Comparative Phylogenetics of Papilio Butterfly Wing Shape and Size Demonstrates Independent Hindwing and Forewing Evolution.

The complex forces that shape butterfly wings have long been a subject of experimental and comparative research. Butterflies use their wings for flight, camouflage, mate recognition, warning, and mimicry. However, general patterns and correlations among wing shape and size evolution are still poorly understood. We collected geometric morphometric measurements from over 1400 digitized museum specimens of Papilio swallowtails and combined them with phylogenetic data to test two hypotheses: 1) forewing shape and size evolve independently of hindwing shape and size and 2) wing size evolves more quickly than wing shape. We also determined the major axes of wing shape variation and discovered that most shape variability occurs in hindwing tails and adjacent areas. We conclude that forewing shape and size are functionally and biomechanically constrained, whereas hindwings are more labile, perhaps in response to disruptive selective pressure for Batesian mimicry or against predation. The development of a significant, re-usable, digitized data resource will enable further investigation on tradeoffs between flight performance and ecological selective pressures, along with the degree to which intraspecific, local-scale selection may explain macroevolutionary patterns. [Batesian mimicry; Lepidoptera; geometric morphometrics; museum specimens.].

Phylogenomics resolves major relationships and reveals significant diversification rate shifts in the evolution of silk moths and relatives.

BACKGROUND: Silkmoths and their relatives constitute the ecologically and taxonomically diverse superfamily Bombycoidea, which includes some of the most charismatic species of Lepidoptera. Despite displaying spectacular forms and diverse ecological traits, relatively little attention has been given to understanding their evolution and drivers of their diversity. To begin to address this problem, we created a new Bombycoidea-specific Anchored Hybrid Enrichment (AHE) probe set and sampled up to 571 loci for 117 taxa across all major lineages of the Bombycoidea, with a newly developed DNA extraction protocol that allows Lepidoptera specimens to be readily sequenced from pinned natural history collections. RESULTS: The well-supported tree was overall consistent with prior morphological and molecular studies, although some taxa were misplaced. The bombycid Arotros Schaus was formally transferred to Apatelodidae. We identified important evolutionary patterns (e.g., morphology, biogeography, and differences in speciation and extinction), and our analysis of diversification rates highlights the stark increases that exist within the Sphingidae (hawkmoths) and Saturniidae (wild silkmoths). CONCLUSIONS: Our study establishes a backbone for future evolutionary, comparative, and taxonomic studies of Bombycoidea. We postulate that the rate shifts identified are due to the well-documented bat-moth "arms race". Our research highlights the flexibility of AHE to generate genomic data from a wide range of museum specimens, both age and preservation method, and will allow researchers to tap into the wealth of biological data residing in natural history collections around the globe.

A Combined test using both cell sediment and supernatant cell-free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients.

INTRODUCTION: The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell-free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients. METHODS: A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction. RESULTS: Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group. CONCLUSIONS: A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.

SurePath® LBC improves the diagnostic accuracy of intrahepatic and hilar cholangiocarcinoma.

INTRODUCTION: The current study aimed to compare cytology using SurePath® (SP)-LBC and biliary tissue histology (BTH) for the diagnosis of biliary disease. METHODS: Between January 2014 and December 2016, 57 patients underwent endoscopic retrograde cholangiopancreatography for the diagnosis of biliary disease. Biliary cytological samples were processed using SP-LBC and subsequently BTH was performed. A final diagnosis was confirmed by surgery (23 malignant cases) and clinical follow-up (34 benign and malignant cases): 18 extrahepatic cholangiocarcinoma; 17 intrahepatic/hilar cholangiocarcinoma (intra/H-CC); eight other malignant disease; and 14 benign biliary disease. The diagnoses made using SP-LBC and BTH were classified into four categories: (1) benign; (2) indeterminate; (3) suspicious for malignancy/malignant; and (4) inadequate. In addition, diagnostic accuracy was compared between SP-LBC and BTH. RESULTS: Although 23% (13/57) of BTH samples were classified as inadequate, all SP-LBC cases were classified as adequate. Among 43 malignant cases, 11 normal, four indeterminate and 28 suspicious for malignancy/malignant were found using SP-LBC (26%, 9% and 65%, respectively), in contrast to 10 inadequate, nine normal, 10 indeterminate and 14 suspicious for malignancy/malignant observed using BTH (23%, 21%, 23%, and 33%, respectively). The identification of malignant cells was strikingly different between SP-LBC and BTH. Furthermore, limited to intra/H-CC, accuracy was significantly higher using SP-LBC than using BTH (P < .001). CONCLUSIONS: SP-LBC of the biliary tract is a useful and reliable method for diagnosing biliary malignant disease and has an advantage over BTH for detecting malignant cells and accurately diagnosing intra/H-CC.

