Optimal diuretic strategies in heart failure.

Heart failure (HF) is one of the major causes of morbidity and mortality in the world. According to a 2019 American Heart Association report, about 6.2 million American adults had HF between 2013 and 2016, being responsible for almost 1 million admissions. As the population ages, the prevalence of HF is anticipated to increase, with 8 million Americans projected to have HF by 2030, posing a significant public health and financial burden. Acute decompensated HF (ADHF) is a syndrome characterized by volume overload and inadequate cardiac output associated with symptoms including some combination of exertional shortness of breath, orthopnea, paroxysmal nocturnal dyspnea (PND), fatigue, tissue congestion (e.g., peripheral edema) and decreased mentation. The pathology is characterized by hemodynamic abnormalities that result in autonomic imbalance with an increase in sympathetic activity, withdrawal of vagal activity and neurohormonal activation (NA) resulting in increased plasma volume in the setting of decreased sodium excretion, increased water retention and in turn an elevation of filling pressures. These neurohormonal changes are adaptive mechanisms which in the short term are associated with increased contractility of the left ventricular (LV) and improvement in cardiac output. But chronically, the failing heart is unable to overcome the excessive pressure and volume leading to worsening HF. The primary symptomatic management of ADHF includes intravenous (IV) diuresis to help with decongestion and return to euvolemic status. Even though diuretics have not been shown to provide any mortality benefit, they have been clinically proven to be of significant benefit in the acute decompensated phase, as well as in chronic management of HF. Loop diuretics remain the mainstay of therapy for symptomatic management of HF with use of thiazide diuretics for synergistic effect in the setting of diuretic resistance. Poor diuretic efficacy has been linked with higher mortality and increased rehospitalizations.

Geographic Inequalities in Cardiovascular Mortality in the United States: 1999 to 2018.

OBJECTIVE: To evaluate the trends in cardiovascular, ischemic heart disease (IHD), stroke, and heart failure mortality in the stroke belt in comparison with the rest of the United States. PATIENTS AND METHODS: We evaluated the nationwide mortality data of all Americans from the Centers for Disease Control and Prevention Wide-Ranging Online Data for Epidemiologic Research database from 1999 to 2018. Cause-specific deaths were identified in the stroke belt and nonstroke belt populations using International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes. The relative percentage gap was estimated as the absolute difference computed relative to nonstroke belt mortality. Piecewise linear regression and age-period-cohort modeling were used to assess, respectively, the trends and to forecast mortality across the 2 regions. RESULTS: The cardiovascular mortality rate (per 100,000 persons) was 288.3 (95% CI, 288.0 to 288.6; 3,684,273 deaths) in the stroke belt region and 251.2 (95% CI, 251.0 to 251.3; 13,296,164 deaths) in the nonstroke belt region. In the stroke belt region, age-adjusted mortality rates due to all cardiovascular causes (average annual percentage change [AAPC] in mortality rates, -2.4; 95% CI, -2.8 to -2.0), IHD (AAPC, -3.8; 95% CI, -4.2 to -3.5), and stroke (AAPC, -2.8; 95% CI, -3.4 to -2.1) declined from 1999 to 2018. A similar decline in cardiovascular (AAPC, -2.5; 95% CI, -3.0 to -2.0), IHD (AAPC, -4.0; 95% CI, -4.3 to -3.7), and stroke (AAPC, -2.9; 95% CI, -3.2 to -2.2) mortality was seen in the nonstroke belt region. There was no overall change in heart failure mortality in both regions (PAAPC>.05). The cardiovascular mortality gap was 11.8% in 1999 and 15.9% in 2018, with a modest reduction in absolute mortality rate difference (~7 deaths per 100,000 persons). These patterns were consistent across subgroups of age, sex, race, and urbanization status. An estimated 101,953 additional cardiovascular deaths need to be prevented from 2020 to 2025 in the stroke belt to ameliorate the gap between the 2 regions. CONCLUSION: Despite the overall decline, substantial geographic disparities in cardiovascular mortality persist. Novel approaches are needed to attenuate the long-standing geographic inequalities in cardiovascular mortality in the United States, which are projected to increase.

