is a key regulatory feature for chromatin that is established by opposing enzymatic activities of lysine acetyltransferases (KATs/HATs) and deacetylases (KDACs/HDACs). Esa1, like its human homolog Tip60, is an essential MYST family enzyme that acetylates histones H4 and H2A and other nonhistone substrates. Here we report that the essential requirement for ESA1 in Saccharomyces cerevisiae can be bypassed upon loss of Sds3, a noncatalytic subunit of the Rpd3L deacetylase complex. By studying the esa1∆ sds3∆ strain, we conclude that the essential function of Esa1 is in promoting the cellular balance of acetylation. We demonstrate this by fine-tuning acetylation through modulation of HDACs and the histone tails themselves. Functional interactions between Esa1 and HDACs of class I, class II, and the Sirtuin family define specific roles of these opposing activities in cellular viability, fitness, and response to stress. The fact that both increased and decreased expression of the ESA1 homolog TIP60 has cancer associations in humans underscores just how important the balance of its activity is likely to be for human well-being.