Search

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.

Background: Standard care for lower extremity injuries with exposed bone or tendon is vascularized tissue transfer. In patients with deep and extensive burn injuries, these options are often not available or for various reasons are technically difficult. The advent of Integra, a permanent dermal replacement matrix, provides an important alternative to the traditional reconstructive choices., Methods: The authors report seven patients, with a total of nine lower extremity injuries, from the Los Angeles County and University of Southern California Hospital Burn Unit treated from September of 2001 to January of 2003 who presented with complex burn injuries involving their lower extremities with tendon, open joints, and/or bone exposure., Results: All seven patients were treated initially with tangential excision of the burn eschar and placement of Integra. After engraftment, epidermal coverage was restored with a thin split-thickness skin graft. This treatment provided stable wound coverage, and none of the patients have required further surgical procedures to maintain wound closure., Conclusions: The use of a bioengineered, cell-free dermal matrix expands reconstructive options and allows salvage of extremities that might otherwise have been amputated or required prolonged, staged procedures. The authors' experience shows that Integra provides a sound alternative option for providing durable coverage of vital lower extremity structures. It should be considered as part of the lower extremity wound coverage algorithm.

Nucleotide excision repair is a versatile repair pathway that counteracts the deleterious effects of various DNA lesions. In nucleotide excision repair, there is a transcription-coupled repair (TCR) pathway that focuses on DNA damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPA-binding protein 2), containing tetratricopeptide repeats, has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 has been shown to interact with Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and is involved in TCR and transcription. Here we purified XAB2 as a multimeric protein complex consisting of hAquarius, XAB2, hPRP19, CCDC16, hISY1, and PPIE, which are involved in pre-mRNA splicing. Knockdown of XAB2 with small interfering RNA in HeLa cells resulted in a hypersensitivity to killing by UV light and a decreased recovery of RNA synthesis after UV irradiation and regular RNA synthesis. Enhanced interaction of XAB2 with RNA polymerase IIo or XPA was observed in cells treated with DNA-damaging agents, indicating DNA damage-responsive activity of the XAB2 complex. These results indicated that the XAB2 complex is a multifunctional factor involved in pre-mRNA splicing, transcription, and TCR.

2-(2-Chloro-6-fluorophenyl)acetamides having 2,2-difluoro-2-aryl/heteroaryl-ethylamine P3 and oxyguanidine P1 substituents are potent thrombin inhibitors (K(i)=0.9-33.9 nM). 2-(5-Chloro-pyridin-2-yl)-2,2-difluoroethylamine was the best P3 substituent, yielding the most potent inhibitor (K(i)=0.7 nM). Replacing the P3 heteroaryl group with a phenyl ring or replacing the difluoro substitution with dimethyl or cyclopropyl groups in the linker reduced the affinity for thrombin significantly. The aminopyridine P1s also provided an increase in potency.

Unlabelled: Fibroblast growth factor (FGF) signaling and beta-catenin activation have been shown to be crucial for early embryonic liver development. This study determined the significance of FGF10-mediated signaling in a murine embryonic liver progenitor cell population as well as its relation to beta-catenin activation. We observed that Fgf10(-/-) and Fgfr2b(-/-) mouse embryonic livers are smaller than wild-type livers; Fgf10(-/-) livers exhibit diminished proliferation of hepatoblasts. A comparison of beta-galactosidase activity as a readout of Fgf10 expression in Fgf10(+/LacZ) mice and of beta-catenin activation in TOPGAL mice, demonstrated peak Fgf10 expression from E9 to E13.5 coinciding with peak beta-catenin activation. Flow cytometric isolation and marker gene expression analysis of LacZ(+) cells from E13.5 Fgf10(+/LacZ) and TOPGAL livers, respectively, revealed that Fgf10 expression and beta-catenin signaling occur distinctly in stellate/myofibroblastic cells and hepatoblasts, respectively. Moreover, hepatoblasts express Fgfr2b, which strongly suggests they can respond to recombinant FGF10 produced by stellate cells. Fgfr2b(-/-)/TOPGAL(+/+) embryonic livers displayed less beta-galactosidase activity than livers of Fgfr2b(+/+)/TOPGAL(+/+) littermates. In addition, cultures of whole liver explants in Matrigel or cell in suspension from E12.5 TOPGAL(+/+)mice displayed a marked increase in beta-galactosidase activity and cell survival upon treatment with recombinant FGF10, indicating that FGFR (most likely FGFR2B) activation is upstream of beta-catenin signaling and promote hepatoblast survival., Conclusion: Embryonic stellate/myofibroblastic cells promote beta-catenin activation in and survival of hepatoblasts via FGF10-mediated signaling. We suggest a role for stellate/myofibroblastic FGF10 within the liver stem cell niche in supporting the proliferating hepatoblast.

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to-plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100-200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses.

