Precise observation of C. elegans dynamic behaviours under controlled thermal stimulus using a mobile phone-based microscope.

We investigated the temperature-dependent locomotion of Caenorhabditis elegans by using the mobile phone-based microscope. We developed the customized imaging system with mini incubator and smartphone to effectively control the thermal stimulation for precisely observing the temperature-dependent locomotory behaviours of C. elegans. Using the mobile phone-based microscope, we successfully followed the long-term progress of specimens of C. elegans in real time as they hatched and explored their temperature-dependent locomotory behaviour. We are convinced that the mobile phone-based microscope is a useful device for real time and long-term observations of biological samples during incubation, and can make it possible to carry out live observations via wireless communications regardless of location. In addition, this microscope has the potential for widespread use owing to its low cost and compact design.

Multi-chord IR-visible two-color interferometer on KSTAR.

Major parts of an IR-visible two-color interferometer (TCI) on KSTAR have been upgraded for the multi-chord operation: (1) a diode-pumped-solid-state (DPSS) laser (660 nm) replacing the former HeNe laser (633 nm), (2) vacuum-compatible vibration isolator with titanium retro-reflectors, and (3) full digital phase comparator for multi-chord real-time density signals. The commercial compact DPSS laser suits the multiple chord configuration with its strong beam power (500 mW) and long coherent length (>100 m). Ti retro-reflectors are mounted on vacuum-compatible vibration isolators. The isolators are essential for the visible beams to avoid any fringe skips due to their short wavelength, considering the speed of the mechanical vibration (up to hundreds of µm). Field-programmable-gate-array (FPGA) modules count the entire fringes fast enough with a signal output rate up to 1.25 MHz, solving the fringe skip issues. The FPGA module enables the full digital processing of the phase comparator with a CORDIC algorithm after the sampling rate of 160 MS/s for the 40 MHz intermediate frequency of each beam. The full digital signals are transferred to the main plasma control system in real-time. Stable single-input-single-output operation of the KSTAR density control was demonstrated with the TCI. The real-time density profile control is also promising in the near future, with multiple actuators such as pellets and gas puffings.

The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins.

PKR is a serine/threonine protein kinase induced by interferon treatment and activated by double-stranded RNAs. As a result of activation, PKR becomes autophosphorylated and catalyzes phosphorylation of the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2). While studying the regulation of PKR in virus-infected cells, we found that a cellular 58-kDa protein (P58) was recruited by influenza virus to downregulate PKR and thus avoid the kinase's deleterious effects on viral protein synthesis and replication. We now report on the cloning, sequencing, expression, and structural analysis of the P58 PKR inhibitor, a 504-amino-acid hydrophilic protein. P58, expressed as a histidine fusion protein in Escherichia coli, blocked both the autophosphorylation of PKR and phosphorylation of the alpha subunit of eIF-2. Western blot (immunoblot) analysis showed that P58 is present not only in bovine cells but also in human, monkey, and mouse cells, suggesting the protein is highly conserved. Computer analysis revealed that P58 contains regions of homology to the DnaJ family of proteins and a much lesser degree of similarity to the PKR natural substrate, eIF-2 alpha. Finally, P58 contains nine tandemly arranged 34-amino-acid repeats, demonstrating that the PKR inhibitor is a member of the tetratricopeptide repeat family of proteins, the only member identified thus far with a known biochemical function.

Direct synthesis of well-ordered and unusually reactive MnSBA-15 mesoporous molecular sieves with high manganese content.

