Suppression of Extensive Neurofilament Phosphorylation Rescues α-Internexin/Peripherin-Overexpressing PC12 Cells from Neuronal Cell Death

Intermediate filament (IF) overproduction induces abnormal accumulation of neuronal IF, which is a pathological indicator of some neurodegenerative disorders. In our study, α-Internexin- and peripherin-overexpressing PC12 cells (pINT-EGFP and pEGFP-peripherin) were used as models to study neuropathological pathways responsible for neurodegenerative diseases. Microarray data revealed that Cdk5-related genes were downregulated and Cdk5 regulatory subunit-associated protein 3 (GSK-3α and GSK-3β) were upregulated in pINT-EGFP cells. Increased expression of phosphorylated neurofilament and aberrant activation of Cdk5 and GSK-3β were detected in both pEGFP-peripherin and pINT-EGFP cells by Western blotting. In addition, pharmacological approaches to retaining viability of pINT-EGFP and pEGFP-peripherin cells were examined. Treatment with Cdk5 inhibitor and GSK-3β inhibitor significantly suppressed neuronal death. Dynamic changes of disaggregation of EGFP-peripherin and decrease in green fluorescence intensity were observed in pEGFP-peripherin and pINT-EGFP cells by confocal microscopy after GSK-3β inhibitor treatment. We conclude that inhibition of Cdk5 and GSK-3β suppresses neurofilament phosphorylation, slows down the accumulation of neuronal IF in the cytoplasm, and subsequently avoids damages to cell organelles. The results suggest that suppression of extensive neurofilament phosphorylation may be a potential strategy for ameliorating neuron death. The suppression of hyperphosphorylation of neuronal cytoskeletons with kinase inhibitors could be one of potential therapeutic treatments for neurodegenerative diseases.     

BRCA1 and BRCA2 germline mutations in Malaysian women with early-onset breast cancer without a family history

Background

In Asia, breast cancer is characterised by an early age of onset: In Malaysia, approximately 50% of cases occur in women under the age of 50 years. A proportion of these cases may be attributable, at least in part, to genetic components, but to date, the contribution of genetic components to breast cancer in many of Malaysia''s ethnic groups has not been well-characterised.

Methodology

Given that hereditary breast carcinoma is primarily due to germline mutations in one of two breast cancer susceptibility genes, BRCA1 and BRCA2, we have characterised the spectrum of BRCA mutations in a cohort of 37 individuals with early-onset disease (≤40 years) and no reported family history. Mutational analysis of BRCA1 and BRCA2 was conducted by full sequencing of all exons and intron-exon junctions.

Conclusions

Here, we report a total of 14 BRCA1 and 17 BRCA2 sequence alterations, of which eight are novel (3 BRCA1 and 5 BRCA2). One deleterious BRCA1 mutation and 2 deleterious BRCA2 mutations, all of which are novel mutations, were identified in 3 of 37 individuals. This represents a prevalence of 2.7% and 5.4% respectively, which is consistent with other studies in other Asian ethnic groups (4–9%).     

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

Background

Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular α-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs).

Methodology/Principal Findings

We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (∼1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions.

Conclusion/Significance

Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets.     

Subcellular localization of prion proteins and the 37 kDa/67 kDa laminin receptor fused to fluorescent proteins

The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-DeltaSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-DeltaSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules.     

N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. In the present study we tested a novel thiol compound, N-acetylcysteine amide (AD4), the amide form of N-acetyl cysteine (NAC) for its antioxidant effects. Using flow-cytometry, we showed that in vitro treatment of blood cells from beta-thalassemic patients with AD4 elevated the reduced glutathione (GSH) content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their ROS. These effects resulted in a significant reduced sensitivity of thalassemic RBC to hemolysis and phagocytosis by macrophages. Intra-peritoneal injection of AD4 to beta-thalassemic mice (150 mg/kg) reduced the parameters of oxidative stress (p<0.001). Our results show the superiority of AD4, compared to NAC, in reducing oxidative stress markers in thalassemic cells both in vitro and in vivo.     

