Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.     

Expression of the FLT3-ITD oncogene sensitizes murine progenitor B-cell line BAF3 to cytotoxic action of binase

Acute myeloid leukemia makes up about 30% of all leukemia cases in adults. Mutations in the genes of the receptor tyrosine kinases KIT and FLT3, along with chromosomal translocations, are frequently found in leukemic cells. In the current work, we show that the transgenic B-cells BAF3/FLT3-ITD are significantly more sensitive to cytotoxic action of the ribonuclease binase than original BAF3 cells. BAF3/FLT3-ITD cells differ from BAF3 in expression of the FLT3-ITD oncogene, which results in the alteration of normal signaling pathways. We observed a similar effect previously when studying binase cytotoxic action in cells Kasumi-1 and FDC-P1-N822K, in which the activated oncogene KIT-N822K was expressed. An elevated cytotoxicity of binase to the cells that express the FLT3-ITD oncogene indicates that, as in case of the FDC-P1 cells transduced by the KIT oncogene, the expression of an activated oncogene determines the cell’s sensitivity to the binase action.     

Can Consumers Make Affordable Care Affordable? The Value of Choice Architecture

Tens of millions of people are currently choosing health coverage on a state or federal health insurance exchange as part of the Patient Protection and Affordable Care Act. We examine how well people make these choices, how well they think they do, and what can be done to improve these choices. We conducted 6 experiments asking people to choose the most cost-effective policy using websites modeled on current exchanges. Our results suggest there is significant room for improvement. Without interventions, respondents perform at near chance levels and show a significant bias, overweighting out-of-pocket expenses and deductibles. Financial incentives do not improve performance, and decision-makers do not realize that they are performing poorly. However, performance can be improved quite markedly by providing calculation aids, and by choosing a “smart” default. Implementing these psychologically based principles could save purchasers of policies and taxpayers approximately 10 billion dollars every year.     

Role for cER and Mmr1p in anchorage of mitochondria at sites of polarized surface growth in budding yeast

Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function.     

Distribution and habitat characteristics of Manayunkia speciosa and infection prevalence with the parasite Ceratomyxa shasta in the Klamath River, Oregon-California

A survey for Manayunkia speciosa, the freshwater polychaete host for the myxozoan parasite Ceratomyxa shasta, was conducted from 2003 to 2005 as part of an integrated study of the epizootiology of ceratomyxosis in Klamath River salmonids. Substrata samples (n = 257) were collected in a variety of habitats from Klamath Lake to the mouth of the Klamath River to document occurrence and relative abundance of the polychaete by habitat type and to estimate the prevalence of C. shasta within selected polychaete populations. Populations of M. specios a were identified throughout the Klamath River within pools (51.6%), eddy-pools (47.0%), and runs (40.0%). Large populations of M. speciosa were consistently found at the inflow to the main-stem reservoirs where densities were correlated with distance from the inflow into the reservoir. Using polymerase chain reaction assay and composite samples, 12 of 71 populations identified were tested for C. shasta, revealing a mean infection prevalence of 0.27%. An area of elevated infection prevalence (4.9 and 8.3%) was identified with 2 populations below a barrier to salmonid migration, which explains the high infectious spore densities demonstrated in concurrent studies and observations of C. shasta-induced mortality in Klamath River fall Chinook salmon (Oncorhynchus tshawytscha).     

Favorable balance of anti-oxidant/pro-oxidant systems and ablated oxidative stress in Brown Norway rats in renal ischemia-reperfusion injury

Oxidative stress is important in the pathogenesis of renal ischemia-reperfusion (IR) injury; however whether imbalances in reactive oxygen production and disposal account for susceptibility to injury is unclear. The purpose of this study was to compare necrosis, apoptosis, and oxidative stress in IR-resistant Brown Norway rats vs. IR-susceptible Sprague-Dawley (SD) rats in an in vivo model of renal IR injury. As superoxide (O₂^·−) interacts with nitric oxide (NO) to form peroxynitrite, inducible NO synthase (iNOS) and nitrotyrosine were also examined. Renal IR was induced in SD and BN rats by bilateral clamping of renal arteries for 45 min followed by reperfusion for 24 h (SD 24 and BN 24, respectively). BN rats were resistant to renal IR injury as evidenced by lower plasma creatinine and decreased acute tubular necrosis. TUNEL staining analysis demonstrated significantly decreased apoptosis in the BN rats vs. SD rats after IR. Following IR, O₂^·− levels were also significantly lower in renal tissue of BN rats vs. SD rats (P < 0.05) in conjunction with a preservation of the O₂^·− dismutating protein, CuZn superoxide dismutase (CuZn SOD) (P < 0.05). This was accompanied by an overall decrease in 4-hydroxynonenal adducts in the BN but not SD rats after IR. BN rats also displayed lower iNOS expression (P < 0.05) resulting in lower tissue NO levels and decreased nitrotyrosine formation (P < 0.01) following IR. Collectively these results show that the resistance of the BN rat to renal IR injury is associated with a favorable balance of oxidant production vs. oxidant removal. This work was supported in part by a Medical College of Wisconsin-Research Affairs Committee Grant to V. Nilakantan, and by divisional funds to V. Nilakantan and B.D. Shames.     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.     

