The Fontan Udenafil Exercise Longitudinal Trial: Subgroup Analysis.

The Pediatric Heart Network's Fontan Udenafil Exercise Longitudinal (FUEL) Trial (Mezzion Pharma Co. Ltd., NCT02741115) demonstrated improvements in some measures of exercise capacity and in the myocardial performance index following 6 months of treatment with udenafil (87.5 mg twice daily). In this post hoc analysis, we evaluate whether subgroups within the population experienced a differential effect on exercise performance in response to treatment. The effect of udenafil on exercise was evaluated within subgroups defined by baseline characteristics, including peak oxygen consumption (VO2), serum brain-type natriuretic peptide level, weight, race, gender, and ventricular morphology. Differences among subgroups were evaluated using ANCOVA modeling with fixed factors for treatment arm and subgroup and the interaction between treatment arm and subgroup. Within-subgroup analyses demonstrated trends toward quantitative improvements in peak VO2, work rate at the ventilatory anaerobic threshold (VAT), VO2 at VAT, and ventilatory efficiency (VE/VCO2) for those randomized to udenafil compared to placebo in nearly all subgroups. There was no identified differential response to udenafil based on baseline peak VO2, baseline BNP level, weight, race and ethnicity, gender, or ventricular morphology, although participants in the lowest tertile of baseline peak VO2 trended toward larger improvements. The absence of a differential response across subgroups in response to treatment with udenafil suggests that the treatment benefit may not be restricted to specific sub-populations. Further work is warranted to confirm the potential benefit of udenafil and to evaluate the long-term tolerability and safety of treatment and to determine the impact of udenafil on the development of other morbidities related to the Fontan circulation.Trial Registration NCT0274115.

The influence of insurance status on treatment and outcomes in oral cavity cancer: an analysis on 46,373 patients.

The purpose of this study was to determine the influence of insurance status on treatment and outcomes in oral cavity cancer. Patients were identified in the National Cancer Database (NCDB). Data were collected and analyzed using χ2 tests, Kaplan-Meier methods, and multivariable Cox regression models. Those uninsured or on Medicaid were more likely to be younger (P<0.001), minority race (P<0.001), have a lower median household income (P<0.001), lower educational attainment (P<0.001), not undergo primary resection (P<0.001), present with higher T (P<0.001),N (P<0.001), and M (P<0.001) stage of disease, and have a higher tumor grade (P<0.001). On univariate analysis, those with private insurance had significantly better overall survival than those uninsured (hazard ratio (HR) 1.481), under Medicaid (HR 2.006), or on Medicare (HR 1.921). On multivariable Cox regression analysis, insurance status remained an independent prognosticator even after accounting for multiple demographic, socioeconomic, treatment, and clinicopathological factors. These data suggest that insurance status is associated with treatment and outcomes in patients with oral cavity cancer. Being uninsured or on Medicaid was found to be associated with a higher risk of a poorer prognosis when compared to private insurance, and the data suggest the need to expand comprehensive medical coverage and optimize access to adequate medical care in vulnerable patient populations.

Prognostic value of negative interim 2-[¹8F]-fluoro-2-deoxy-d-glucose PET/CT in diffuse large B-cell lymphoma.