The mesoporous MnSBA-15 materials with different n(Si)/n(Mn) ratios of 4, 8, 20, and 50 have been synthesized, for the first time, using manganese nitrate tetrahydrate and Pluronic 123 triblock polymer [(EO)20(PO)70(EO)20] by simply adjusting the molar ratio of water to hydrochloric acid (n(H2O)/n(HCl)) under direct hydrothermal conditions. For the effect of structural and textural properties with incorporation of manganese, the MnSBA-15 has been synthesized with different synthesis temperatures at the fixed molar ratios of n(Si)/n(Mn) = 4 and n(H2O)/n(HCl) = 295 in the synthesis gel. The hydrothermal and thermal stabilities of MnSBA-15 have also been investigated. The calcined MnSBA-15 materials prepared have been characterized by ICP-AES, XRD, N2 adsorption, ESR, FE-SEM, and TEM. The ICP-AES studies show a higher amount of manganese incorporation on the silica pore walls, as MnSBA-15 with a n(Si)/n(Mn) ratio up to 2.2 can be successfully prepared at a fixed n(H2O)/n(HCl) molar ratio of 295 by adjusting the ratios of n(Si)/n(Mn) in the synthesis gel. The structural and textural properties of calcined MnSBA-15 prepared can be found by the results of XRD and N2 adsorption. The investigation of ESR results clearly describe the effect of structure and Mn species coordination on the SBA-15 silica pore walls while the uniform pore diameter and rope-like hexagonal mesoporous structure of MnSBA-15 can be identified by TEM and FE-SEM images. With increasing synthesis temperature, an increase the unit cell parameter, pore size, and pore volume and a decrease the specific surface area and pore wall thickness of MnSBA-15 can be obviously noted by the results of XRD and N2 adsorption. The hexagonal MnSBA-15 materials prepared could be tested as catalysts in epoxidation of trans-stilbene to produce trans-stilbene oxide under various optimal conditions while their catalytic properties could also be compared to the results of MnMCM-41 and ZrMnMCM-41.

Immunological identification of cholesterol ester hydrolase in the steroidogenic tissues, adrenal glands and testis.

Monoclonal antibodies were generated against the purified pancreatic cholesterol ester hydrolase (CEH, EC 3.1.1.13) to examine the expression of CEH in various bovine tissues. The presence of CEH isozyme antigenically indistinguishable from pancreatic enzyme in the steroidogenic tissues, adrenal glands and testis has been first demonstrated here using the immunoprecipitation method. These results suggest that CEH isozyme, similar to pancreatic CEH, might be involved in the cholesterol metabolism in the steroidogenic tissue.

Phospholipase D1 is located and activated by protein kinase C alpha in the plasma membrane in 3Y1 fibroblast cell.

The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase C alpha (PKC alpha) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKC alpha (GFP-PKC alpha) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKC alpha-stimulated PLD activity segregated with a plasma membrane marker, even though a significant amount was co-fractionated with markers for endoplasmic reticulum (ER) and Golgi. Upon treatment with phorbol myristate acetate (PMA), PKC alpha translocated from the cytosolic fraction to the membrane fraction to which PLD1 also localized. GFP-PLD1 was found in the plasma membrane as well as a in a perinuclear compartment consistent with ER and Golgi and in other dispersed vesicular structures in the cytoplasm. However, most of GFP-PKC alpha was translocated from the cytosol to the plasma membrane after treatment with PMA. From these results, we concluded that the plasma membrane is the major site of PLD1 activation by PKC alpha in 3Y1 cells.

Phorbol myristate acetate-dependent association of protein kinase C alpha with phospholipase D1 in intact cells.

A phospholipase D1 (PLD1) was purified from rat brain by the use of antibody-coupled protein A Sepharose. We found that protein kinase C alp (PKCalpha) stimulated PLD1 activity in the presence of phorbol myristate acetate (PMA). PMA-dependent association of PKCalpha with PLD1 was verified in NIH-3T3 fibroblast cells, and COS7 cells transiently expressing PLD1 as well as in vitro suggesting that the activation of PLD1 resulted from direct association of PKCalpha with PLD1.

Comparison of radionuclide and enzymatic estimate of infarct size in patients with acute myocardial infarction.

A comparison was made of the estimated size of the myocardial infarction occurring in 26 patients with a first infarction using creatine kinase (CK) enzyme release between radionuclide gated blood pool measurement of total and regional ventricular function and thallium-201 scintigraphic measurement of myocardial perfusion defects. Creatine kinase estimates of infarct size (enzymatic infarct size) correlated closely with the percent of abnormal contracting regions, left ventricular ejection fraction and thallium-201 estimates of percent of abnormal perfusion area (r = 0.78, 0.69 and 0.74, respectively, p less than 0.01). A close correlation also existed between percent abnormal perfusion area and percent of abnormal contracting regions (r = 0.81, p less than 0.01) and left ventricular ejection fraction (r = 0.69, p less than 0.01). Enzymatic infarct size was larger in anterior (116 +/- 37 CK-g-Eq) than inferior (52 +/- 29 CK-g-Eq) myocardial infarction (p less than 0.01) and was associated with significantly more left ventricular functional impairment as determined by left ventricular ejection fraction (33 +/- 7 versus 60 +/- 10%) (p less than 0.01) and percent abnormal perfusion area (58 +/- 14 versus 13 +/- 12) (p less than 0.01). No significant correlation was observed between enzymatic infarct size and right ventricular ejection fraction. These different methods of estimating infarct size correlated closely with each other in these patients with a first uncomplicated myocardial infarction.