Obesity and Serial NT-proBNP Levels in Guided Medical Therapy for Heart Failure With Reduced Ejection Fraction: Insights From the GUIDE-IT Trial.

Background Obese patients have lower NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels. The prognostic implications of achieving NT-proBNP levels ≤1000 pg/mL in obese patients with heart failure (HF) receiving biomarker-guided therapy are not completely known. We evaluated the prognostic implications of obesity and having NT-proBNP levels (≤1000 pg/mL) in the GUIDE-IT (Guiding Evidence-Based Therapy Using Biomarker-Intensified Treatment in HF) trial participants. Methods and Results The risk of adverse cardiovascular events (HF hospitalization or cardiovascular mortality) was assessed using multivariable-adjusted Cox proportional hazard models based on having NT-proBNP ≤1000 pg/mL (taken as a time-varying covariate), stratified by obesity status. The study outcome was also assessed on the basis of the body mass index at baseline. The predictive ability of NT-proBNP for adverse cardiovascular events was assessed using the likelihood ratio test. Compared with nonobese patients, obese patients were mostly younger, Black race, and more likely to be women. NT-proBNP levels were 59.0% (95% CI, 39.5%-83.5%) lower among obese individuals. The risk of adverse cardiovascular events was lower in obese (hazard ratio [HR], 0.48; 95% CI, 0.29-0.59) and nonobese (HR, 0.32; 95% CI, 0.19-0.57) patients with HF who had NT-proBNP levels ≤1000 pg/mL, compared with those who did not. There was no interaction between obesity and having NT-proBNP ≤1000 pg/mL on the study outcome (P>0.10). Obese patients had a greater risk of developing adverse cardiovascular events (HR, 1.39; 95% CI, 1.01-1.90) compared with nonobese patients. NT-proBNP was the strongest predictor of adverse cardiovascular event risk in both obese and nonobese patients. Conclusions On-treatment NT-proBNP level ≤1000 pg/mL has favorable prognostic implications, irrespective of obesity status. NT-proBNP levels were the strongest predictor of cardiovascular events in both obese and nonobese individuals in this trial. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01685840.

Geographic Variation in Cardiovascular Health Among American Adults.

OBJECTIVE: To evaluate the contemporary geographic trends in cardiovascular health in the United States and its relationship with geographic distribution of cardiovascular mortality. METHODS: By use of a retrospective cross-sectional design, the 2011-2017 Behavioral Risk Factor Surveillance System (BRFSS) was queried to determine the age-adjusted prevalence of cardiovascular health index (CVHI) metrics (sum of ideal blood pressure, blood glucose concentration, lipid levels, body mass index, smoking, physical activity, and diet). Cardiovascular health was estimated as both continuous (0 to 7 points) and categorical (ideal, intermediate, poor) variables from the BRFSS. Age-adjusted cardiovascular mortality for 2017 was obtained from the Centers for Disease Control and Prevention WONDER database. RESULTS: Among 1,362,529 American adult participants of the BRFSS 2011-2017 and all American residents in 2017, the CVHI score increased from 3.89±0.004 in 2011 to 3.96±0.005 in 2017 (Ptrend<.001) nationally, with modest improvement across all regions (Ptrend<.05 for all). Ideal cardiovascular health prevalence improved in the northeastern (Ptrend=.03) and southern regions (Ptrend=.002). In 2017, the prevalence of coronary heart disease (6.8%; 95% CI, 6.5% to 7.1%) and stroke (3.7%; 95% CI, 3.4% to 3.9%) was highest in the southern region. The CVHI score (3.81±0.01) and the prevalence of ideal cardiovascular health (12.2%; 95% CI, 11.7% to 12.7%) were lowest in the southern United States. This corresponded to the higher cardiovascular mortality in the southern region (233.0 [95% CI, 232.2- to 33.8] per 100,000 persons). CONCLUSION: Despite a modest improvement in CVHI, only 1 in 6 Americans has ideal cardiovascular health with significant geographic differences. These differences correlate with the geographic distribution of cardiovascular mortality. An urgent unmet need exists to mitigate the geographic disparities in cardiovascular morbidity and mortality.