AIM: To assess the prognostic value of negative interim combined 2-[(18)F]-fluoro-2-deoxy-d-glucose ((18)F-FDG) positron-emission tomography/computed tomography (PET/CT) in patients with diffuse large B-cell lymphoma (DLBCL). MATERIALS AND METHODS: Ninety-two patients with histologically proven DLBCL were enrolled. All of the patients underwent (18)F-FDG PET/CT at diagnosis, and interim PET/CT after the second cycle of chemotherapy with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisolone (R-CHOP). Negative interim PET/CT was defined as the disappearance of all abnormal (18)F-FDG uptake compared to the pretreatment PET/CT image, as determined by visual assessment. The clinical outcome of patients was estimated as progression-free survival (PFS), and the prognostic significance of clinicopathological and imaging parameters were assessed using the Cox proportional hazards model. RESULTS: Thirty-six patients (39.1%) showed lymphoma progression within a median follow-up of 30.8 months. According to univariate analysis, Ann Arbor stage, serum lactate dehydrogenase level, Eastern Cooperative Oncology Group scale, International Prognostic Index (IPI) score, and maximum standardised uptake values on initial PET/CT were significant prognostic factors for PFS (all p<0.05). Among these parameters, only the IPI score was an independent predictor for PFS (p=0.044). Survival of patients with a high IPI score (≥3) was poorer than those with a low IPI score (0-2; p<0.001). CONCLUSION: Despite a negative interim (18)F-FDG PET/CT, approximately 39% of DLBCL patients showed progression during follow-up. Although the negative PET/CT was obtained during chemotherapy, it is important to closely follow-up patients, especially those with a high IPI score.

Distribution of Porcine Endogenous Retrovirus in Different Organs of the Hybrid of a Landrace and a Jeju Domestic Pig in Korea.

Xenotransplantation offers a solution to the shortage of available organs for transplantation, and the pig represents an ideal source of such organs. However, porcine endogenous retrovirus (PERV), whose genome is integrated in pigs, has been suggested to pose a potential risk of xenotransmission. Expression of PERVs in different organs of pigs was carefully measured at DNA, mRNA, and protein levels, providing information valuable for the application of pig organs in xenotransplantation. An analysis of PERV DNA showed that a very similar number of PERV copies was present in the genome of all organs, whereas mRNA and protein levels of PERV varied depending on the organ, with kidney, liver, and spleen expressing high levels of both mRNA and protein. In contrast, mRNA and protein levels were dissimilar in the lung and brain, where mRNA levels were low but protein levels were high. This discrepancy indicates that mRNA levels are not always reflected in protein expression. In addition, the difference between mRNA and protein highlights the importance of choosing the proper analysis method for diagnosing viral infection. In summary, this study provides insight into the distribution of PERV in various organs at the DNA, mRNA, and protein levels, and also informs the proper selection of tissues or organs for future clinical xenotransplantation.

Salivary gland scintigraphy in Sjögren's syndrome. Comparison of the diagnostic performance of visual and semiquantitative analysis.

UNLABELLED: The aim of this study was to compare the diagnostic utility of visual versus semi-quantitative analysis of salivary gland scintigraphy in the diagnosis of Sjögren's syndrome (SS). PATIENTS, METHODS: 99mTc-pertechnetate salivary gland scintigraphy was performed in 145 patients (133 women, 12 men) with clinically suspicious SS. The images were interpreted with visual and semiquantitative methods and the diagnostic performances for SS were compared using uptake and excretory functional parameters. RESULTS: In total, 76 patients (52.4%) were finally diagnosed with SS. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of visual analysis for the diagnosis of SS were 88.2%, 48.6%, 65.1%, 79.1%, and 69.2%, respectively. Semiquantitative values, the area under the ROC curve for uptake ratio and percentage excretion in the right salivary glands were significantly greater than 0.5 (p < 0.05). However, the percentage excretion in the left salivary glands did not show a statistically significant diagnostic ability for SS. The diagnostic ability of visual assessment was greater than that of the semiquantitative method in terms of evaluating uptake and excretory function in the submandibular glands. CONCLUSION: Visual analysis of salivary gland scintigraphy showed greater diagnostic utility than semiquantitative assessment in the diagnosis of SS, especially in the submandibular glands.

Diagnostic performance of 18F-FDG PET/CT, ultrasonography and MRI. Detection of axillary lymph node metastasis in breast cancer patients.