Pre-treatment CD8+ tumour-infiltrating lymphocyte density predicts distant metastasis after definitive treatment in patients with stage III/IV hypopharyngeal squamous cell carcinoma.

OBJECTIVES: Although inflammatory markers, such as the neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and local immune markers have been shown to have prognostic utility, limited information is available regarding inflammatory and pre-existing tumour-infiltrating lymphocyte density and their association with prognosis in patients with hypopharyngeal squamous cell carcinoma. We investigated the prognostic ability of inflammatory markers and tumour-infiltrating lymphocyte density in stage III and stage IV hypopharyngeal squamous cell carcinoma patients receiving definitive treatment. DESIGN: Retrospective cohort study. SETTING: Kurume University Hospital. PARTICIPANTS: Ninety-six stage III or stage IV hypopharyngeal squamous cell carcinoma patients treated at the Kurume University Hospital between 2000 and 2014. MAIN OUTCOME MEASURES: Inflammatory markers and pre-treatment tumour-infiltrating lymphocyte density were examined from recorded haematologic data and immunohistochemical analysis. RESULTS: Multivariate analyses showed that the CD8+ tumour-infiltrating lymphocyte density was an independent predictive factor for distant metastasis and overall survival, whereas inflammatory markers, including the neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio, were not correlated with distant metastasis or overall survival. CONCLUSIONS: Higher pre-treatment CD8+ tumour-infiltrating lymphocyte density is a useful predictive biomarker for reduced distant metastasis and better prognosis.

Cytological nuclear atypia classification can predict prognosis in patients with endometrial cancer.

OBJECTIVE: Endometrial cancer is one of the leading causes of malignancy in females. Nuclear findings are important for patients with cancer, and can provide valuable information to treating oncologists. We investigated whether nuclear findings were a useful prognostic factor in patients with endometrial cancer. METHOD: We investigated 71 cases of endometrial carcinoma with paired histology and cytology at Kurume University Hospital. We classified endometrial endometrioid adenocarcinoma (EEC) G1 and G2 as type I carcinomas, and uterine papillary serous carcinoma (UPSC), clear cell carcinoma (CC) and EEC G3 as type II carcinomas. For the establishment of the cytological nuclear atypia classification, we examined the following nuclear factors on the cytological smears: mitotic figures, prominent nucleoli, nuclear area and anisonucleosis. RESULTS: There was a significant difference in mitotic figures (P < 0.001) and anisonucleosis (P = 0.026) in cytological smears between type I and type II carcinomas. Based on these findings, we categorized cytological nuclear atypia into three groups, nuclear atypia-1 (57.7%), nuclear atypia-2 (19.7%) and nuclear atypia-3 (22.5%), and this classification system correlated well with prognosis in patients with endometrial cancer (P < 0.001). Furthermore, this classification system was able to extract patients with a good prognosis from those with high-grade carcinomas, such as UPSC+CC+EEC G3, and patients with a poor prognosis from those with EEC G1. CONCLUSIONS: Our system of cytological nuclear atypia classification based on endometrial cytology can predict patient prognosis. Cytological nuclear atypia classification and histological typing may be useful for the treatment and follow-up of patients with endometrial cancer, and should be routinely incorporated into cytological reports.

Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer.

BACKGROUND: Recent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death-ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC. PATIENTS AND METHODS: The expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry. RESULTS: Expression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients. CONCLUSIONS: High expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.