Mortality Due to Mitral Regurgitation Among Adults in the United States: 1999-2018.

OBJECTIVE: To evaluate the nationwide trends in mortality due to mitral regurgitation (MR) among American adults from 1999 to 2018. PATIENTS AND METHODS: Trends in mortality due to MR were assessed using retrospective cross-sectional analyses of nationwide mortality data from death certificates of all American residents between January 1, 1999, and December 31, 2018, using the Centers for Disease Control and Prevention Wide-Ranging Online Data for Epidemiologic Research database. Piecewise linear regression was used to evaluate the trends in the overall population and in subgroups. RESULTS: Among 45,982 deaths due to MR during the study period, higher mortality rates were seen in older White females from the western United States. In 1999, the crude and age-adjusted mortality rates were 27.4 (95% CI, 26.3 to 28.4) and 27.5 (95% CI, 26.4 to 28.5) per 1,000,000 persons, respectively. By 2018, these rates declined to 18.0 (95% CI, 17.3 to 18.7) and 17.7 (95% CI, 17.0 to 18.4) per 1,000,000 persons, respectively (P<.001 for trend for both). Crude mortality rates declined from 1999 to 2012 (annual percentage change [APC], -4.1 (95% CI, -4.6 to -3.6) but then increased after 2012 (APC, 2.6 [95% CI, 0.8 to 4.4; P<.001 for change in trend]). The age-adjusted mortality rates declined from 1999 to 2012 (APC, -3.9 [95% CI, -4.4 to -3.4]) but subsequently increased after 2012 (APC, 1.4 [95% CI, -0.4 to 3.2; P<.001 for change in trend]). The observed decrease was consistent across age, sex, race, geographic region, and urbanization subgroups (P<.05 for all). CONCLUSION: Mortality due to MR in American adults declined at an annual rate of approximately 4% until 2012 and has since then increased by about 1.5% annually. These mortality trends warrant further investigation.

Geographic Variation in Racial Disparities in Health and Coronavirus Disease-2019 (COVID-19) Mortality.

OBJECTIVE: To evaluate the race-stratified state-level prevalence of health determinants and the racial disparities in coronavirus disease 2019 (COVID-19) cumulative incidence and mortality in the United States. PATIENTS AND METHODS: The age-adjusted race-stratified prevalence of comorbidities (hypertension, diabetes, dyslipidemia, and obesity), preexisting medical conditions (pulmonary disease, heart disease, stroke, kidney disease, and malignant neoplasm), poor health behaviors (smoking, alcohol abuse, and physical inactivity), and adverse socioeconomic factors (education, household income, and health insurance) was computed in 435,139 American adult participants from the 2017 Behavioral Risk Factor Surveillance System survey. Correlation was assessed between health determinants and the race-stratified COVID-19 crude mortality rate and infection-fatality ratio computed from respective state public health departments in 47 states. RESULTS: Blacks had a higher prevalence of comorbidities (63.3%; 95% CI, 62.4% to 64.2% vs 55.1%; 95% CI, 54.7% to 55.5%) and adverse socioeconomic factors (47.0%; 95% CI, 46.0% to 47.9% vs 30.9%; 95% CI, 30.6% to 31.3%) than did whites. The prevalence of preexisting medical conditions was similar in blacks (30.4%; 95% CI, 28.8% to 32.1%) and whites (30.8%; 95% CI, 30.2% to 31.4%). The prevalence of poor health behaviors was higher in whites (57.2%; 95% CI, 56.3% to 58.0%) than in blacks (50.2%; 95% CI,46.2% to 54.2%). Comorbidities and adverse socioeconomic factors were highest in the southern region, and poor health behaviors were highest in the western region. The cumulative incidence rate (per 100,000 persons) was 3-fold higher in blacks (1546.4) than in whites (540.4). The crude mortality rate (per 100,000 persons) was 2-fold higher in blacks (83.2) than in whites (33.2). However, the infection-fatality ratio (per 100 cases) was similar in whites (6.2) and blacks (5.4). Within racial groups, the geographic distribution of health determinants did not correlate with the state-level COVID-19 mortality and infection-fatality ratio (P>.05 for all). CONCLUSION: Racial disparities in COVID-19 are largely driven by the higher cumulative incidence of infection in blacks. There is a discordance between the geographic dispersion of COVID-19 mortality and the regional distribution of health determinants.