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Fibroblast growth factor 8 (FGF8) is expressed at the mid-hindbrain boundary and is an important signal emanating from the isthmic organizer. Wnt1 is expressed in the caudal midbrain juxtaposed to Fgf8 expression and has been implicated in its regulation. In this study, we examine the requirement for continuous Wnt signaling in the maintenance of Fgf8 expression at the isthmus. We demonstrate that prior to HH10, ongoing Wnt signaling is required to maintain the normal pattern of isthmic Fgf8 expression in ovo. Similarly, in explant assays, sustained Wnt signaling is essential to maintain Fgf8 expression in rhombomere 1. The mechanism by which Wnt signaling regulates isthmic Fgf8 expression is likely to be a maintenance response rather than an inductive effect. Finally, we show that Wnt maintenance of Fgf8 expression is dependent upon positive feedback by FGF signaling itself, and that rhombomere 1 does not receive instructive cues from the posterior hindbrain. In summary, these findings establish that a sustained reciprocal interaction between Wnt and FGF signaling is essential to maintain isthmic identity.

Prion diseases are transmissible neurodegenerative disorders. It is widely accepted that prions are the infectious agents responsible for disease transmission, and the sequence homology between the infectious prion and the host prion protein determines its transmission efficiency across species. However, previous studies have often reported different results regarding seeding efficiency, the efficiency of initiating amyloid propagation by adding pre-existing amyloid fibrils as seed. In the present study, we used synthetic peptides as a simple system to determine the sequence-dependent transmission barrier between hamster and mouse. We found that the heterologous seeding efficiency of hamster and mouse prion peptides was 4 times less than that of homologous seeding. Moreover, residue 139 was not the only residue in determining seeding efficiency. When the seed had Ile at this position, the homology at this position between seed and monomer determined the seeding efficiency. When the seed had Met at this position, homology at residues 109 and 112 determined the seeding efficiency.

Our objectives were to ascertain the safety (cardiotoxicity) and efficacy of non-pegylated liposomal doxorubicin [Myocet (M)] compared with conventional doxorubicin (A) in patients with metastatic breast cancer (MBC) who had received adjuvant anthracycline treatment and were at high risk of developing iatrogenic cardiomyopathy. This retrospective analysis is based on data pooled from two prospective phase III comparative randomized clinical trials comparing Myocet versus conventional doxorubicin in combination with cyclophosphamide and as single agents, respectively, for the treatment of MBC. The outcome measures reviewed in this analysis were overall response, time to treatment failure, time to disease progression, overall survival and cardiotoxicity. The analysis was carried out by strata according to patients' previous exposure to adjuvant anthracyclines. Kaplan-Meier, log-rank chi-test, Cox proportional-hazards and Cochran-Mantel-Haenszel statistics were used for the analysis. Sixty-eight patients were included in this analysis: 29 and 39 patients from Studies 1 and 2, respectively, had received adjuvant anthracycline treatment. Study 1, with n=297, compared M 60 mg/m (M60) plus cyclophosphamide (C) 600 mg/m (C600) versus A 60 mg/m (A60) plus C600 as first-line treatment for MBC. Twenty-nine patients had received prior adjuvant doxorubicin, of whom, after randomization, 14 received M60+C600 and 15 received A60+C600 for the treatment of MBC. Study 2, with n=224, compared M 75 mg/m (M75) with A 75 mg/m (A75) as first-line treatment for MBC disease. Thirty-nine patients had received prior adjuvant doxorubicin, of whom, after randomization, 18 received M75 and 21 received A75 for their MBC. Hence, 32 patients received M-containing regimens and 36 received A-containing regimens for the treatment of MBC. Median age in both groups was 54 years. The groups were well balanced in terms of demographic characteristics. Overall response rates were 31% and 11% for M-treated patients and A-treated patients, respectively (Cochran-Mantel-Haenszel P=0.04, odds ratio=4.0). Median time to progression was 4.5 versus 3.4 months [log-rank P=0.66, hazard ratio (HR)=1.14], median time to treatment failure was 4.2 versus 2.1 months (log-rank P=0.01, HR=2.06) and median survival time was 16 versus 15 months (log-rank P=0.71, HR=1.12). Cardiac events occurred in 22% of M-treated patients [one congestive heart failure (CHF)] versus 39% of A-treated patients (three CHFs) (log-rank, P=0.001). Median lifetime dose at onset of cardiotoxicity was 780 mg/m for M versus 580 mg/m for A (log-rank P=0.001, HR=4.8). This retrospective analysis shows that treatment based on non-pegylated liposomal doxorubicin (Myocet) significantly reduced the risk of cardiotoxicity in patients with MBC who had received prior adjuvant doxorubicin. Furthermore, anti-tumor activity and time to treatment failure were significantly improved compared with patients who received treatment based on conventional doxorubicin for their MBC. This analysis revisits the therapeutic option of including doxorubicin in the treatment of MBC patients who have had prior adjuvant anthracycline exposure.