Convergent evolution of morphology and habitat use in the explosive Hawaiian fancy case caterpillar radiation.

Species occurring in unconnected, but similar habitats and under similar selection pressures often display strikingly comparable morphology, behaviour and life history. On island archipelagos where colonizations and extinctions are common, it is often difficult to separate whether similar traits are a result of in situ diversification or independent colonization without a phylogeny. Here, we use one of Hawaii's most ecologically diverse and explosive endemic species radiations, the Hawaiian fancy case caterpillar genus Hyposmocoma, to test whether in situ diversification resulted in convergence. Specifically, we examine whether similar species utilizing similar microhabitats independently developed largely congruent larval case phenotypes in lineages that are in comparable, but isolated environments. Larvae of these moths are found on all Hawaiian Islands and are characterized by an extraordinary array of ecomorphs and larval case morphology. We focus on the 'purse cases', a group that is largely specialized for living within rotting wood. Purse cases were considered a monophyletic group, because morphological, behavioural and ecological traits appeared to be shared among all members. We constructed a phylogeny based on nuclear and mitochondrial DNA sequences from 38 Hyposmocoma species, including all 14 purse case species and 24 of non-purse case congeners. Divergence time estimation suggests that purse case lineages evolved independently within dead wood and developed nearly identical case morphology twice: once on the distant Northwest Hawaiian Islands between 15.5 and 9 Ma and once on the younger main Hawaiian Islands around 3.0 Ma. Multiple ecomorphs are usually found on each island, and the ancestral ecomorph of Hyposmocoma appears to have lived on tree bark. Unlike most endemic Hawaiian radiations that follow a clear stepwise progression of colonization, purse case Hyposmocoma do not follow a pattern of colonization from older to younger island. We postulate that the diversity of microhabitats and selection from parasitism/predation from endemic predators may have shaped case architecture in this extraordinary endemic radiation of Hawaiian insects.

A DNA Extraction Method for Insects From Sticky Traps: Targeting a Low Abundance Pest, Phthorimaea absoluta (Lepidoptera: Gelechiidae), in Mixed Species Communities.

Invasive insects can cause catastrophic damage to ecosystems and cost billions of dollars each year due to management expenses and lost revenue. Rapid detection is an important step to prevent invasive insects from spreading, but improvements in detection capabilities are needed for bulk collections like those from sticky traps. Here we present a bulk DNA extraction method designed for the detection of Phthorimaea absoluta Meyrick (Lepidoptera: Gelechiidae), an invasive moth that can decimate tomato crops. We test the extraction method for insect specimens on sticky traps, subjected to different temperature and humidity conditions, and among mock insect communities left in the field for up to 21 d. We find that the extraction method yielded high success (>92%) in recovering target DNA across field and lab trials, without a decline in recovery after three weeks, across all treatments. These results may have a large impact on tomato growing regions where P. absoluta is in the early stages of invasion or not yet present. The extraction method can also be used to improve detection capabilities for other bulk insect collections, especially those using sticky traps, to the benefit of pest surveys and biodiversity studies.

Caspase-independent cell killing by Fas-associated protein with death domain.

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.

Necrotic death pathway in Fas receptor signaling.

A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association.

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.

Functional activation of Jak1 and Jak3 by selective association with IL-2 receptor subunits.

The interleukin-2 receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta, and IL-2R gamma chains, the last of which is also used in the receptors for IL-4, IL-7, and IL-9. Stimulation with IL-2 induces the tyrosine phosphorylation and activation of the Janus kinases Jak1 and Jak3. Jak1 and Jak3 were found to be selectively associated with the "serine-rich" region of IL-2R beta and the carboxyl-terminal region of IL-2R gamma, respectively. Both regions were necessary for IL-2 signaling. Furthermore, Jak3-negative fibroblasts expressing reconstituted IL-2R became responsive to IL-2 after the additional expression of Jak3 complementary DNA. Thus, activation of Jak1 and Jak3 may be a key event in IL-2 signaling.