Angiostatin inhibits acute lung injury in a mouse model.

Acute lung injury is marked by profound influx of activated neutrophils, which have delayed apoptosis, along with fluid accumulation that impairs lung function and causes high mortality. Inflammatory and antimicrobial molecules, such as reactive oxygen species from activated neutrophils with prolonged lifespan, cause tissue damage and contribute to lung dysfunction. Angiostatin, an endogenous antiangiogenic molecule, is expressed in the lavage fluid of patients with acute respiratory distress syndrome and modifies neutrophil infiltration in a mouse model of peritonitis. Our aim was to investigate the therapeutic role of angiostatin in acute lung injury. We analyzed bronchoalveolar lavage and lung tissues from C57BL/6 mouse model of Escherichia coli LPS-induced acute lung injury to assess the effects of angiostatin treatment. Subcutaneous angiostatin administered at 5 h after LPS treatment reduces histological signs of inflammation, protein accumulation, lung Gr1+ neutrophils, myeloperoxidase activity, and expression of phosphorylated p38 MAPK in lung tissues and peripheral blood neutrophils, while increasing the number of apoptotic cells in the lungs without affecting the levels of macrophage inflammatory protein-1 α, IL-1ß, keratinocyte chemoattractant, and monocyte chemoattractant protein-1 in lavage and lung homogenates at 9 and 24 h after LPS treatment. In contrast, angiostatin administered intravenously 5 h after LPS treatment did not reduce histological sign of inflammation, BAL cell recruitment, and protein concentration at 9 h of LPS treatment. We conclude that angiostatin administered subcutaneously after LPS challenge inhibits acute lung inflammation up to 24 h after LPS treatment.

Comparison of the response to experimentally induced short-term inflammation in the temporomandibular and metacarpophalangeal joints of horses.

OBJECTIVE: To investigate the relationship between inflammatory responses of the temporomandibular joint (TMJ) and the metacarpophalangeal (MCP) joint in clinically normal horses. ANIMALS: 7 mature horses. PROCEDURES: In each horse, 1 TMJ and 1 MCP joint were injected with lipopolysaccharide (LPS; 0.0025 µg). The contralateral TMJ and MCP joint were injected with saline (0.9% NaCl) solution. Synovial fluid samples were collected from all 4 joints over 24 hours after injection. Concentrations of interleukin-6, tumor necrosis factor-α, transforming growth factor-ß, and total protein were measured via immunoassay. Horses were assessed for clinical signs of joint inflammation at each time point. RESULTS: Concentrations of interleukin-6 were not significantly different between LPS-injected MCP joints and TMJs at any time point. Transforming growth factor-ß concentrations were significantly increased in MCP joints, compared with concentrations in TMJs, at 12 and 24 hours after injection. Tumor necrosis factor-α concentrations were significantly higher in LPS-injected TMJs than in LPS-injected MCP joints at 1 and 6 hours after injection. Total protein concentration did not differ significantly between LPS-injected MCP joints and TMJs. Injection of LPS induced clinical inflammation at all time points; additionally, 2 MCP joints (but no TMJs) had an inflammatory response to injection of saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: The inflammatory response to LPS appeared to be attenuated more quickly in TMJs than in MCP joints of horses. The difference in response suggested that a lack of clinical osteoarthritis in the TMJ of horses could be attributable to a difference in cytokine response.

Expression and activity of N-myristoyltransferase in lung inflammation of cattle and its role in neutrophil apoptosis.