Possible role of pectin-containing mucilage and dew in repairing embryo DNA of seeds adapted to desert conditions

BACKGROUND AND AIMS: Repair of damage to DNA of seed embryos sustained during long periods of quiescence under dry desert conditions is important for subsequent germination. The possibility that repair of embryo DNA can be facilitated by small amounts of water derived from dew temporarily captured at night by pectinaceous surface pellicles was tested. These pellicles are secreted during early seed development and form mucilage when hydrated. METHODS: Seeds of Artemisia sphaerocephala and Artemisia ordosica were collected from a sandy desert. Their embryos were damaged by gamma radiation to induce a standard level of DNA damage. The treated seeds were then exposed to nocturnal dew deposition on the surface of soil in the Negev desert highlands. The pellicles were removed from some seeds and left intact on others to test the ability of mucilage to support repair of the damaged DNA when night-time humidity and temperature favoured dew formation. Repair was assessed from fragmentation patterns of extracted DNA on agarose gels. KEY RESULTS: For A. sphaerocephala, which has thick seed pellicles, DNA repair occurred in seeds with intact pellicles after 50 min of cumulative night dew formation, but not in seeds from which the pellicles had been removed. For A. ordosica, which has thin seed pellicles, DNA repair took at least 510 min of cumulative night dewing to achieve partial recovery of DNA integrity. The mucilage has the ability to rehydrate after daytime dehydration. CONCLUSIONS: The ability of seeds to develop a mucilaginous layer when wetted by night-time dew, and to repair their DNA under these conditions, appear to be mechanisms that help maintain seed viability under harsh desert conditions.     

Integrative control of coronary resistance vessel tone by endothelin and angiotensin II is altered in swine with a recent myocardial infarction

Several studies have indicated an interaction between the renin-angiotensin (ANG II) system and endothelin (ET) in the regulation of vascular tone. Previously, we have shown that both ET and ANG II exert a vasoconstrictor influence on the coronary resistance vessels of awake normal swine. Here, we investigated whether the interaction between ANG II and ET exists in the control of coronary resistance vessel tone at rest and during exercise using single and combined blockade of angiotensin type 1 (AT(1)) and ET(A)/ET(B) receptors. Since both circulating ANG II and ET levels are increased after myocardial infarction (MI), we investigated if the interaction between these systems is altered after MI. In awake healthy swine, coronary vasodilation in response to ET(A)/ET(B) receptor blockade in the presence of AT(1) blockade was similar to vasodilation produced by ET(A)/ET(B) blockade under control conditions. In awake swine with a 2- to 3-wk-old MI, coronary vasodilator responses to individual AT(1) and ET(A)/ET(B) receptor blockade were virtually abolished, despite similar coronary arteriolar AT(1) and ET(A) receptor expression compared with normal swine. Unexpectedly, in the presence of AT(1) blockade (which had no effect on circulating ET levels), ET(A)/ET(B) receptor blockade elicited a coronary vasodilator response. These findings suggest that in normal healthy swine the two vasoconstrictor systems contribute to coronary resistance vessel control in a linear additive manner, i.e., with negligible cross-talk. In contrast, in the remodeled myocardium, cross-talk between ANG II and ET emerges, resulting in nonlinear redundant control of coronary resistance vessel tone.     

白沙蒿粘液瘦果萌发中聚糖内切水解酶的活动特性

对白沙蒿( Artemisia sphaerocephaha Krasch.)种子萌发不同阶段的种胚提取物中几种可能降解果皮外层粘液物质的多糖内切酶进行了检测.研究结果表明:多聚半乳糖醛酸酶在干燥胚中已存在并具较高的活性,但其活性随着种子吸涨及萌发而降低.此酶并不释放到种胚外.β-甘露聚糖内切酶随着种子的吸涨而增加并被萌发的种胚释放到种子外.纤维素酶在种子萌发的过程中不表现出活性.提取物中的所有多糖内切酶都无法降解果皮外层的粘液物质.     

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect. This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD.