Citations

Background and Purpose— Anecdotal evidence exists for at least 2 subpopulations of intracranial saccular aneurysms, namely, those that may form rapidly and rupture when small versus those that enlarge slowly and may rupture particularly when ≥10 mm in diameter. We sought to determine whether the endothelial nitric oxide synthase (eNOS) T-786C single nucleotide polymorphism (SNP), implicated in cardiovascular disease susceptibility, could facilitate differentiation between small (≤5 mm) versus large (≥10 mm) ruptured aneurysms. Methods— In accordance with institutional guidelines, clinical data were recorded prospectively and genomic DNA was isolated from blood samples obtained from 52 aneurysmal subarachnoid hemorrhage (SAH) patients (cases) and 90 randomly selected controls. Samples were assayed for eNOS gene promoter T-786C SNP with the use of gene microarray technology. Statistical analyses included multiple logistic regression. Results— Although there was no difference in genotype distributions between cases and controls, all 13 patients with large aneurysms were ( T / C ) heterozygous for the polymorphism, while 9 of 22 patients (41%) with small aneurysms were ( T / T or C / C ) homozygous ( P =0.01). The mean (±SD) ruptured aneurysm diameter among all heterozygotes (8.5±5.2 mm) was significantly greater than that for C / C (6.0±2.3 mm) or T / T (4.7±1.8 mm) homozygotes ( P =0.04). With the use of multivariate analysis, heterozygosity remained significantly associated with aneurysm size ≥10 mm ( P =0.03). Conclusions— The eNOS T-786C SNP distinguishes genetically between small and large ruptured aneurysms. Although not predictive of SAH in the population at large, our data suggest that among persons with known intracranial aneurysms, eNOS T-786C genotype may be a factor influencing the size at which an aneurysm ruptures, a finding that should be taken into consideration along with other anatomic features of the aneurysm.

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is “downstream” of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell–derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle–isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell–derived cardiomyocytes. Stem cell–derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell–derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell–derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.

Purpose: Preoperative chemotherapy is the conventional primary treatment in locally advanced breast cancer (LABC). We investigated the safety and efficacy of primary twice-weekly paclitaxel and concurrent radiation (RT) before modified radical mastectomy followed by adjuvant doxorubicin-based chemotherapy. Patients and Methods: Stage IIB (T3N0) to III LABC patients were eligible. Primary chemoradiation consisted of paclitaxel, 30 mg/m2 delivered intravenously for 1 hour twice weekly for a total of 8 to 10 weeks, and concurrent RT (45 Gy at 1.8 Gy/fraction). Modified radical mastectomy was performed at least 2 weeks after completion of chemoradiation or on recovery of skin toxicity. Postoperatively, patients who responded to paclitaxel and RT received four cycles of doxorubicin/paclitaxel, whereas patients who did not respond received doxorubicin/cytoxan. Results: Forty-four patients were accrued. Toxicity from paclitaxel/RT included grade 3 skin desquamation (7%), hypersensitivity (2%), and stomatitis (2%). Postsurgery complications occurred in six patients (14%). The only grade 4 toxicity of postmastectomy chemotherapy was hematologic (10%). Grade 3 toxicities were leukopenia (24%), infection (22%), peripheral neuropathy (17%), arthralgia and pain (17%), stomatitis (12%), fatigue (10%), esophagitis (5%), and nausea (2%). Overall clinical response rate to preoperative paclitaxel and RT was 91%. Thirty-four percent of patients achieved a pathologic response in the mastectomy specimen: 16% pathologic complete responses (clearance of invasive cancer in the breast and axillary contents) and 18% pathologic partial responses (< 10 residual microscopic foci of invasive breast cancer). Conclusion: Twice-weekly paclitaxel with concurrent RT is a feasible and effective primary treatment for LABC. Future studies should compare primary chemoradiation to chemotherapy in LABC.

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (K ATP ) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted K ATP channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for K ATP channels in the heart.

Background and Purpose — Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model. Methods — Recombinant adenoviral vectors (5×10 9 plaque-forming units/animal) encoding genes for eNOS (AdeNOS) and β-galactosidase (AdLacZ) or vehicle were injected into the cerebrospinal fluid (CSF) of dogs on day −1 (ie, 24 hours before the first intra-CSF injection of blood on day 0). Cerebral angiography was performed at day 0 (baseline) and day 7 (immediately before death), and tissues were harvested for additional studies. Results — Western analysis and immunohistochemistry detected recombinant eNOS exclusively in cerebral arteries isolated from AdeNOS-transduced dogs, and in this group of animals CSF NO concentrations were significantly elevated by day 2. Analysis of day 7 versus day 0 cerebral angiograms for each group revealed significant spasm at the basilar artery midpoint in AdLacZ-transduced and nontransduced dogs but not in AdeNOS-transduced dogs. Isometric force recording of basilar arteries isolated from AdeNOS-transduced dogs showed significantly augmented relaxations to bradykinin and reduced contractions to endothelin-1. Conclusions — Our results suggest that expression of recombinant eNOS in the adventitia of cerebral arteries may contribute toward protection against post-SAH vasospasm.

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca2+-binding protein that regulates diverse vital cell functions, including Ca2+ storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt−/− embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt−/− EBs, despite normal expression levels of cardiac transcription factors. Crt−/− EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt−/− phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca2+ with EGTA or BAPTA, or by inhibiting Ca2+/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca2+ concentration ([Ca2+]c) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt−/− cardiac cells in the absence of ionomycin-triggered [Ca2+]c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca2+-dependent checkpoint critical for early events during cardiac myofibrillogenesis.

Exchange of information between the nucleus and cytosol depends on the metabolic state of the cell, yet the energy-supply pathways to the nuclear compartment are unknown. Here, the energetics of nucleocytoplasmic communication was determined by imaging import of a constitutive nuclear protein histone H1. Translocation of H1 through nuclear pores in cardiac cells relied on ATP supplied by mitochondrial oxidative phosphorylation, but not by glycolysis. Although mitochondria clustered around the nucleus, reducing the distance for energy transfer, simple nucleotide diffusion was insufficient to meet the energetic demands of nuclear transport. Rather, the integrated phosphotransfer network was required for delivery of high-energy phosphoryls from mitochondria to the nucleus. In neonatal cardiomyocytes with low creatine kinase activity, inhibition of adenylate kinase-catalyzed phosphotransfer abolished nuclear import. With deficient adenylate kinase, nucleoside diphosphate kinase, which secures phosphoryl exchange between ATP and GTP, was unable to sustain nuclear import. Up-regulation of creatine kinase phosphotransfer, to mimic metabolic conditions of adult cardiac cells, rescued H1 import, suggesting a developmental plasticity of the cellular energetic system. Thus, mitochondrial oxidative phosphorylation coupled with phosphotransfer relays provides an efficient energetic unit in support of nuclear transport.

The apolipoprotein E ( APOE ) gene is the only genetic risk factor that has so far been linked to risk for late-onset Alzheimer's disease (LOAD). However, 50 percent of Alzheimer's disease cases do not carry an APOE4 allele, suggesting that other risk factors must exist. We performed a two-stage genome-wide screen in sibling pairs with LOAD to detect other susceptibility loci. Here we report evidence for an Alzheimer's disease locus on chromosome 10. Our stage one multipoint lod score (logarithm of the odds ratio for linkage/no linkage) of 2.48 (266 sibling pairs) increased to 3.83 in stage 2 (429 sibling pairs) close to D10S1225 (79 centimorgans). This locus modifies risk for Alzheimer's disease independent of APOE genotype.