N-myristoyltransferase (NMT) attaches a 14 carbon fatty acid, myristic acid, to the N-terminal glycine residue of proteins. NMT exists in two isoforms NMT1 and NMT2. Myristoylated proteins play critical roles in protein-protein interactions, cell signaling and oncogenesis. Although elevated expression of NMT1 has been described in colorectal carcinoma, its expression and roles in normal and inflamed lungs of the cattle are unknown. Therefore, we investigated the expression and activity of NMT in a bovine model of lung inflammation induced with Mannheimia hemolytica and in vitro in neutrophils and macrophages. Western blots revealed increased expression of NMT1 in lungs from infected animals compared to control animals. Total NMT activity was reduced in inflamed lungs compared to control animals (p < 0.05) along with increased expression of enolase, a putative inhibitor of NMT. NMT1 staining was observed in the septum, vascular endothelium and the epithelium in the lungs from control as well as infected calves. NMT1 expression was intense in neutrophils in the necrotic areas in the inflamed lungs. Immuno-electron microscopy localized NMT1 in cytoplasm and nuclei of endothelium, pulmonary intravascular macrophages and airway epithelium. Total NMT activity and NMT1 expression were increased in neutrophils and macrophages exposed to Escherichia coli LPS in vitro. NMT knockdown increased apoptosis in activated neutrophils. This is the first report demonstrating expression of NMT in normal and inflamed lungs and a novel role for NMT in regulation of neutrophil lifespan.

The role of RGD-tagged helical rosette nanotubes in the induction of inflammation and apoptosis in human lung adenocarcinoma cells through the P38 MAPK pathway.

The rosette nanotubes (RNTs) are a class of biologically inspired, self-assembling, metal-free, hydrophilic nanotubes, which hold tremendous potential as targeted drug delivery vehicles. We investigated the cell signaling events caused by lysine-functionalized RNTs (K-RNT) co-assembled with Arg-Gly-Asp-Ser-Lys-functionalized RNTs (RGDSK-RNT) for induction of inflammation and apoptosis in human adenocarcinoma (Calu-3) cells. When co-assembled in a ratio of 1:10 microM these composite RNTs (referred to as RGDSK/K-RNTs) rapidly induced phosphorylation of P38 mitogen-activated protein kinase (MAPK) within 2 min. Higher concentrations of RGDSK/K-RNTs (>10:100 microM) resulted in a P38 MAPK-dependent increase in secretion of TNF-alpha. RGDSK/K-RNTs (1:10-40:400 microM) also caused a concentration- and P38 MAPK-dependent increase in caspase-3 activity and DNA fragmentation in Calu-3 cells at 18 h of exposure. Over-expression of pro-apoptotic genes including caspase-3, BAK1, CIDEB, TP53BP2, FAS, TNF and FASLG supported pro-apoptotic behaviors of these RNTs. We conclude that RGDSK/K-RNTs induce phosphorylation of P38 MAPK, which regulate secretion of TNF-alpha, activation of caspase-3 and apoptosis in Calu-3 cells. These results suggest that the RNTs could be used as a drug to induce apoptosis in cancer cells or as a versatile platform to deliver a variety of biologically active molecules for cancer therapy.

Macrophage inflammatory response to self-assembling rosette nanotubes.

Rosette nanotubes (RNTs) are a new class of nanomaterials with significant therapeutic potential. However, societal concerns related to the potential adverse health effects of engineered nanomaterials drew attention towards the investigation of their interaction with the human U937 macrophage cell line. The cells are treated with medium only (control), lysine (50 microg mL(-1)), lysine-functionalized RNTs (RNT-K; 1, 5, and 50 microg mL(-1)), Min-U-Sil quartz microparticles (80 microg mL(-1)), or lipopolysaccharide (1 microg mL(-1)). The supernatant and cells are assayed for cell viability, cytokine protein, and mRNA expression at 1, 6, and 24 h post-treatment. The results indicate that RNT-K activate transcription of proinflammatory genes (interleukin-8 and tumor necrosis factor-alpha (TNF-alpha)) within 1 h, but this effect is not accompanied by protein secretion into the supernatant. The effect of the length of RNTs on human U937 macrophage viability is also investigated. Although both short and long RNT-K exhibit time-dependent effects on TNF-alpha transcription, only the short RNT-K (5 microg mL(-1)) increase TNF-alpha concentration at 6 h relative to the long RNT-K. Moreover, RNT-K (1 and 5 microg mL(-1)) have no effect on cell viability by 24 h. These data indicate that RNT-K do not induce a robust inflammatory response or cytotoxicity in the U937 human macrophage cell line, and therefore could be used for biomedical applications.