Intravitreal anti-vascular endothelial growth factor monotherapy for large submacular hemorrhage secondary to neovascular age-related macular degeneration.

PURPOSE: To evaluate the efficacy of anti-vascular endothelial growth factor (VEGF) monotherapy for large submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (nAMD). METHODS: A total of 49 treatment-naive patients (49 eyes) with large SMH (more than five disc areas (DAs)) secondary to nAMD were retrospectively included. All patients were treated with an initial series of 3 monthly intravitreal anti-VEGF injections, followed by as-needed injections. At the 12-month follow-up, changes in best-corrected visual acuity (BCVA), hemorrhage area, central foveal thickness, and development of vitreous hemorrhage after treatment were evaluated. RESULTS: The mean SMH area was 13.9 ± 8.8 disk areas (DAs) and mean symptom duration was 7.25 ± 5.9 days at baseline. The mean number of injections was 4.49 ± 1.61. Twelve months after treatment, the mean BCVA significantly improved from 1.14 ± 0.61 logarithm of the minimum angle of resolution (logMAR; 20/276, Snellen equivalent) to 0.82 ± 0.53 logMAR (20/132; P = 0.002). Twenty-four eyes (49%) showed improvement of more than three lines of BCVA at 12 months after treatment. Baseline BCVA (odds ratio (OR), 5.119; 95% confidence interval (CI), 1.993-9.545; P = 0.004), duration of symptoms (OR, 0.727; 95% CI, 0.332-0.952; P = 0.024), hemorrhage area (OR, 0.892; 95% CI, 0.721-0.965; P = 0.011), and baseline central foveal thickness (OR, 0.881; 95% CI, 0.722-0.945; P = 0.032) were significantly associated with good visual acuity 12 months after treatment. CONCLUSIONS: Intravitreal anti-VEGF monotherapy is a valuable treatment option for large SMH secondary to nAMD.

cDNA cloning of the 210-kDa paraneoplastic pemphigus antigen reveals that envoplakin is a component of the antigen complex.

Although the 210 and 190-kDa proteins are the most frequently detected antigens reacting with sera of patients with paraneoplastic pemphigus (PNP) in immunoblot analysis, there is still uncertainty as to the nature of these PNP antigens. To isolate and characterize a cDNA clone encoding the 210-kDa PNP antigen, we screened a human keratinocyte lambda gt 11 cDNA expression library by the immunoperoxidase method with serum IgG from a PNP patient. The IgG used for the immunoscreening of a keratinocyte cDNA expression library recognized 210- and 190-kDa antigens by immunoblotting. A single clone, called here the PNP clone, producing a fusion protein that reacted strongly with the patient's IgG, was further characterized. Only the PNP patient's IgG, but not IgG from a normal control, pemphigus foliaceus, or pemphigus vulgaris patients, bound the plaques of this positive clone. Furthermore, PNP IgG affinity purified on plaques of this clone, but not unrelated clones, bound to keratinocyte cell surfaces by immunofluorescence and reacted with the 210-kDa PNP antigen by immunoblotting. EcoRI digestion of the clone's cDNA insert demonstrated a 1.4-kbp fragment. This cDNA insert was placed into a M13 mp 18 vector and sequenced. Sequence analysis revealed that the cDNA insert of the PNP clone encodes a part of the central rod domain and the COOH-terminal C domain of envoplakin, a newly defined precursor of the cornified envelope that is homologous to desmoplakin. This result demonstrates that the 210-kDa PNP antigen is envoplakin and PNP is an autoimmune disease that produces autoantibodies against intermediate filament-associated proteins in desmosomes and hemidesmosomes, desmoplakin, bullous pemphigoid antigen 1 (BPAG 1), and envoplakin.

Activation of phospholipase D1 by direct interaction with ADP-ribosylation factor 1 and RalA.