UNLABELLED: The aim of this study was to evaluate the diagnostic abilities of 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography(PET/CT) compared with those of ultrasonography and magnetic resonance imaging (MRI) for axillary lymph node staging in breast cancer patients. PATIENTS, METHODS: Preoperative 18F-FDG PET/non-contrast CT, ultrasonography and MRI were performed in 215 women with breast cancer. Axillary lymph node dissection was performed in all patients and the diagnostic performance of each modality was evaluated using histopathologic assessments as the reference standard. ROC curves were compared to evaluate the diagnostic ability of several imaging modalities (i. e., ultrasonography, MRI and 18F-FDG PET/CT). RESULTS: In total, 132 patients (61.4%) had axillary lymph node metastasis. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the detection of axillary lymph node metastasis were 72.3%, 77.3%, 66.7%, 81.6%, 75.3% for ultrasonography, 67.5%, 78.0%, 65.9%, 79.2%, 74.0% for MRI, and 62.7%, 88.6%, 77.6%, 79.1%, 78.6% for 18F-FDG PET/CT, respectively. There was no significant difference in diagnostic ability among the imaging modalities (i.e., ultrasonography, MRI and 18F-FDG PET/CT). The diagnostic ability of 18F-FDG PET/CT was significantly improved by combination with MRI (p = 0.0002) or ultrasonography (p < 0.0001). The combination of 18F-FDG PET/CT with ultrasonography had a similar diagnostic ability to that of all three modalities combined (18F-FDG PET/CT+ultrasonography+MRI, p = 0.05). CONCLUSION: The diagnostic performance of 18F-FDG PET/CT for detection of axillary node metastasis was not significantly different from that of ultrasonography or MRI in breast cancer patients. Combining 18F-FDG PET/CT with ultrasonography or MRI could improve the diagnostic performance compared to 18F-FDG PET/CT alone.

Bone marrow mesenchymal stem cell transplantation in acute brain trauma. Improvement of brain glucose metabolism in a rat model.

AIM: This study was performed to evaluate the effects of intravenously transplanted rat bone-marrow derived mesenchymal stem cells (rBMSCs) in an acute brain trauma model using serial 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in rat models. ANIMALS, METHODS: Trauma models were made using a controlled cortical impact injury device. The stem cell treatment group was treated with intravenous injections of BMSCs, and models without stem cell therapy comprised the control group. Serial 18F-FDG PET images were obtained 1, 7, 14, 21, and 28 days after trauma. The difference in 18F-FDG uptake between day 1 and each time point after trauma was analyzed with SPM2 (uncorrected p < 0.005). RESULTS: The stem cell treatment group demonstrated significantly higher 18F-FDG uptake in the right parietal region at 14 days after trauma than at 1 day after trauma. An increase in glucose metabolism in the right parietal cortex appeared on days 21 and 28 after trauma in the group without stem cell treatment. The 18F-FDG uptake in the brain was improved over a broader area, including the right parietal and right primary somatosensory cortex, on days 21 and 28 after trauma in the stem cell treatment group compared with the group without stem cell treatment. CONCLUSION: BMSC therapy in trauma models led to improved glucose metabolism. This result might support the therapeutic effect of stem cells in brain trauma.

A comparison of cerebral glucose metabolism in Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) share many similar aspects, and making a clinical diagnosis of one disorder over the other relies heavily on an arbitrary criterion, so-called 1-year rule. This study was designed to search for any difference of metabolic patterns in these two disorders using F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) images. We enrolled 16 patients with PD, 13 patients with PDD, and seven patients with DLB. FDG PET was performed, and images were reconstructed by iterative reconstruction using the computed tomography (CT) images, and were normalized to a standard template. Statistical comparison between groups were performed on a voxel-by-voxel basis using t-statistics (two-sample t-test). Compared with the patients with PD, both PDD and DLB patients showed similar patterns of decreased metabolism in bilateral inferior and medial frontal lobes, and right parietal lobe (P(uncorrected) < 0.001). In a direct comparison, DLB patients had significant metabolic decrease (p(uncorrected) < 0.005) in the anterior cingulate compared with those with PDD. These findings support the concept that PDD and DLB have similar underlying neurobiological characteristics, and that they can be regarded as a spectrum of Lewy body disorders.

Formation of an icosahedral structure during the freezing of gold nanoclusters: surface-induced mechanism.