Rosette nanotubes show low acute pulmonary toxicity in vivo.

Nanotubes are being developed for a large variety of applications ranging from electronics to drug delivery. Common carbon nanotubes such as single-walled and multi-walled carbon nanotubes have been studied in the greatest detail but require solubilization and removal of catalytic contaminants such as metals prior to being introduced to biological systems for medical application. The present in vivo study characterizes the degree and nature of inflammation caused by a novel class of self-assembling rosette nanotubes, which are biologically inspired, naturally water-soluble and free of metal content upon synthesis. Upon pulmonary administration of this material we examined responses at 24 h and 7d post-exposure. An acute inflammatory response is triggered at 50 and 25 microg doses by 24 h post-exposure but an inflammatory response is not triggered by a 5 microg dose. Lung inflammation observed at a 50 microg dose at 24 h was resolving by 7d. This work suggests that novel nanostructures with biological design may negate toxicity concerns for biomedical applications of nanotubes. This study also demonstrates that water-soluble rosette nanotube structures represent low pulmonary toxicity, likely due to their biologically inspired design, and their self-assembled architecture.

Role of pulmonary intravascular macrophages in endotoxin-induced lung inflammation and mortality in a rat model.

BACKGROUND: Bile-duct ligated (BDL) rats recruit pulmonary intravascular macrophages (PIMs) and are highly susceptible to endotoxin-induced mortality. The mechanisms of this enhanced susceptibility and mortality in BDL rats, which are used as a model of hepato-pulmonary syndrome, remain unknown. We tested a hypothesis that recruited PIMs promote endotoxin-induced mortality in a rat model. METHODS: Rats were subjected to BDL to induce PIM recruitment followed by treatment with gadolinium chloride (GC) to deplete PIMs. Normal and BDL rats were treated intravenously with E. coli lipopolysaccharide (LPS) with or without GC pre-treatment followed by collection and analyses of lungs for histopathology, electron microscopy and cytokine quantification. RESULTS: BDL rats recruited PIMs without any change in the expression of IL-1beta, TNF-alpha and IL-10. GC caused reduction in PIMs at 48 hours post-treatment (P < 0.05). BDL rats treated intravenously with E. coli LPS died within 3 hours of the challenge while the normal LPS-treated rats were euthanized at 6 hours after the LPS treatment. GC treatment of rats 6 hours or 48 hours before LPS challenge resulted in 80% (1/5) and 100% (0/5) survival, respectively, at 6 hours post-LPS treatment. Lungs from BDL+LPS rats showed large areas of perivascular hemorrhages compared to those pre-treated with GC. Concentrations of IL-1beta, TNF-alpha and IL-10 were increased in lungs of BDL+LPS rats compared to BDL rats treated with GC 48 hours but not 6 hours before LPS (P < 0.05). CONCLUSION: We conclude that PIMs increase susceptibility for LPS-induced lung injury and mortality in this model, which is blocked by a reduction in their numbers or their inactivation.

Low inflammatory activation by self-assembling Rosette nanotubes in human Calu-3 pulmonary epithelial cells.

