Evidence for transcription and potential translation of the human 1.9 kb HindIII repetitive element.

Recombinant cDNA clones corresponding to the human 1.9kb HindIII repetitive element have been isolated from a cDNA library of liver cytoplasmic polyadenylated RNA. These cDNAs share 95% homology with the reported genomic DNA sequence and a similar amount of homology at the amino acid level with putative coding sequences (see preceding article by Mottez et al). They were isolated as two of four false positives from a human cDNA library in lambda gt11 and were selected with an antibody to an unrelated enzyme. These results provide direct evidence that this repetitive element is transcribed to form poly(A)+ RNA which could be translatable. Also, these observations may add to our understanding of the sources of false positives which are frequently observed in screens of cDNA libraries with antibodies as probes.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.

The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.     

Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.     

Spinoreticular neurons that receive group III input are inhibited by MLR stimulation.

In decerebrate paralyzed cats, we examined the responses of 18 spinoreticular neurons to electrical stimulation of the mesencephalic locomotor region. The activity of each of the spinoreticular neurons was recorded extracellularly from laminae IV through VI of the L(7) and S(1) spinal cord. In addition, each of the 18 spinoreticular neurons received group III afferent input from the tibial nerve. Spinoreticular projections were established for each of 18 neurons by antidromic invasion of the ventro lateral medulla at the P11 though P14 levels. The onset latencies and current thresholds for antidromic invasion from the ventro lateral medulla averaged 15.0 +/- 3.8 ms and 117 +/- 11 microA, respectively. Electrical stimulation of the mesencephalic locomotor region attenuated the spontaneous activity or the responses of each of the spinoreticular neurons to tibial nerve stimulation at currents that recruited group III afferents. Our data support the notion that thin-fiber muscle afferent input to the ventrolateral medulla is gated by a central command to exercise.     

Neuronal ER Stress Impedes Myeloid-Cell-Induced Vascular Regeneration through IRE1α Degradation of Netrin-1

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Highlights? Canonical ER stress pathways are activated in central neurons during hypoxia/ischemia ? The ER stress endoribonuclease IRE1α degrades the neurovascular guidance cue netrin-1 ? Neuronal-derived netrin-1 activates a reparative proangiogenic program in microglial cells ? Neuronal ER stress prevents reparative angiogenesis in the ischemic neural retina     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response.

Severe hyperhomocysteinemia is associated with endothelial cell injury that may contribute to an increased incidence of thromboembolic disease. In this study, homocysteine induced programmed cell death in human umbilical vein endothelial cells as measured by TdT-mediated dUTP nick end labeling assay, DNA ladder formation, induction of caspase 3-like activity, and cleavage of procaspase 3. Homocysteine-induced cell death was specific to homocysteine, was not mediated by oxidative stress, and was mimicked by inducers of the unfolded protein response (UPR), a signal transduction pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum. Dominant negative forms of the endoplasmic reticulum-resident protein kinases IRE1alpha and -beta, which function as signal transducers of the UPR, prevented the activation of glucose-regulated protein 78/immunoglobulin chain-binding protein and C/EBP homologous protein/growth arrest and DNA damage-inducible protein 153 in response to homocysteine. Furthermore, overexpression of the point mutants of IRE1 with defective RNase more effectively suppressed the cell death than the kinase-defective mutant. These results indicate that homocysteine induces apoptosis in human umbilical vein endothelial cells by activation of the UPR and is signaled through IRE1. The studies implicate that the UPR may cause endothelial cell injury associated with severe hyperhomocysteinemia.