The freezing behavior of gold nanoclusters was studied by employing molecular dynamics simulations based on the semiempirical embedded-atom method. Investigations of the gold nanoclusters revealed that, just after freezing, ordered nanosurfaces with a fivefold symmetry were formed with interior atoms remaining in the disordered state. Further lowering of temperatures induced nanocrystallization of the interior atoms that proceeded from the surface towards the core region, finally leading to an icosahedral structure. These dynamic processes explain why the icosahedral cluster structure is dominantly formed in spite of its energetic metastability.

Transplacental transfer and age-related levels of serum IgG antibodies to the capsular polysaccharides of Streptococcus pneumoniae types 14 and 19 in Korea.

Little is known about the prevalence of naturally acquired IgG antibodies to the capsular polysaccharides of Streptococcus pneumoniae (pneumococcal IgG) in Korea. In the present study, we investigated transplacental transfer and age-related levels of pneumococcal IgG to provide background seroepidemiologic data for S. pneumoniae in Korea. One hundred thirty eight sera were assayed by ELISA for IgG to pneumococcal polysaccharide capsular serotypes 14 and 19, the predominant serotypes for under 15 yr of age in Korea. The subjects were divided into 7 subgroups according to age. The cord/maternal geometric mean titer of pneumococcal were 4.47+/-5.88/5.21 +/- 5.88 for serotype 14, and 4.68 +/- 5.55/6.55 +/- 6.92 for serotype 1 9 (mean +/- standard deviation, microg/mL). After birth, the geometric mean titers of pneumococcal IgG for serotypes 14 and 19 expressed in microg/mL were 1.18+/-2.12 and 1.41+/-2.17 in the 0-6 months group, 0.27+/-0.19 and 0.69+/-0.93 in 7-12 months, 0.21+/-0.22 and 0.64+/-1.32 in 1-2 yr, 0.69+/-0.78 and 2.65+/-2.46 in 3-6 yr, 2.52+/-2.72 and 8.29+/-4.24 in 7-10 yr, respectively. In conclusion, reduced transplacental transfer and very low serum concentrations of pneumococcal IgG may contribute to the susceptibility of neonates, infants, and young children to S. pneumoniae infection.

Dipyridamole myocardial SPECT with low heart rate response indicates cardiac autonomic dysfunction in patients with diabetes.

BACKGROUND: Because dipyridamole is used to assess heart rate (HR) variability, we investigated whether a low HR response during dipyridamole single photon emission computed tomography (SPECT) in patients with diabetes indicates the presence of cardiac autonomic neuropathy (CAN). METHODS AND RESULTS: Subjects were 61 non-insulin-dependent diabetes patients without perfusion defects, myocardial infarction, or arrhythmia who underwent thallium 201 SPECT imaging. The control group comprised 28 subjects without diabetes. HR was measured during infusion of dipyridamole at a rate of 0.14 mg/kg/min, and peak-baseline ratios of 1.20 or less were defined as low. CAN severity was classified by standard autonomic function tests as severe (n = 22), mild (n = 19), or none (n = 20). HR ratios were significantly attenuated in patients with diabetes compared with those in control subjects (1.22 +/- 0.12 vs 1.32 +/- 0.12, P <.001). Among the patients with diabetes, HR ratios decreased as CAN severity increased from none (1.32 +/- 0.10) to mild (1.23 +/- 0.12, P <.05) to severe (1.13 +/- 0.08, P <.005). There was good correlation between HR ratio and R-R interval ratio to deep breathing and to Valsalva, and patients with low HR ratios showed an attenuated response to both tests (all P <.001). The sensitivity and specificity of HR ratios in the detection of CAN were 77% and 74% for severe CAN and 63% and 90% for mild-to-severe CAN, respectively. CONCLUSIONS: In patients with diabetes who have normal dipyridamole SPECT results, an attenuated HR response observed during stress indicates a high likelihood of CAN. Further work that assesses these results in diabetes patients with coronary artery disease is warranted.

Usefulness of diabetic retinopathy as a marker of risk for thallium myocardial perfusion defects in non-insulin-dependent diabetes mellitus.