Phospholipase D1 (PLD1) is known to be activated by ADP-ribosylation factor 1 (ARF1). We report here that ARF1 co-immunoprecipitates with PLD1 and that the ARF1-dependent PLD activation is induced by the direct interaction between ARF1 and PLD1. We found that RalA, another member of the small GTP-binding proteins, synergistically enhances the ARF1-dependent PLD activity with an EC50 of about 30 nM. Using in vitro binding assay, we show that ARF1 and RalA directly interact with different sites of PLD1. The results suggest that the independent interactions of RalA and ARF1 with PLD1 are responsible for the synergistic activation.

Triphasic perfusion computed tomography in acute middle cerebral artery stroke: a correlation with angiographic findings.

OBJECTIVE: To evaluate the usefulness of triphasic perfusion computed tomography (TPCT) in diagnosing middle cerebral artery (MCA) occlusion and in assessing the perfusion deficit and collateral circulation in patients with acute ischemic stroke. BACKGROUND: Conventional angiography is the criterion standard for the diagnosis of MCA occlusion and for the assessment of perfusion deficit and collateral blood supply. The risk of hemorrhagic transformation after recanalization of occluded arteries by thrombolytic therapy is considered high when pretherapeutic residual flow is markedly reduced. PATIENTS AND METHODS: In 8 patients within 3 hours of onset of acute MCA stroke, precontrast computed tomographic scans were taken, and then TPCT was performed after power-injector controlled intravenous administration of contrast media. Sequential images of early, middle, and late phases were obtained. The whole procedure took 5 minutes. Perfusion deficit on TPCT was graded as "severe" or "moderate," depending on the state of collateral flow. Digital subtraction angiography (DSA) was performed in all patients within 6 hours of acute stroke. Direct intra-arterial urokinase infusion was begun immediately after the angiographic superselection of the MCA occlusion site in 6 of the 8 patients within 7 hours of onset (range, 4.3-6.2 hours). RESULTS: The DSA findings showed occlusion of the MCA stem (n = 1) and at the bifurcation (n = 4). The sites of proximal MCA occlusion could be identified on the early and middle images of TPCT in all 5 patients. On DSA findings, all 8 patients had a zone of perfusion deficit with markedly slow leptomeningeal collaterals and a zone of perfusion deficit with no collaterals. The zone of severe perfusion deficit on TPCT corresponded to the zone of perfusion deficit with no or few collaterals on angiography, and the zone of moderate perfusion deficit on TPCT corresponded to that of perfusion deficit with markedly slow leptomeningeal collaterals. Early parenchymal hypoattenuation on precontrast computed tomography was confined to the zone of severe perfusion deficit on TPCT. The initial National Institutes of Health Stroke Scale score correlated better with the total extent of severe perfusion deficit and moderate perfusion deficit on TPCT than that of severe perfusion deficit alone. After direct intra-arterial thrombolysis within 7 hours of onset, symptomatic hemorrhagic transformation did not develop in 4 patients with small severe perfusion deficit (33% or less of the presumed MCA territory). However, the remaining 2 patients with large severe perfusion deficit (more than 50% of the presumed MCA territory) deteriorated to death with hemorrhagic transformation. CONCLUSIONS: Triphasic perfusion computed tomography is useful for diagnosing proximal MCA occlusion and assessing perfusion deficit and collateral circulation as reliably as DSA. The zone of severe perfusion deficit on TPCT may be presumed to be the ischemic core, and that of moderate perfusion deficit, the penumbra zone. Triphasic perfusion computed tomography may be used as a rapid and noninvasive tool to make thrombolysis safer.

Initial and follow-up brain MRI findings and correlation with the clinical course in Wilson's disease.

We performed pretreatment brain MRIs in 25 patients with neurologically symptomatic Wilson's disease (WD) and clinical and MRI follow-up in 16 of them. All 25 pretreatment MRIs revealed abnormalities, with abnormal high-signal intensity (HSI) in bilateral thalami being the most common (92%). HSI lesions in the brainstem (84%) and the basal ganglia (72%) were also common. Brain atrophy was present in 88% of the 25 patients. In the follow-up period of 5 to 24 months, during which the patients were treated with D-penicillamine, both HSI lesions and neurologic symptoms improved in 88% of the 16 patients, but the brain atrophy did not change.

Rapid and simple measurement of serotonin N-acetyltransferase activity by liquid biphasic diffusion assay.