Rosette nanotubes (RNT) are a new class of metal-free organic nanotubes synthesized through self-assembly. Because of the wide range of potential biomedical applications associated with these materials, it is necessary to evaluate their potential in vitro toxicity. Here the cytotoxicity of a lysine-functionalized nanotube (RNT-K) in a human Calu-3 pulmonary epithelial cell line is investigated. The cells were treated with media only (control), lysine (50 mg mL(-1)), RNT-K (1, 5, and 50 microg mL(-1)), Min-U-Sil quartz microparticles (QM; 80 microg mL(-1)), and lipopolysaccharide (LPS; 1 microg mL(-1)). The supernatants were analyzed at 1, 6, and 24 h after treatment for the expression of three proinflammatory mediators: IL-8, TNF-alpha and EMAP-II. Cellular viability determined with the Trypan blue assay is significantly reduced in the QM and high-dose RNT-treated groups. TNF-alpha and EMAP-II are undetectable by enzyme-linked-immunosorbent assay (ELISA) in the supernatant of all groups. Although IL-8 concentrations do not differ between treatments, its concentrations increase with time within each of the groups. Quantitative reverse-transcriptase polymerase chain reaction (qRTPCR) of IL-8 mRNA shows increased expression in the high-dose RNT-treated groups at both 1 and 6 h, while an adhesion molecule, ICAM-1 mRNA, shows the greatest increase at 6 h in the QM-treated group. In summary, RNT-K neither reduces cell viability at moderate doses nor does it induce a time-dependent inflammatory response in pulmonary epithelial cells in vitro.

Correct targeting of plant ARF GTPases relies on distinct protein domains.

Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N-terminal myristoyl group but to different target membranes. Specifically, we investigated how ARF1 is targeted to the Golgi and post-Golgi structures, whereas ARFB accumulates at the plasma membrane. While the subcellular localization of ARFB appears to depend on multiple domains including the C-terminal half of the GTPase, the correct targeting of ARF1 is dependent on two domains: an N-terminal ARF1 domain that is necessary for the targeting of the GTPase to membranes and a core domain carrying a conserved MxxE motif that influences the relative distribution of ARF1 between the Golgi and post-Golgi compartments. We also established that the N-terminal ARF1 domain alone was insufficient to maintain an interaction with membranes and that correct targeting is a protein-specific property that depends on the status of the GTP switch. Finally, an ARF1-ARFB chimera containing only the first 18 amino acids from ARF1 was shown to compete with ARF1 membrane binding loci. Although this chimera exhibited GTPase activity in vitro, it was unable to recruit coatomer, a known ARF1 effector, onto Golgi membranes. Our results suggest that the targeting of ARF GTPases to the correct membranes may not only depend on interactions with effectors but also relies on distinct protein domains and further binding partners on the Golgi surface.

High-aspect ratio nanoparticles in nanotoxicology.

In this Learned Discourse, we introduce nanotoxicology and note that little information is available on the role of aspect ratio in nanomaterial toxicity. Aspect ratio has implications for clearance from the lungs and for phagocytic cells such as the macrophage. Whether the role of aspect ratio on the nanoscale presents unique toxicological considerations remains unknown. In the case of rosette nanotubes, other physicochemical factors, such as solubility and the absence of metal content, might outweigh any possible effects incurred by aspect ratio. However, more systematic toxicity studies are required to determine the role of specific nanomaterial properties relative to biological responses.

A heat-activated MAP kinase (HAMK) as a mediator of heat shock response in tobacco cells.

A heat-activated MAP kinase (HAMK), immunologically related to the extracellular signal-regulated kinase (ERK) super-family of protein kinases, has been identified in BY2 cells of tobacco. The activation of HAMK at 37 degrees C was transient and detected within 2 min and reached a maximum level within 5 min. Ca(2+) chelators and channel blockers, and the known inhibitors of MEK, a MAP kinase kinase, prevented the heat activation of HAMK. This suggests that HAMK activation is part of a heat-triggered MAP kinase cascade that requires Ca(2+) influx. The heat shock protein HSP70 accumulated at 37 degrees C, but not when HAMK activation was prevented with the inhibitors of MEK or with Ca(2+) chelators or channel blockers. As previously shown for heat activation of HAMK, heat-induced accumulation of HSP70 requires membrane fluidization and reorganization of cytoskeleton. We concluded that heat-triggered HAMK cascade might play an essential role in the launching of heat shock response and hsp gene expression in tobacco cells.