Among 236 non-insulin-dependent diabetics with clinically suspected coronary artery disease, the rate of thallium-201 myocardial perfusion defects was significantly higher in subjects with (40.6%) than without (22.1%) diabetic retinopathy. Retinopathy was associated with a higher risk of perfusion defects in subjects with cardiac and noncardiac chest pain, and may thus be a useful marker for selecting patients in whom thallium scintigraphy screening is warranted.

The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm.

Paraxial mesoderm in vertebrates gives rise to all trunk and limb skeletal muscles, the trunk skeleton, and portions of the trunk dermis and vasculature. We show here that germline deletion of mouse pMesogenin1, a bHLH class gene specifically expressed in developmentally immature unsegmented paraxial mesoderm, causes complete failure of somite formation and segmentation of the body trunk and tail. At the molecular level, the phenotype features dramatic loss of expression within the presomitic mesoderm of Notch/Delta pathway components and oscillating somitic clock genes that are thought to control segmentation and somitogenesis. Subsequent patterning and specification steps for paraxial mesoderm also fail, leading to a complete absence of all trunk paraxial mesoderm derivatives, which include skeletal muscle, vertebrae, and ribs. We infer that pMesogenin1 is an essential upstream regulator of trunk paraxial mesoderm development and segmentation.

The bHLH class protein pMesogenin1 can specify paraxial mesoderm phenotypes.

A new bHLH gene from mouse that we call pMesogenin1 (referring to paraxial mesoderm-specific expression and regulatory capacities) and its candidate ortholog from Xenopus were isolated and studied comparatively. In both organisms the gene is specifically expressed in unsegmented paraxial mesoderm and its immediate progenitors. A striking feature of pMesogenin1 expression is that it terminates abruptly in presumptive somites (somitomeres). Somitomeres rostral to the pMesogenin1 domain strongly upregulate expression of pMesogenin's closest known paralogs, MesP1 and MesP2 (Thylacine1/2 in Xenopus). Subsequently, the most rostral somitomere becomes a new somite and expression of MesP1/2 is sharply downregulated before this transition. Thus, expression patterns of these bHLH genes, together with that of an additional bHLH gene in the mouse, Paraxis, collectively define discrete but highly dynamic prepatterned subdomains of the paraxial mesoderm. In functional assays, we show that pMesogenin1 from either mouse or frog can efficiently drive nonmesodermal cells to assume a phenotype with molecular and cellular characteristics of early paraxial mesoderm. Among genes induced by added pMesogenin1 is Xwnt-8, a signaling factor that induces a similar repertoire of marker genes and a similar cellular phenotype. Additional target genes induced by pMesogenin1 are ESR4/5, regulators known to play a significant role in segmentation of paraxial mesoderm (W. C. Jen et al., 1999, Genes Dev. 13, 1486-1499). pMesogenin1 differs from other known mesoderm-inducing transcription factors because it does not also activate a dorsal (future axial) mesoderm phenotype, suggesting that pMesogenin1 is involved in specifying paraxial mesoderm. In the context of the intact frog embryo, ectopic pMesogenin1 also actively suppressed axial mesoderm markers and disrupted normal formation of notochord. In addition, we found evidence for cross-regulatory interactions between pMesogenin1 and T-box transcription factors, a family of genes normally expressed in a broader pattern and known to induce multiple types of mesoderm. Based on our results and results from prior studies of related bHLH genes, we propose that pMesogenin1 and its closest known relatives, MesP1/2 (in mouse) and Thylacine1/2 (in Xenopus), comprise a bHLH subfamily devoted to formation and segmentation of paraxial mesoderm.

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

The mouse and human genes encoding the recognition component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.

Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.

Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.

Interfacial properties as stability predictors of lecithin-stabilized perfluorocarbon emulsions.