Interaction of GAPR-1 with lipid bilayers is regulated by alternative homodimerization

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis related proteins group 1 (PR-1). GAPR-1 is a peripheral membrane-binding protein that strongly associates with lipid-enriched microdomains at the cytosolic leaflet of Golgi membranes. Little is known about the mechanism of GAPR-1 interaction with membranes. We previously suggested that dimerization plays a role in the function of GAPR-1 and here we report that phytic acid (inositol hexakisphosphate) induces dimerization of GAPR-1 in solution. Elucidation of the crystal structure of GAPR-1 in the presence of phytic acid revealed that the GAPR-1 dimer differs from the previously published GAPR-1 dimer structure. In this structure, one of the monomeric subunits of the crystallographic dimer is rotated by 28.5°. To study the GAPR-1 dimerization properties, we investigated the interaction with liposomes in a light scattering assay and by flow cytometry. In the presence of negatively charged lipids, GAPR-1 caused a rapid and stable tethering of liposomes. [D81K]GAPR-1, a mutant predicted to stabilize the IP6-induced dimer conformation, also caused tethering of liposomes. [A68K]GAPR-1 however, a mutant predicted to stabilize the non-rotated dimer conformation, is capable of binding to liposomes but did not cause liposome tethering. Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR-1 dynamics as a potential mechanism to modulate GAPR-1 function.     

Thromboembolic risks of recombinant factor VIIa Use in warfarin-associated intracranial hemorrhage: a case–control study

Background

Recombinant factor VIIa (rFVIIa) may be used for rapid hemostasis in life-threatening hemorrhage. In warfarin-associated intracerebral hemorrhage (wICH), FVIIa use is controversial and may carry significant thromboembolic risks. We compared incidence of baseline thromboembolic risk factors and thromboembolism rates in wICH patients treated with additional rFVIIa to those treated with standard therapy of fresh frozen plasma (FFP) and vitamin K alone.

Methods

We identified 45 consecutive wICH patients treated with additional rFVIIa over 5-year period, and 34 consecutive wICH patients treated with standard therapy alone as comparison group. We compared the incidence of post-hemorrhage cardiac and extra-cardiac thromboembolic complications between two treatment groups, and used logistic regression to adjust for significant confounders such as baseline thromboembolic risk factors. We performed secondary analysis comparing the quantity of FFP transfused between two treatment cohorts.

Results

Both rFVIIa-treated and standard therapy-treated wICH patients had a high prevalence of pre-existing thromboembolic diseases including atrial fibrillation (73% vs 68%), deep venous thrombosis (DVT) or pulmonary embolism (PE) (22% vs 18%), coronary artery disease (CAD) (38% vs 32%), and abnormal electrocardiogram (EKG) (78% vs 85%). Troponin elevation following wICH was prevalent in both groups (47% vs 41%). Clinically significant myocardial infarction (MI), defined as troponin > 1.0 ng/dL, occurred in 13% of rFVIIa-treated and 6% of standard therapy-treated patients (p=0.52). Past history of CAD (p=0.0061) and baseline abnormal EKG (p=0.02) were independently associated with clinically significant MI following wICH while rFVIIa use was not. The incidences of DVT/PE (2% vs 9%; p=0.18) and ischemic stroke (2% vs 0%; p=0.38) were similar between two treatment groups. Recombinant FVIIa-treated patients had lower mean INR at 3 (p=0.0001) and 6 hours (p<0.0001) and received fewer units of FFP transfusion (3 vs 5; p=0.003).

Conclusions

Pre-existing thromboembolic risk factors as well as post-hemorrhage troponin elevation are prevalent in wICH patients. Clinically significant MI occurs in up to 13% of wICH patients. rFVIIa use was not associated with increased incidence of clinically significant MI or other venous or arterial thromboembolic events in this wICH cohort.