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991.
The K–Cl cotransporter (KCC) functions in maintaining chloride and volume homeostasis in a variety of cells. In the process of cloning the mouse KCC3 cDNA, we came across a cloning mutation (E289G) that rendered the cotransporter inactive in functional assays in Xenopus laevis oocytes. Through biochemical studies, we demonstrate that the mutant E289G cotransporter is glycosylation-deficient, does not move beyond the endoplasmic reticulum or the early Golgi, and thus fails to reach the plasma membrane. We establish through co-immunoprecipitation experiments that both wild-type and mutant KCC3 with KCC2 results in the formation of hetero-dimers. We further demonstrate that formation of these hetero-dimers prevents the proper trafficking of the cotransporter to the plasma membrane, resulting in a significant decrease in cotransporter function. This effect is due to interaction between the K–Cl cotransporter isoforms, as this was not observed when KCC3-E289G was co-expressed with NKCC1. Our studies also reveal that the glutamic acid residue is essential to K–Cl cotransporter function, as the corresponding mutation in KCC2 also leads to an absence of function. Interestingly, mutation of this conserved glutamic acid residue in the Na+-dependent cation-chloride cotransporters had no effect on NKCC1 function in isosmotic conditions, but diminished cotransporter activity under hypertonicity. Together, our data show that the glutamic acid residue (E289) is essential for proper trafficking and function of KCCs and that expression of a non-functional but full-length K–Cl cotransporter might results in dominant-negative effects on other K–Cl cotransporters.  相似文献   
992.

Background

Data on suppression of HIV replication in the CNS and on the subsequent risk of neurocognitive impairment using monotherapy with boosted protease inhibitors are limited.

Methods

Ours was an exploratory cross-sectional study in patients on lopinavir/ritonavir-based monotherapy (LPV/r-MT) or standard triple therapy (LPV/r-ART) for at least 96 weeks who maintained a plasma viral load <50 copies/mL. HIV-1 RNA in CSF was determined by HIV-1 SuperLow assay (lower limit of detection, 1 copy/mL). Neurocognitive functioning was assessed using a recommended battery of neuropsychological tests covering 7 areas. Neurocognitive impairment (NCI) was determined and also a global deficit score (GDS) for study comparisons.

Results

Seventeen patients on LPV/r-MT and 17 on LPV/r-ART were included. Fourteen (82.4%) patients on LPV/r-MT and 16 (94.1%) on LPV/r-ART had HIV-1 RNA <1 copy/mL in CSF (p = 0.601). NCI was observed in 7 patients on LPV/r-MT and in 10 on LPV/r-ART (41% vs 59%; p = 0.494). Mean (SD) GDS was 0.22 (0.20) in patients on LPV/r-MT and 0.47 (0.34) in those on LPV/r-ART (p = 0.012).

Conclusions

Suppression of HIV in CSF is similar in individuals with durable plasma HIV-1 RNA suppression who are receiving LPV/r-MT or LPV/r-ART for at least 96 weeks. Findings for HIV-1 replication in CSF and neurocognitive status indicate that this strategy seems to be safe for CNS functioning.  相似文献   
993.
Feline leukemia virus (FeLV) is a naturally transmitted gammaretrovirus that infects domestic cats. FeLV-945, the predominant isolate associated with non-T-cell disease in a natural cohort, is a member of FeLV subgroup A but differs in sequence from the FeLV-A prototype, FeLV-A/61E, in the surface glycoprotein (SU) and long terminal repeat (LTR). Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in pathogenesis indistinguishable from that of FeLV-A/61E, namely, thymic lymphoma of T-cell origin. In contrast, substitution of both FeLV-945 LTR and SU into FeLV-A/61E resulted in multicentric lymphoma of non-T-cell origin. These results implicated the FeLV-945 SU as a determinant of pathogenic spectrum. The present study was undertaken to test the hypothesis that FeLV-945 SU can act in the absence of other unique sequence elements of FeLV-945 to determine the disease spectrum. Substitution of FeLV-A/61E SU with that of FeLV-945 altered the clinical presentation and resulted in tumors that demonstrated expression of CD45R in the presence or absence of CD3. Despite the evident expression of CD45R, a typical B-cell marker, T-cell receptor beta (TCRβ) gene rearrangement indicated a T-cell origin. Tumor cells were detectable in bone marrow and blood at earlier times during the disease process, and the predominant SU genes from proviruses integrated in tumor DNA carried markers of genetic recombination. The findings demonstrate that FeLV-945 SU alters pathogenesis, although incompletely, in the absence of FeLV-945 LTR. Evidence demonstrates that FeLV-945 SU and LTR are required together to fully recapitulate the distinctive non-T-cell disease outcome seen in the natural cohort.  相似文献   
994.