The purpose of this study was to determine whether the addition of small quantities of minor lecithin components (phosphatidylinositol, phosphatidic acid, lysophosphatidylethanolamine, and cholesterol) and Pluronic F68 to lecithin could improve the stability of lecithin-stabilized perfluorocarbon emulsions. Attempts were made to correlate emulsion stability with interfacial properties (tension and charge). Dynamic interfacial tension was determined using a Teflon Wilhelmy plate method [reported previously (1)]. Emulsions were prepared by microfluidization. Microelectrophoresis was used to measure emulsion droplet charge, and photon correlation spectroscopy and Coulter analysis were used to determine emulsion stability as a function of droplet size. Thermal kinetic accelerated stability testing was conducted. Various droplet size parameters were used to compare emulsion stabilities, and an overall stability ranking, based on these parameters, was obtained for each emulsion. Small quantities of additives altered emulsion stability and these data were correlated with interfacial properties and initial droplet diameters. The addition of cholesterol to lecithin resulted in the most stable perfluorocarbon emulsion.

Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome.

MRF4 (herculin/Myf-6) is one of the four member MyoD family of transcription factors identified by their ability to enforce skeletal muscle differentiation upon a wide variety of nonmuscle cell types. In this study the mouse germline MRF4 gene was disrupted by targeted recombination. Animals homozygous for the MRF4bh1 allele, a deletion of the functionally essential bHLH domain, displayed defective axial myogenesis and rib pattern formation, and they died at birth. Differences in somitogenesis between homozygous MRF4bh1 embryos and their wild-type littermates provided evidence for three distinct myogenic regulatory programs (My1-My3) in the somite, which correlate temporally and spatially with three waves of cellular recruitment to the expanding myotome. The first program (My1), marked initially by Myf-5 expression and followed by myogenin, began on schedule in the MRF4bh1/bh1 embryos at day 8 post coitum (E8). A second program (My2) was highly deficient in homozygous mutant MRF4 embryos, and normal expansion of the myotome failed. Moreover, expression of downstream muscle-specific genes, including FGF-6, which is a candidate regulator of inductive interactions, did not occur normally. The onset of MyoD expression around E10.5 in wild-type embryos marks a third myotomal program (My3), the execution of which was somewhat delayed in MRF4 mutant embryos but ultimately led to extensive myogenesis in the trunk. By E15 it appeared to have largely compensated for the defective My2 program in MRF4 mutants. Homozygous MRF4bh1 animals also showed improper rib pattern formation perhaps due to the absence of signals from cells expressing the My2 program. Finally, a later and relatively mild phenotype was detected in intercostal muscles of newborn animals.

Involvement of JunD in transcriptional activation of the orphan receptor gene nur77 by nerve growth factor and membrane depolarization in PC12 cells.

nur77, an immediate-early gene that encodes an orphan nuclear receptor, is rapidly and transiently induced by nerve growth factor (NGF) stimulation or membrane depolarization in the rat pheochromocytoma-derived cell line PC12. The Nur77 protein can act as a potent transcription activator and may function to regulate the expression of downstream genes in response to extracellular stimuli. We show here that activation of nur77 by NGF treatment and membrane depolarization is signalled through distinct pathways. These distinct signals appear to converge on the same transcription factors acting on the same promoter elements. We show that nur77 activation by both processes requires two cis-acting AP1-like elements, NAP1 and NAP2, which contain the core sequence TGCGTCA centered at 67 and 38 nucleotides upstream of the transcription start site. The NAP elements can confer inducibility by NGF and membrane depolarization on an otherwise unresponsive heterologous promoter. We identified JunD as a key mediator of nur77 activation by reason of the following observations. (i) JunD, but not CREB or other members of the Fos/Jun family, is a component of NAP binding activity in PC12 cell nuclear extracts. (ii) JunD, but not other Fos/Jun family members, specifically transactivates the nur77 promoter through the NAP elements (iii) A dominant-negative mutant of JunD effectively abolishes the activation of nur77 by either NGF treatment or membrane depolarization. These data draw a contrast between the regulation of nur77 with that of c-fos, in which the sequence requirements for activation by NGF treatment and membrane depolarization appear separable, and CREB appears to play a role in activation by both NGF and membrane depolarization.