Efficacy of autologous fat injection laryngoplasty with an adenoviral vector expressing hepatocyte growth factor in a canine model.

OBJECTIVE: The effectiveness of autologous fat injection laryngoplasty may be reduced by resorption of injected fat tissue. The aim of the present study was to clarify the efficacy of fat injection laryngoplasty using autologous fat plus a replication-defective adenoviral vector expressing hepatocyte growth factor, regarding reduction of injected fat tissue resorption. MATERIAL AND METHODS: Four female beagle dogs were used in this study. After sedation, a direct laryngoscope was introduced to enable visualisation of the larynx. In each dog, harvested autologous fat plus an adenoviral vector expressing hepatocyte growth factor was injected into the right true vocal fold, and harvested fat plus an adenoviral vector expressing no gene was injected into the left true vocal fold. A total laryngectomy was performed one year after the intracordal fat injection. Coronal sections of the resected whole larynges were made and the following parameters assessed using light and electron microscopy: size of fat area; number of vasculoendothelial cells surrounding adipocytes; and shape of injected adipocytes in the vocal fold. RESULTS: The fat area was significantly larger and the number of vasculoendothelial cells surrounding adipocytes significantly greater in the intracordal fat injection containing adenoviral vector expressing hepatocyte growth factor, compared with the control intracordal fat injection containing adenoviral vector expressing no gene. When viewed under electron microscopy, the injected adipocytes were observed to have grafted better in the intracordal fat injection with hepatocyte growth factor adenoviral vector, compared with the control intracordal fat injection with adenoviral vector expressing no gene. CONCLUSIONS: Injection into the vocal fold of autologous fat containing an adenoviral vector expressing hepatocyte growth factor can reduce subsequent resorption of injected fat.

Critical role of biklf in erythroid cell differentiation in zebrafish.

Hematopoietic cells arise from ventral mesoderm in different vertebrates, but the mechanisms through which various factors contribute to the hematopoietic processes, including erythrogenesis, remain incompletely understood. The Krüppel-like transcription factor Biklf is preferentially expressed in blood islands throughout zebrafish embryogenesis, marking the region of future erythropoiesis [1]. In this paper, we show that expression of biklf is significantly suppressed in the blood-less mutants vampire and m683 in which primitive hematopoiesis is impaired. Knockdown of biklf using morpholino-based antisense oligonucleotides (biklf-MO) led to a potent reduction in the number of circulating blood cells and deficiency in hemoglobin production. Consistently, we found that the expression of beta(e3)globin is strongly suppressed in biklf-MO-injected embryos, while gata1 expression is partly inhibited at the 10-somite stage. In addition, analysis of reporter constructs driven by the GATA1 and beta-globin promoters showed that Biklf can positively regulate both genes. These results indicate that Biklf is required for erythroid cell differentiation in zebrafish.

Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling.

The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.

Inhibition of Fas-induced apoptosis by Bcl-2.

Jurkat cells express Fas, and rapidly undergo apoptosis in response to Fas ligand or an agonistic anti-Fas antibody. This apoptotic pathway is mediated by a cascade of caspases. In this report, we show that Fas activation induced the processing of caspase 8 in Jurkat cells with a time frame similar to the activation of caspase 3 and the proteolysis of nuclear proteins. Jurkat cell transformants that overexpress Bcl-2 were partially but not completely resistant to the Fas-induced apoptosis. Little processing of caspase 8 was observed upon Fas activation in these transformants. Furthermore, although caspase 8 was recruited to Fas upon Fas activation in the parental Jurkat cells, the recruitment of caspase 8 to Fas was inhibited in the transformants overexpressing Bcl-2. These results suggest that Bcl-2 inhibits Fas-induced apoptosis by preventing the formation of the death-inducing signaling complex that is composed of Fas, FADD/MORT1, and caspase 8.

Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts.

OBJECTIVES: The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND: The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS: Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS: In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS: The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.

Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes.

OBJECTIVES: The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes. BACKGROUND: Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells. However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility. METHODS: Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent. RESULTS: Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner. The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate). These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters. The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes. Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes. KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects. CONCLUSIONS: These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.