We report here a rapid, simple, and accurate method to assay for serotonin N-acetyltransferase (NAT) activity. This assay relies on the selective diffusion of radiolabeled acetyltryptamine into a water-immiscible scintillation fluid. Unlike organic solvent extraction, thin-layer chromatography, or high performance liquid chromatography, the separation of acetyltryptamine from acetyl CoA and tryptamine is not required in the method. Moreover, the limit of sensitivity is less than 4 pmol of N-acetyltryptamine formed per sample. Enhancement of NAT activity upon beta-adrenergic receptor stimulation in the rat pineal gland was clearly detected with this method. In addition, the NAT activity measurements obtained with this method agreed quantitatively in the pineal gland and other brain tissues with the conventional organic solvent extraction method. The results suggest that this liquid biphasic diffusion assay is applicable to the detection of NAT activity in tissues and cells.

Ultrasound diagnosis of nonobstetric disease during pregnancy.

The use of ultrasound in obstetrics is well established; however, its use in nonobstetric disease during pregnancy has not been emphasized. A diagnostic work-up during pregnancy is complicated by the fact that many of the usual tests require radiation to the fetus. This paper presents 3 cases in which ultrasonic scanning contributed to the diagnosis of nonobstetric disease during pregnancy. Postitive findings included enlarged edematous pancreas, gallstones, and splenomegaly. In 1 case, the finding of a normal gallbladder was helpful. When usual diagnostic procedures such as oral cholecystogram, retrograde endoscopic pancreatography, and nuclear medicine scans are perhaps contraindicated during pregnancy, the ultrasound scan is the diagnostic test of choice.

Ultrasound air contrast transfusion technique: an aid to managing the Rh-affected fetus with ascites.

The successful management of an Rh-sensitized pregnant patient is reported in whom fetal intrauterine transfusions were utilized despite the presence of fetal ascites before the 26th week of gestation. A new ultrasound air contrast technique is described that confirms the correct intraabdominal placement of the transfused blood. The technique is not dependent on the amount of blood introduced, nor is it masked by fetal ascites.

Ultrasound demonstration of wandering spleen.

The correct preoperative diagnosis of a wandering spleen is infrequently made either clinically or with conventional radiologic studies. Since ultrasound can readily identify the spleen, it may assist in making this diagnosis. We describe three patients to illustrate the advantages and usefulness of ultrasound. In one postmastectomy patient, the suspected abdominal metastasis was shown to be a displaced spleen, thus obviating the necessity for an exploratory laparotomy. In the other two patients, ultrasound diagnosis was helpful in clinical treatment by localizing the wandering spleen. Ultrasound evaluation is particularly suited for patients with obscure abdominal masses because it is safe, simple, and fast.

Effect of a novel saponin adjuvant derived from Quillaja saponaria on the immune response to recombinant hepatitis B surface antigen.

Adjuvant activity of saponins extracted from the South American tree Quillaja saponaria has been demonstrated with many antigens. Recently, four saponin fractions (designated as QS-7, QS-17, QS-18, and QS-21) with adjuvant activity were purified by reverse phase chromatography. In particular, efficacy of the less toxic QS-21 fraction has been demonstrated with several recombinant viral antigens including HIV gp120. Here, we report a novel saponin fraction (designated as QS-L1) derived from Quillaja saponaria. Unlike previously identified saponins, QS-L1 had a different chemical structure and showed adjuvant activity only when administered in the presence of alum-precipitated antigen. Interestingly, the QS-L1 greatly increased not only a humoral immune response but also cellular immune response to recombinant hepatitis B virus surface antigen (HBsAg). Furthermore, QS-L1 showed lower toxicity in vivo and in vitro than the previously identified saponin fraction, QS-21. Finally, we examined the chemical structure of the QS-L1 using mass spectroscopic analysis, carbohydrate composition analysis and NMR spectroscopic analysis. Thus, our results indicated that this novel QS-L1 saponin fraction had several desirable properties required for an effective adjuvant.

Ultrasonographic diagnosis of fetal hydronephrosis in utero.

Ultrasonography offers a noninvasive method to visualize antenatal or neonatal anatomy which will lead to early detection and treatment of abnormalities. In these 2 cases, ultrasound allowed prompt diagnosis of hydronephrosis despite normal physical examination and laboratory values at birth.