Background

We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning.

Methods

EDTA-anticoagulated plasma and serum samples were obtained before conditioning and about once per week after transplantation until day 100. Samples were aliquoted and stored at −80°C within 3 hours after collection until measurement of cytokines. IL-7 and IL-15 levels were measured by ELISAs.

Results

Median IL-7 plasma levels remained below 6 pg/L throughout the first 100 days, although IL-7 plasma levels were significantly higher on days 7 (5.1 pg/mL, P = 0.002), 14 (5.2 pg/mL, P<0.001), and 28 (5.1 pg/mL, P = 0.03) (but not thereafter) than before transplantation (median value of 3.8 pg/mL). Median IL-15 serum levels were significantly higher on days 7 (12.5 pg/mL, P<0.001), 14 (10.5 pg/mL, P<0.001), and 28 (6.2 pg/mL, P<0.001) than before transplantation (median value of 2.4 pg/mL). Importantly, IL-7 and IL-15 levels on days 7 or 14 after transplantation did not predict grade II–IV acute GVHD.

Conclusions

These data suggest that IL-7 and IL-15 levels remain relatively low after nonmyeloablative transplantation, and that IL-7 and IL-15 levels early after nonmyeloablative transplantation do not predict for acute GVHD.  相似文献   
995.

Background

Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat.

Methods

Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA.

Results

Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R2 = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R2 = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine.

Conclusions

Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss.  相似文献   
996.
Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.  相似文献   
997.
Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.  相似文献   
998.
Mortalities related to the detection of Ostreid Herpesvirus 1 (OsHV-1) have been previously reported in France among larvae and spat of the Pacific oyster Crassostrea gigas. Adult oysters appear less sensitive to herpesvirus infections, although OsHV-1 has been detected in adults without signs of disease or mortality. This suggests that the virus is able to persist in its host and that adult oysters may be able to control OsHV-1 infection. Little is known about antiviral substances in invertebrates. The present work concerns the research of antiviral substances in adult oyster C. gigas, where putative antiviral activities were monitored using 3 strategies: (1) in metabolites with variable polarity, (2) in peptidic extracts and (3) in crude haemolymph. In vitro antiviral assays were based on inhibition of Herpes simplex virus type 1 (HSV-1) replication in Vero cell monolayers. All extracts presented no cytotoxicity. Antiviral activity was detected in the fresh filtered haemolymph (EC50:425 microg ml(-1)) and seasonal variation of the haemolymph antiviral activity was monitored.  相似文献   
999.
Spontaneous synchronization of coupled circadian oscillators   总被引:1,自引:0,他引:1       下载免费PDF全文
In mammals, the circadian pacemaker, which controls daily rhythms, is located in the suprachiasmatic nucleus (SCN). Circadian oscillations are generated in individual SCN neurons by a molecular regulatory network. Cells oscillate with periods ranging from 20 to 28 h, but at the tissue level, SCN neurons display significant synchrony, suggesting a robust intercellular coupling in which neurotransmitters are assumed to play a crucial role. We present a dynamical model for the coupling of a population of circadian oscillators in the SCN. The cellular oscillator, a three-variable model, describes the core negative feedback loop of the circadian clock. The coupling mechanism is incorporated through the global level of neurotransmitter concentration. Global coupling is efficient to synchronize a population of 10,000 cells. Synchronized cells can be entrained by a 24-h light-dark cycle. Simulations of the interaction between two populations representing two regions of the SCN show that the driven population can be phase-leading. Experimentally testable predictions are: 1), phases of individual cells are governed by their intrinsic periods; and 2), efficient synchronization is achieved when the average neurotransmitter concentration would dampen individual oscillators. However, due to the global neurotransmitter oscillation, cells are effectively synchronized.  相似文献   
1000.
Reactive oxygen species (ROS) generated by Helicobacter pylori infection have been suggested to be important factors in induction of gastric malignancies. Utilizing electron spin resonance spectrometry, H. pylori-dependent radical formation and hydroxyl- and superoxide-anion radical scavenging activity was investigated. In contrast to previous reports, we found that H. pylori does not produce ROS, but displays superoxide scavenging activity. This scavenging activity was increased in cag-positive H. pylori strains when compared to strains lacking an intact cag pathogenicity island, and was dependent on enzyme activity. We hypothesize that the increased scavenging activity of cag-positive H. pylori strains is an adaptation to the increased inflammatory response associated with the cag-positive genotype of H. pylori.  相似文献   
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