Background: Current data suggest that 30%-50% of patients with syncope leave the hospital without a defined etiology of their syncopal event. Recent studies have shown significant differences in both presentation and therapy in women with coronary disease and congestive heart failure., Methods: To assess the impact of sex on the frequency of syncope and the rate of identifying a specific etiology among elderly patients who present to the emergency department (ED), a retrospective chart review was performed during a 1-year period. Consecutive patients older than 65 years presenting with syncope to a large urban teaching hospital were enrolled. Inclusion criterion was documented loss of consciousness with no other obvious etiology of loss of consciousness such as stroke or seizure. Charts were screened for presenting history, ethnicity, sex, comorbid conditions, living circumstances, outpatient medication, and cardiac risk factors and outcomes including acute coronary syndromes, myocardial infarction, and death., Results: During 1 year of observation, 7496 women (60%) of 12,401 patients over the age of 65 presented to our ED, and 219 (2.9%) of these met inclusion criteria for syncope. In comparison, 4905 (40%) men presented to the ED, 104 (2.1%) of whom had syncope (relative risk of syncope for women vs men, 1.38; 95% confidence interval, 1.09-1.74). Men were more likely to have comorbid conditions including coronary artery disease (p < or = .01), prior myocardial infarction (p < or = .03), and diabetes mellitus (p < or = .03) than were women. No sex differences were noted in living circumstances, such as living alone, with assistance, or in an institution. Forty-two percent of patients received no diagnosis on discharge from the hospital. Sixty-nine of 147 women admitted (47%) had no etiology of their syncope as compared to 27 (32%) of men. The relative risk for no diagnosis in women versus men was 1.67 (95% confidence interval, 1.07-2.61)., Conclusions: Considerable numbers of patients presenting to EDs with syncope remain without a diagnosis. Women, despite being less likely to have concomitant coronary artery disease or diabetes, are significantly more likely to present to an ED with syncope, yet less likely to be discharged with a defined etiology.

The reincorporation of lipids into monolayers at the air-water interface after collapse is important to the maintenance of low surface tensions on subsequent expansion and compression cycles. For single component, anionic dipalmitoylphosphatidylglycerol monolayers, the fraction of recovered lipid is proportional to the subphase ionic strength. The collapse mechanism and structure of the collapsed materials appear unchanged with ionic strength. A simple electrostatic barrier model shows that the fractional recovery depends exponentially on the Debye length; this is verified by experiment. This simple model suggests possible catalytic roles for the cationic lung surfactant specific proteins SP-B and SP-C that induce structural changes in the monolayer that may act as charge-neutralizing docking sites for surfactant in the subphase, leading to faster and more efficient recovery.

Background: Most patients admitted to the hospital from the emergency department (ED) with syncope do not have a myocardial infarction (MI), yet a common practice is to draw serial cardiac enzymes., Methods: To assess the value of serial cardiac enzymes in elderly patients who present to the ED with syncope, a retrospective chart review was performed on consecutive patients aged 65 and older with syncope in an urban teaching hospital ED between July 1, 1998 and June 30, 1999. Charts were screened for presenting history, cardiac risk factors, testing, and outcomes including acute coronary syndromes, MI, death, and patients returning to the ED or admitted within 72 hours of discharge., Results: 319 patients met the study criteria of syncope with confirmed loss of consciousness in the absence of seizure or stroke. 141 of 228 admitted patients (62%) had creatine phosphokinase (CPK) drawn and 5% of these had Troponin I (TnI). 3 of 141 patients, or 2.1% (95% CI [confidence interval]: 0.04%-6.09%), had positive cardiac enzymes during their hospitalization. CPK was positive in all 3, and TnI, drawn in 1 patient, was also positive. Two of these patients had chest discomfort and ST segment and T-wave abnormalities on electrocardiogram (ECG) in addition to a syncopal event. The third patient had dementia and could not recall the details surrounding her syncopal event. In addition, her baseline ECG demonstrated a left bundle branch block, limiting ECG interpretation., Conclusions: Cardiac enzymes may be of little additional value if drawn routinely on elderly patients with syncope who are admitted to the hospital from the ED, unless they have other signs or symptoms suggestive of myocardial ischemia.

Latent infections by herpes simplex virus are characterized by repression of productive-cycle gene expression. Several hypotheses to explain this repression involve inhibition of expression of the immediate-early gene activator ICP0 during latency. To address these hypotheses, we developed quantitative reverse transcriptase-PCR assays that detected spliced and intron-containing ICP0 transcripts in mouse ganglia latently infected with wild-type virus. In these ganglia, the numbers of spliced ICP0 transcripts correlated better with the numbers of transcripts from the immediate-early gene encoding ICP4 than with those from the early gene encoding thymidine kinase. There were fewer spliced than intron-containing ICP0 transcripts on average, with considerable ganglion-to-ganglion variation. We then investigated whether ICP0 expression in latently infected ganglia is reduced by the latency-associated transcripts (LATs) and whether splicing of ICP0 transcripts is inhibited by the product of open reading frame (ORF) P. A LAT deletion mutation which essentially eliminates expression of the major LATs did not appreciably increase levels of ICP0 transcripts. LAT deletion mutants did, however, appear to express reduced levels of intron-containing ICP0 transcripts. ORF P mutations did not alter levels of ICP0 transcripts in a manner consistent with inhibition of ICP0 splicing by ORF P. Although these results argue against antisense inhibition of ICP0 expression by LATs or inhibition of ICP0 splicing by ORF P, they are consistent with the possibilities of a block between immediate-early and early gene expression and regulation of spliced versus intron-containing ICP0 transcripts in latently infected ganglia.